kiran et al, j. global trends pharm sci, 2021; 12 (1

Kiran et al, J. Global Trends Pharm Sci, 2021; 12 (1): 9095 - 9108

9095 © Journal of Global Trends in Pharmaceutical Sciences

“PROTECTIVE ROLE OF EXCOECARIA AGALLOCHA L AGAINST

STREPTOZOTOCIN INDUCED DIABETES AND RELATED DIABETIC

NEUROPATHY COMPLICATION”

G. Kiran1, Ganesh N. Sharma2, Birendra Shrivastava3, A. M. S. Sudhakar Babu4

1Deparatment of Pharmacology, Vishwa Bharathi College of Pharmaceutical Sciences, Perecherla, Guntur, Andhra Pradesh, India.

2,3Department of Pharmacology, Jaipur National University, Jaipur, Rajasthan, India.

4Department of Pharmaceutics, Veda College of Pharmacy, Addanki, Praksham, India.

*Corresponding author E-mail: [email protected]

ARTICLE INFO ABSTRACT

Key words:

Excoecaria agallocha,

streptozotocin STZ , lipoprotein-cholesterol,

Lipoprotein,

hyperalagesia, TNF-α,

TGF-β and IL-1β

The present study was designed to investigate the effect of different

extracts of Excoecaria agallocha L. against diabetes mellitus and its

related complications. Diabetes was induced by intraperitoneal

administration of streptozotocin STZ (60 mg/kg) for the development of

diabetic nephropathy and neuropathy. Development of neuropathy was

evident from marked hyperalgesia (thermal as well as mechanical) and

tactile allodynia along with reduced MNCV. Elevated level of nitrite, TNF-

α, TGF-β and IL-1β were also assessed in sciatic nerve of diabetic

neuropathy animals. Treatment with different extracts of Excoecaria

agallocha L significantly attenuated elevated thermal and mechanical

hyperalgesia. Elevated tactile allodynia assessed using flexible Von Frey

filaments was also reversed by extracts. Treatment with both the extracts

significantly attenuated the parameters of oxidative stress in sciatic nerve

of diabetic neuropathic rats. Also, level of nitrite, TNF-α, TGF-β and IL1β

was significantly increased in sciatic nerve of diabetic neuropathy

animals,that were ameliorated by treatment with Excoecaria agallocha L.

extracts.Histopathological changes in sciatic nerve of diabetic rats were

also reversed by the treatment. These findings suggested that the

Excoecaria agallocha L. may be used to manage the diabetes mellitus and

its related complications such as diabetic neuropathy.

INTRODUCTION

Diabetes mellitus is a metabolic

disorder characterized by a loss of glucose

homeostasis with disturbances of carbohydrate,

fat and protein metabolism resulting from

defects in insulin secretion, insulin action, or

both. Without enough insulin, the cells cannot

absorb sufficient glucose from the blood; hence

blood glucose levels increase, and result as

hyperglycemia. If the blood glucose level

remains high over a long period, it can result in

long-term damage to organs, such as kidneys,

liver, eyes, nerves, heart and blood vessels.

Complications in some of these organs can lead

to death also. Diabetes mellitus (sometimes

called "sugar diabetes") is a condition that

occurs when the body can't use glucose (a type

of sugar) normally. Glucose is the main source

of energy for the body's cells. The levels of

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glucose in the blood are controlled by a

hormone called insulin, which is made by the

pancreas.

Induction of Diabetic Neuropathy:

Diabetes was induced by administration of

Streptozotocin (STZ; 60mg/kg i.p.) prepared in

fresh citrate buffer (pH 4.5), 15 minutes after

nicotinamide (230 mg/kg, i.p.). Determining

fasting blood glucose (FBG) level after 72 h of

STZ injection confirmed development of

diabetes. The rats with FBG level ≥ 250 mg/dl

were included in the study. Different doses of

the extracts (200 and 400 mg/kg) were selected

on the basis of oral acute toxicity studies

reported in the literature. Development of

diabetic neuropathy was assessed by using

behavioral parameters on 60th day. Diabetic

neuropathy was evident from marked

Hyperalgesia (thermal as well as mechanical)

and tactile allodynia along with reduced

MNCV.After 60days of streptozotocin

induction, treatment with extract and standard

was continued for next 30 days.

Body weight, blood glucose and serum

insulin estimation: Body weight of each

animal was measured at the start of study and

animals with similar weight were grouped

together. Body weight of each group was

measured periodically till end of study. FBG

level was estimated at interval of 15 days by

using commercial enzymatic kits procured

from Reckon Diagnostics Pvt. Ltd. INDIA

throughout the study. Serum insulin was

determined by Insulin ELISA kit (DRG,

Germany) in blood collected into tubes with

anticoagulant.

Estimation of Thermal Hyperalgesia:

Tail-immersion test: Lower 5 cm portion of

the tail of experimental rat was immersed in a

cup containing hot water (55 ± 0.5˚C). Rat

responds by withdrawing its tail or struggles

(cut-off 12 s). Thus Hyperalgesia was indicated

by reduced reaction time for tail withdrawal.

Hot-plate test: Eddy’s Hot- Plate method was

used to assess central/peripheral. Experimental

animals were individually placed on electrically

heated surface (55 ± 1˚C) and animal responded

by jumping, paw licking to avoid the heat. Cut-

off time was 15 s to avoid paw damage.

Assessment of Mechanical Hyperalgesia:

Randall-Selitto analgesiometer: Paw pressure

threshold was determined using Randall-Selitto

analgesiometer (Ugo Basile, Italy). Hind paw

of the rat were subjected to increasing pressure

at a rate of 10 gm/s and the cut-off pressure

was 250 g to avoid tissue injury. Withdrawal of

animal paw is indicated as Hyperalgesia state

expressed as grams. Experiment was repeated 5

times at an interval of 15 minutes.

Assessment of tactile Allodynia: Von Frey

filaments range (0.4-64 g; Ugo Basile, Italy,

Woodland Hills, CA) were used to evaluate

tactile withdrawal threshold of the hind paw of

experimental animals. Rats were placed in

restraining cages with mesh floor and filaments

were applied perpendicularly on plantar surface

of hind paw with sufficient force to bend the

filament for 6s. Withdrawal or paw flinching

was implicated as positive response.

Experiment was repeated 5 times at an interval

of 5minutes.

Motor Nerve Conduction Velocity (MNCV): Experiment was performed in diabetic rats as

previously given by Morani and Bodhankar,

2008 with slight modification. Rats were

anesthetized with thiopental sodium (50 mg/kg,

i.p.) and the dorsal side of rat’s paw was shaved

Sciatic and tibial nerves were stimulated

respectively by 200 μs square wave pulse

through needle electrodes (1.0-1.5 mA, 2.0

mV/D). Responses were recorded using data

acquisition system (AD Instrument Pvt. Ltd.,

Lab Chart 7.3, and Australia). The MNCV was

determined using the following formula:

Biochemical analysis: For biochemical

estimations, a 10% (w/v) tissue homogenate

(sciatic nerve) was prepared in phosphate

buffered saline (pH 7.4) using a Teflon

homogenizer. The homogenate was centrifuged

at 1000 gm for 10 minutes at 4˚C to remove

nuclei and unbroken cells. The pellet was

discarded and clear supernatant thus obtained

was used to assay thiobarbituric acid reactive

substances (TBARS) and level of antioxidant

enzymes, viz. superoxide dismutase (SOD) and

reduced glutathione (GSH).



Statistical analysis: Statistical analysis was

performed using Graph Pad Prism 6. Values

were expressed as mean ± SEM and one way

analysis of variance (ANOVA) was used for

statistical analysis.

RREESSUULLTTSS AANNDD DDIISSCCUUSSSSIIOONN::

Diabetic Neuropathy: Excoecaria agallocha

extract (AEEA and HAEA) were evaluated for

their effect in diabetic neuropathy at different

doses of 200 and 400 mg/kg.

Effect of Excoecaria agallocha (AEEA and

HAEA) on BW: Administration of AEEA at a

dose of 200 and 400 mg/kg increased the BW

significantly by 15.41 and 21.16 %

respectively. HAEA increased the BW by

16.31and 18.68 % respectively at dose of 200

and 400 mg/kg. Gabapentin at dose of 30

mg/kg increased the BW by 4.51 % (Figure

48).

A significant alleviated FBG level was

observed in DPN rats treated with AEEA and

HAEA at 200 mg/kg and 400 mg/kg from

281±10.69 to 124±10.78 mg/dl and 121±10.77

to 80 ±10.70 mg/dl respectively. Whereas

Gabapentin, a standard drug for neuropathic

pain reduced FBG level from 316 ± 10.98 to 74

± 10.12 mg/ dl (Figure 51).

Administration of 200 and 400 mg/kg

dose of AEEA and HAEA for 30days produced

significant increase in serum insulin level

[AEEA (11.4 ±1.50, 18.3±0.13 μIU/ mL

respectively); HAEA (17.4 ± 1.35, 19.1±1.36

μIU/m respectively]. Gabapentin at dose of

30mg/kg increased the serum insulin level to

20.6 ± 1.39 μIU/ mL (Figure 52).

Treatment with AEEA and HAEA for 30 days

showed marked increase in nociceptive

threshold as compared to DPN control group

dose dependently. Treatment with AEEA at

200 and 400 mg/kg increase the reaction time

i.e. 6.9± 0.67s, and 10.3±0.23s respectively.

HAEA also increase the reaction time to

8.9±0.48s, and 14.9 ± 0.46s respectively.

Gabapentin at dose of 30 mg/kg increased the

reaction time to 14.9 ± 0.63 s. (Figure 53).

Effect of Excoecaria agallocha extracts

(AEEA and HAEA) on thermal

Hyperalgesia using hot plate method:

Administration of AEEA and HAEA to

diabetic rats showed dose dependent increase in

nociceptive threshold in comparison to DPN

control group. Treatment with AEEA at 200

and 400 mg/kg increase the pain threshold i.e.

6.9 ± 0.43 s, and 9.1 ± 0.76 s respectively

whereas with HAEA, 8.9 ± 0.70 s, and 10.1 ±

0.85 s respectively. Gabapentin at dose of 30

mg/kg increased the pain threshold to 9.8

±0.54 s. (Figure 54). Induction of DPN with

STZ (i.p.) significantly (p <0.001) reduced paw

withdrawal threshold by 60th days in DPN

control rats (19.6 ± 1.34 gm) in comparison to

normal control rats (61.8 ± 1.28 gm).

Effect of Excoecaria agallocha extracts

(AEEA and HAEA) on motor nerve

conduction velocity: Induction of diabetic

neuropathy with STZ administration produced

significant reduction in motor nerve conduction

velocity (MNCV) was observed in DPN control

group on 60th day (22.01 ± 1.32 m/s) in

comparison to normal control (54.4 ± 1.12

m/s).

DISCUSSION:

Diabetes mellitus is a group of

longstanding disorders manifested by

hyperglycemia. Etiology of diabetes is related

to defects in insulin secretion and/or insulin

action that leading to hyperglycemia. It has

been revealed that chronic hyperglycemia, the

hallmark of DM, leads to protein glycation and

formation of advanced glycation products

(AGE), which contribute to both microvascular

and macrovascular complications.

These complications affect the entire

body specially, the eyes, nerves, kidneys, livers,

blood vessels, heart and contribute to morbidity

and mortality of diabetes. In addition,

hyperglycemia can lead to high production of

reactive oxygen species (ROS) and

simultaneous reduction of the antioxidant

defence mechanisms, which can cause

oxidative stress. Diabetic neuropathy is the

prevalent and serious complication of DM and

is one of the main reasons for high morbidity

and mortality. Chronic sensorimotor DPN is

the most frequent type of diabetic neuropathy

and is indicated by reduced NCV in longer

nerves.



Table 1: Diabetic Neuropathy

Sr. No Groups Treatment

1. Group 1 Normal control

2. Group 2 DN control

3. Group 3 DN rats + 200 AEEA

4. Group 4 DN rats + 400 AEEA

5. Group 5 DN rats + 200 HEAA

6. Group 6 DN rats + 400 HEAA

7. Group 7 DN rats + 30 mg/kg Gabapentin

Table 2: Effect of AEEA and HAEA on body weight (gm) in diabetic neuropathy

wistar rats

Days Normal Diabetic AEEA

200

AEEA

400

HAEA

200

HAEA

400

Gabapentin

30

Basal

Day

252 ±

10.34

291 ±

9.78#

329 ±

10.12

336 ±

10.13

332 ±

10.11

318 ±

10.08

334 ± 10.45

60th

Day

279 ±

10.54

283 ±

10.34#

287 ±

10.56*

292 ±

10.29**

280 ±

10.23*

269 ±

10.18*

263 ±

10.65***

75th

Day

292 ±

10.69

267 ±

10.92#

269

±10.67

273 ±

10.69**

265 ±

10.36*

241 ±

10.23***

238 ±

9.54***

90th

Day

298 ±

10.89

227 ±

10.56#

253 ±

10.79

250 ±

10.84**

231 ±

10.45**

210 ±

10.20***

208 ±

10.50***

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared with control

and *P < 0.05; **P < 0.01; and ***P < 0.001 in comparison with diabetic control.

Figure 1: Effect of AEEA and HAEA on body weight (gm) in diabetic neuropathy

wistar rats

Effect of alcohol and Hydro alcohol on body weight in diabetic

neuropathy

400

350

300

250

200

150

100

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin

30

50

0 Basal Day 60th Day 75th Day 90th Day



Table 3: Effect of Excoecaria agallocha extracts (AEEA and HAEA) on food intake:

AEEA (200 and 400 mg/kg) reduced food intake to 53 ± 1.67, and 48 ± 1.71 gm/rat/day respectively,

whereas HAEA at 200 and 400 mg/kg reduced food intake to 57 ± 1.80, and 30 ± 1.12 gm/rat/day

respectively by the end of study in comparison to DPN control rats (70 ± 1.45 gm/rat/day;

p<0.001)(Figure 2).

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared with control

and *P < 0.05; **P < 0.01; and ***P < 0.001 in comparison with diabetic control.

Figure 2: Effect of AEEA and HAEA on food intake in diabetic neuropathy wistar rats

Days Normal Diabetic AEEA 200 AEEA 400 HAEA 200 HAEA 400 Gabapentin 30

60th Day 33 ±

1.67

65 ±

1.67#

64 ±

1.11

46 ±

1.55

51 ±

1.67

31 ±

1.45

31 ± 1.12

75th Day 27 ±

1.78

68 ±

1.34#

58 ±

1.45*

41 ±

1.67**

54 ±

1.78***

35 ±

1.71**

36± 1.34***

90th Day 24 ±

1.89

70 ±

1.45#

53 ±

1.67

48 ±

1.71**

57 ±

1.80***

30 ±

1.12***

35 ±

1.65***

Effect of alcohol and Hydro alcohol on food intake in diabetic

neuropathy 80

70

60

50

40

30

20

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin

30

10

0 60th Day 75th Day 90th Day



Effect of Excoecaria agallocha extracts (AEEA and HAEA) on water intake:

Table 4: Effect of AEEA and HAEA on water intake in diabetic-neuropathy wistar rats


200

AEEA

400

HAEA

200

HAEA

400

Gabapen

tin

30

60th

Day

55 ±

2.20

155 ±

2.78#

138 ±

2.7

131 ±

2.34

146 ±

2.46

139 ±

2.89

139 ±

2.46

75th

Day

54 ±

2.27

161 ±

2.88#

131 ±

2.37

127 ±

3.67*

123 ±

2.98*

116 ±

2.13***

110 ±

2.10***

90th

Day

56 ±

2.67

167 ±

2.99#

126 ±

3.84

90 ±

2.87*

88 ±

2.67**

63 ±

2.32***

60 ±

2.01

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared with control and *P < 0.05;

**P < 0.01; and ***P < 0.001 in comparison with diabetic control.

Figure 3: Effect of AEEA and HAEA on water intake in diabetic neuropathy wistar

rats

Effect of alcohol and Hydro alcohol on water intake in diabetic

neuropathy 180

160

140

120

100

80

60

40

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin

30

20




Table 5: Effect of AEEA and HAEA on FBG in diabetic-neuropathy wistar

rats


200

AEEA

400

HAEA

200

HAEA 400 Gabapentin

30

0 Day 58 ±

8.34

288 ±

10.12#

300 ±

10.12

325 ±

10.28

320 ±

10.50

321 ±

10.12

326 ± 10.98

60th

Day

62 ±

8.45

361 ±

10.78#

342 ±

10.23

289 ±

10.45*

300 ±

10.87*

267 ±

10.34**

260 ±

10.45***

75th

Day

70 ±

8.52

400 ±

10.98#

310 ±

10.47

255 ±

10.56*

256 ±

10.20**

121 ±

10.87***

115 ±

10.76***

90th

Day

61 ±

8.60

434 ±

10.65#

281 ±

10.69

124 ±

10.78*

121 ±

10.77**

80 ±

10.70***

74 ±

10.12***

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared with

control and *P < 0.05; **P < 0.01; and ***P < 0.001 in comparison with diabetic control.

Figure 4: Effect of AEEA and HEAA on FBG in diabetic neuropathy wistar rat

Table 6: Effect of AEEA and HEAA on serum insulin in diabetic-neuropathy wistar

rats


200

AEEA

400

HAEA

200

HAEA

400

Gabapentin

30

60th

Day

24.4 ±

1.35

14.1 ±

1.56#

13.7 ±

1.49

13.5 ±

1.30

14.1 ±

1.11

15.5 ±

1.30

14.2 ± 1.21

90th

Day

21.2 ±

1.56

9.0 ±

1.45#

11.4 ±

1.50

18.3 ±

1.58*

17.4 ±

1.35**

19.1 ±

1.36***

20.6 ±

1.39***

Effect of alcohol and Hydro alcohol on FBG in diabetic

neuropathy

500

450

400

350

300

250

200

150

100

50

0

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin

30 0 Day 60th Day 75th Day 90th Day

glu

cose

leve

l (m

g/d

l)



Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared

with control and *P < 0.05; **P < 0.01; and ***P < 0.001 in comparison with

diabetic control.

Figure 5: Effect of AEEA and HEAA on serum insulin in diabetic neuropathy wistar rats

Effect of Excoecaria agallocha extracts (AEEA and HAEA) on thermal Hyperalgesia using tail immersion method:

Table 7: Effect of AEEA and HAEA on thermal Hyperalgesia using tail immersion

diabetic-neuropathy wistar rats


200

AEEA

400

HAEA

200

HAEA

400

Gabapentin

30

60th

Day

15.87 ±

0.12

3.7 ±

0.21#

4.7 ±

0.11

4.5 ±

0.11

4.6 ±

0.12

4.1 ±

0.10

4.7 ± 0.11

75th

Day

15.76 ±

0.34

3.9 ±

0.45#

6.0 ±

0.23

6.1 ±

0.20*

6.8 ±

0.32**

9.4 ±

0.30***

9.9 ±

0.78***

90th

Day

15.98±

0.56

4.3 ±

0.87#

6.9 ±

0.67*

10.3 ±

0.23*

8.9 ±

0.48**

14.9 ±

0.46***

14.9 ±

0.63***

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared

with control and *P < 0.05; **P < 0.01; and ***P < 0.001 in comparison with

diabetic control.

Effect of alcohol and Hydro alcohol on serum insulin in

diabetic neuropathy 30

25

20

15

10

5

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin 30 0 60th Day 90th Day

insu

lin (

µIU

/ml)



Figure 6: Effect of AEEA and HAEA on thermal Hyperalgesia using tail immersion in

diabetic neuropathy wistar rats

Table 8: Effect of AEEA and HAEA on thermal Hyperalgesia using hot plate in diabetic-

neuropathy wistar rats


200

AEEA

400

HAEA

200

HAEA

400

Gabapentin

30

60th

Day

9.67 ±

0.12

3.1 ±

0.12#

4.0 ±

0.23

4.7 ±

0.11

4.7 ±

0.12

4.1 ±

0.11

4.8 ± 0.11

75th

Day

9.98 ±

0.34

3.9 ±

0.38#

5.3 ±

0.11

6.7 ±

0.54*

6.4 ±

0.34**

8.9 ±

0.42***

8.2 ±

0.29***

90th

Day

10.02±

0.56

4.2 ±

0.78#

6.9 ±

0.43*

9.1 ±

0.76**

8.9 ±

0.70***

10.1 ±

0.85***

9.8 ±

0.54***

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared with control and *P

< 0.05; **P < 0.01; and ***P < 0.001 in comparison with diabetic control.

Figure 7: Effect of AEEA and HAEA on thermal Hyperalgesia using hot plate in

diabetic-neuropathy wistar rats

Effect of alcohol and hydro alcohol on thermal Hyperalagesia

using tail immersion in diabetic neuropathy

18

16

14

12

10

8

6

4

2

0

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin




Table 9: Effect of AEEA and HAEA on mechanical Hyperalgesia using Von Frey

filaments in diabetic-neuropathy wistar rats


200

AEEA

400

HAEA

200

HAEA

400

Gabapentin

30

60th

Day

61.8 ±

1.28

19.6 ±

1.11#

15.8 ±

1.36

16.8 ±

1.27

15.6 ±

1.20

15.6 ±

1.39

15.8 ± 1.12

75th

Day

71.3

±1.32

17.9 ±

1.20#

19.9 ±

1.11

21.4 ±

1.49*

23.9 ±

1.45**

43.6±

1.87***

48.4 ±

1.65***

90th

Day

79.6 ±

1.45

14.9 ±

1.34#

30.7 ±

1.23

51.9 ±

1.18*

48.7 ±

1.76**

59.3 ±

1.49***

61.4 ±

1.45***

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared with

control and *P < 0.05; **P < 0.01; and ***P < 0.001 in comparison with diabetic

control.

Effect of alcohol and Hydro alcohol on thermal Hyperalagesia using

hot plate in diabetic neuropathy

12

10

8

6

4

2

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin

30




Figure 8: Effect of AEEA and HAEA on mechanical Hyperalgesia using Von Frey

filaments in diabetic-neuropathy wistar rats

Table 10: Effect of AEEA and HAEA on mechanical Hyperalgesia using motor nerve

conduction velocity in diabetic-neuropathy wistar rats


200

AEEA

400

HAEA

200

HAEA

400

Gabapentin 30

60th

Day

54.4±

1.12

22.01±

1.32#

22.7±

1.21

21.8±

1.29

22.1±

1.29

23.1±

1.43

21.03± 1.49

75th

Day

55.8±

1.32

18.08±1.11# 28.9±

1.34

31.5±

1.89*

32.4±

1.68**

52.3±

1.67***

51.23±

1.53***

90th

Day

61.3±

1.45

15.70±1.78# 34.6±

1.46*

46.1±

1.90**

44.8±

1.74**

58.2±

1.55***

59.78±

1.67***

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared with control and *P

< 0.05; **P < 0.01; and ***P < 0.001 in comparison with diabetic control.

Figure 9: Effect of AEEA and HAEA on mechanical Hyperalgesia using motor nerve

conduction velocity in diabetic-neuropathy wistar rats

Effect of alcohol and Hydro alcohol on mechanical

Hyperalagesia using von fray filament in diabetic neuropathy

90

80

70

60

50

40

30

20

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin

30

10




Table 11: Effect of AEEA and HAEA on mechanical Hyperalgesia using Randall-

Selitto analgesiometer in diabetic-neuropathy wistar rats


200

AEEA

400

HAEA

200

HAEA

400

Gabapentin

30

60th

Day

158.6 ±

4.12

42.7 ±

2.56#

42.7 ±

2.11

43.1 ±

2.45

47.1 ±

2.80

41.6 ±

2.16

43.31 ± 2.11

75th

Day

169.7 ±

4.34

40.8 ±

2.98#

62.8 ±

2.33

61.4 ±

2.67*

63.6 ±

2.11**

91.3 ±

2.78***

99.20 ±

2.82***

90th

Day

177.5 ±

4.56

41.9 ±

2.76#

75.5 ±

2.81

119.8 ±

2.84**

111.1 ±

2.34**

160.2 ±

2.63***

168.24 ±

2.75***

Values are mean ± SD (n=6).Where a represents #P < 0.001 as compared with control and *P <

0.05; **P < 0.01; and ***P < 0.001 in comparison with diabetic control.

Figure 10: Effect of AEEA and HAEA on mechanical Hyperalgesia using Randall-

Selitto analgesiometer in diabetic-neuropathy wistar rats


Hyperalagesia using randall selitto analgesiometer in

diabetic neuropathy

200

180

160

Normal

140

Diabetic

120

AEEA 200

100


Hyperalagesia using MNCV in diabetic neuropathy

70

60

50

40

30

20

10

Normal

Diabetic AEEA

200

AEEA 400

HAEA 200

HAEA 400

Gabapentin

30




Peripheral nerves. These changes includes

endoneural edema, increased intraneural

pressure, decreased blood flow to nerves,

ischemia, and finally Wallerian like axonal

degeneration and thus decrease in MNCV.

Marked decrease in nociceptive pain threshold

was observed in diabetic neuropathy rats

tested in both hot plate and tail immersion

method. Hyperalgesia was evident after 60 days

of STZ-nicotinamide administration in both hot

plate and tail immersion method. Administration

of Excoecaria agallocha L (200 and 400

mg/kg) developed a dose and time dependant

elevation in pain threshold in comparison to

DPN control rats. On 90th day of study, paw

withdrawal threshold using Randall-Selitto

and tactile allodynia due to light touch was

reduced significantly in comparison to DPN

control. Excoecaria agallocha L reversed

diabetes-induced decrease in paw withdrawal

threshold in both the tests in comparison to

DPN control group (Molehin et al; 2018).

Polyol pathway activation in early course of

diabetes leads to impaired release of neural

Na+/K+-ATPase and myoinositol resulting in

impairment of endoneurial blood flow.

Endoneurial hypoxia caused by impairment of

endoneurial blood flow results in impaired

nerve perfusion, reduced MNCV and thus

abnormal nerve function ensues. MNCV was

reduced in STZ-induced diabetic animals but

treatment with extracts of Excoecaria

agallocha L elevated the reduced MNCV in a

dose dependant manner. Results obtained by

treatment of DPN rats with plant extracts were

prominent in comparison to effect of

Gabapentin (30 mg/kg).

CONCLUSION:

The Results obtained in the present

study suggested that Excoecaria agallocha L.

Moreover, supplementation with Excoecaria

agallocha L. reversed the pain response

assessed by thermal (hot plate and tail

immersion assay) and mechanical

Hyperalgesia (Randall Selitto analgesiometer

and Von Frey filament) and improvement of

MNCV in diabetic neuropathy rats. Thus,

these plants modulated diabetic neuropathic

pain via reducing the formation of AGEs and

amelioration of oxidative/nitrosative stress in

peripheral nerves. Finally, it can be concluded

that Excoecaria agallocha L. ameliorated

diabetes and its complications (diabetic

neuropathy).

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