INTRODUCTION:

Diabetes is a disease associated with elevated blood glucose levels, leading to major complications such as diabetic neuropathy, nephropathy, retinopathy and cardiovascular diseases. More than 100 million of the world’s population has already reached the diabetic mark and the disease now kills more people than AIDS.¹ Albizia procera makes a good cabinet and furniture timber and is also suitable for general construction, agricultural implements, household products, poles, house posts, and packing cases. It is suitable source material for paper pulp, giving satisfactory yields of bleached pulp.². Evaluation of pharmacological activities confirmed Albizia procera as antioxidant, anticancer analgesic, antibacterial, CNS depressant, in-vitro α-amylase, α-glucosidase inhibitor, anti-HIV-1 integrase activity, antidiabetic, antidiarrheal and hepato-protective activities.³

The survey report illustrated that no scientific evidence has been determined for the in vivo anti diabetic activity on Albizia procera barks. Hence the present study was investigated and focused on the specific n-butanol fraction of Albizia procera against streptozotocin induced diabetic rats.

MATERIAL AND METHODS:

The dried barks of Albizia procera was extracted sequentially by hot continuous percolation by soxhlet apparatus using solvents chloroform and ethanol. The ethanol extract was fractionated using liquid-liquid partition using hexane, ethyl acetate, and n- butanol. The n-butanol fraction of Albizia procera has good in vitro antidiabetic activity which was investigated in previous research in this plant. Hence n-butanol fraction of Albizia procera was selected for in vivo antidiabetic activity.

Acclimatization of Animals:

Male Wistar rats (150-175g) were maintained under standard laboratory condition at the centre for experimental animals in Madras Medical College. Animal ethical Committee’s clearance 1917/ ReBi/S/16/CPCSEA/25.10.2016 was obtained for study. After seven days of Acclimatization, the animals were randomly assigned for the acute toxicity groups. Each group containing 3 animals were housed individually in labeled cages with solid plastic sides and floor with stainless steel grid tops. Animals were allowed free access to standard pellet diet and water ad libitum. They were maintained in controlled laboratory conditions of 12hrs dark/light cycle, 22±2ºC temperatures and 45-60% humidity.

Acute Toxicity of N - Butanol Fraction of Albizia Procera in Rats:

Six animals were used for each step of the study. Animals made to fast prior to dosing (food was withdrawn overnight and water was withdrawn 3hrs before drug administration) following the period of fasting. The animals were weighed and the extracts were administered in a single dose, as 1% suspension in Carboxy Methyl Cellulose, by oral intubation food was withheld for further one hour after the administration of drug. The starting dose levels selected for the study with a dose of 5mg/kg and the dose was increased step by step to 50,300,500 and 2000mg/kg body weight. The mortality of the animals dosed at one step will determine the next step.⁴

In vivo Antidiabetic Activity of N–Butanol Fraction of Albizia Procera in Rats:

Diabetes was prompted through single intraperitoneal injection of freshly prepared streptozotocin (STZ) (50 mg/kg b.w.) in 0.1 M citrate buffer (pH = 4.5) to overnight starved rats. Diabetic rats were permitted to drink 20% glucose solution overnight to overcome the initial drug induced hypoglycemic death. The blood glucose level was measured after three days, and rats with glucose levels >250mg/dL were considered as diabetic. At the time of induction, control rats were injected with 0.2mL of vehicle (0.1 Mcitrate buffer, pH 4.5) alone.⁵

Grouping of Animals:

The total of 30 matured normoglycemic Wistar rats of either sex (12–14 weeks of age, weighing about 180-200g) was collected for this experiment. Animals were acclimated for a period of 7 days in our laboratory condition prior to the experiment.

Table 1: Grouping of Animal

Group I	Normal control rat (n=6)
Group II	Streptozotocin Diabetic Rats (n=6)
Group III	Diabetic animal treated with 200mg/kg of (n-butanol fraction of Albizia procera )(APF – NB) (n =6)
Group IV	Diabetic animal treated with 400mg/kg of of (n-butanol fraction of Albizia procera) (APF – NB) (n=6)
Group V	Glibenclamide - 600µg/ml (2.5mg/kg (n=6)

N Butanol fraction and standard drug were administered orally by gastric intubation using a force- feeding needle to the respective group of rats for 21days. After overnight fasting the blood was withdrawn from the tail vein and fasting blood sugar level will be checked by using glucometer 0, 7, 14 and 21day period. Animal body weight is also recorded before and after treatment.^6,7

Biochemical estimation:

Blood was collected from retro-orbital venous plexus of the rats using capillary tubes under isoflurane anesthesia. Serum lipid profile were estimated by standard analytical techniques

Estimation of Lipid Parameters:

On various days blood samples were collected by retro-orbital puncture, under ether anesthesia in STZ induced rat models.⁸ The collected samples were centrifuged for 15 minutes at 2500rpm. Then serum samples were collected and analyzed for serum High Density Lipoprotein Cholesterol (HDL-C), Low Density Lipoprotein Cholesterol (LDL-C)

Estimation of HDL-cholesterol:

CHOD-PAP method was used to estimate the serum HDL cholesterol level. CHOD-PAP method was used to estimate the serum HDL cholesterol level. For this 2ml if serum was taken in a test tube and 0.5ml of precipitation reagent was added.⁸ The mixture was shaken thoroughly and left to stand for 10min at +15 to 25c and then centrifuged for 15min at 4000rpm. Within 2hr after centrifugation, the clear supernatant was used for the determination of HDL-C. One ml of the supernatant was taken in a test tube (T) and 1ml of solution was added to it. In another test tube 0.1ml DW was taken and 1ml reaction solution (B) was added. The mixtures were mixed thoroughly, incubated for 10min at 15-25C or for 5min at 37˚C and measured the absorbance of the sample against reagent blank at 546 nm.^9,10

Estimation of LDL cholesterol:

LDL cholesterol was estimated by using Friedwald’s (1972) formula as follows:

Total cholesterol-HDL-C-Triglyceride

LDL in mg % = --------------------------------------------------

RESULTS:

Acute toxicity study:

The animals were observed individually every 30 minutes after dosing the first 24hrs and thereafter daily for a total of 14 days. The time at which signs of toxicity appear and disappear was observed systematically and recorded for each animal. The absence or presence of compound-related mortality of the animals dosed at one step will determine the next step. Any mortality during the experiment for 14 days was observed and recorded. The n-butanol fraction of Albizia procera was found to be non-toxic up to the dose of 2000mg/kg and did not cause any death of the tested animals and the LD₅₀ value is expected to exceed 2000mg/kg/body weight.

In vivo Antidiabetic Activity of N – Butanol Fraction of Albizia Procera in Rats:

Blood glucose level:

Administration of STZ resulted in significant (P < 0.001) increase in mean blood glucose level on day 7, day 14 and 21. Post treatment with an APF-NB in STZ induced rats significantly reduced (P<0.001) the increase the blood glucose level as compared to group II. N- butanol fraction produces dose dependent effect as the high dose (400mg/kg) shows more significant anti diabetic activity than 200mg/kg (P<0.01) Treatment with glibenclamide after induction of diabetes significantly (P<0.001) reduced the increased blood glucose level as compared to group II (Table 2)

Table 3, lists the changes in the body weight of the control and experimental rats treated with the fraction and glibenclamide. The body weight of the STZ-induced diabetic rats was not lowered than that of the normal control groups after 21 days. But the administration of n butanol fraction of Albizia procera (APF-NB) 400mg/kg, significantly decrease the body weight on 21^st day compared to Group II (diabetic control)

LDL:

As illustrated in table 4, there was a significant rise (P<0.001) in LDL level in STZ induced diabetic rats when compared to normal group. The administration of glibenclamide has significant (P< 0.001) lowering of LDL level at 7^th day. The administration of APF-NB at 400mg/kg have showed a significant lowering (P < 0.001) of LDL level when compared to Group II (STZ induced diabetic rats).

HDL:

As illustrated in table 5, there was a significant decline (P< 0.001) in HDL level in STZ induced diabetic rats when compared to normal group. The administration of glibenclamide has significant (P< 0.001) increase of HDL level compared to group II. The administration of APF-NB, at 400mg/kg have showed a significant increase (P < 0.001) of HDL level on 14^th and 21^st day when compared to Group II (STZ induced diabetic rats).

Table 2: Effect of n-butanol fraction on blood glucose level in STZ induced diabetic rats

Group n = 6	Treatment	0 day	7^th day	14^th day	21^st day
1	Control (STZ induced)	116 ± 5.812	301 ± 1.195 ***	306.5 ± 1.176 ***	307.5 ± 2.141***
2	Positive Control (Glibenclamide) 600 µg/kg	114 ± 4.789	211.5 ± 2.419 ***	141.7 ± 0.714 ***	135.2 ± 1.195***
3	Active fraction (n butanol). 200mg/kg	117 ± 3.027	256.7 ± 2.486 **	240.3 ± 3.073 **	223.3 ± 1.667 **
4	Active fraction (n butanol). 400mg/kg	113.7 ± 3.190	160 ± 3.483 ***	123.0 ± 1.571 ***	123.3 ± 1.667 ***

All values are expressed as mean ± SEM. Non-Significant (ns) normal control, diabetic control, positive control; 400mg/kg (APF-NB), *** P<0.001 vs Control (STZ induced) significant by ANOVA followed by Dunnett’s t – test.

Table 3: Effect of n butanol fraction on body weight in STZ induced diabetic rats

Group n = 6	Treatment	0 day	7^th day	14^th day	21^st day
1	Control (STZ induced)	203.3 ± 6.146	203.3 ± 6.146	204.3 ± 6.667	203.3 ± 6.667
2	Positive Control (Glibenclamide)600 µg/kg	203.3 ± 5.578	205.5 ± 5.384	215.5 ± 4.386	217.5 ± 4.801
3	Active fraction (n butanol). 200mg/kg	200.0 ± 7.303	200.9 ± 6.520	196.3 ± 7.191	193.7 ± 7.032 *
4	Active fraction (n butanol). 400mg/kg	188.3 ± 5.426	187.2 ± 5.357	179.2 ± 5.552 *	173.3 ± 4.660 **

Non-Significant (ns) normal control, diabetic control, positive control; 400mg/kg (APF-NB), ** P<0.01 vs Group II, significant by ANOVA followed by Dunnett’s t – test. * P<0.05, 400mg/kg (APF - NB),

Table 4: Effect of n- butanol fraction on LDL in STZ induced diabetic rats

Group n = 6	Treatment	7^th day	14^th day	21^st day
1	Normal Control	46.67 ± 0.6146	46.33 ± 0.8433	56.33 ± 0.8433
2	Control (STZ induced)	64.25 ± 0.6551 ***	64.22 ± 0.7120 ***	67.33 ± 0.3333***
3	Positive Control (Glibenclamide) 600 µg/kg	36.50 ± 0.4282 ***	46.50 ± 0.4282 ^ns	56.50 ± 0.4282 ^ns
4	Active fraction (n butanol). 200mg/kg	57.17 ± 1.833 ***	57.83 ± 1.558 ***	53.00 ± 0.540 ^ns
5	Active fraction (n butanol). 400mg/kg	37.67 ± 0.6146 ***	38.00 ± 0.7782 ***	40.00 ± 0.4472 ***

The values are expressed as mean ± SEM, n = 6

*** P<0.001, (Positive control) vs normal group, significant by ANOVA followed by Dunnett’s t – test. *** P<0.001, 400mg/kg (APF - NB)

Table 5: Effect of n- butanol fraction on HDL Level in STZ induced diabetic rats

Group n = 6	Treatment	7^th day	14^th day	21^st day
1	Normal Control	46.67 ± 0.6146	47.67 ± 0.6146	48.83 ± 1.014
2	Control (STZ induced)	36.50 ± 0.4282 ***	37.33 ± 0.6146 ***	38.00 ± 0.6831 ***
3	Positive Control (Glibenclamide) 600 µg/kg	64.25 ± 0.655 ***	65.25 ± 0.6551 ***	75.25 ± 0.6551 ***
4	Active fraction (n butanol). 200mg/kg	37.67 ± 0.6146 ***	38.00 ± 0.7303 ***	44.83 ± 1.138 *
5	Active fraction (n butanol). 400mg/kg	57.17 ± 1.833 ***	58.67 ± 1.308 ***	58.67 ± 1.308 ***

The values are expressed as mean ± SEM, n = 6

*** P<0.001, (Positive control) vs normal group, significant by ANOVA followed by Dunnett’s t – test. *** P<0.001, 400mg/kg (APF - NB

DISCUSSION:

In the present study, APF-NB showed significant lowering of blood glucose level, an index of diabetic control. STZ-induced diabetes is one of the widely used animal models that mimic the diabetes mellitus^11,12 The selective destruction of insulin-producing β-cells of the pancreas by STZ is mediated by induction of high levels of DNA strand breaks in these cells,^13,14 causing activation of poly (ADP-ribose) polymerase (PARP), resulting in reduction of cellular NAD+, and cell death.¹⁵ The metabolism of glucose, proteins and lipids is abnormal in diabetes due to insulin secretion defect, leading to various metabolic disorders and complications.¹⁶ In addition, STZ generates potential free radicals such as nitric oxide (NO) by intracellular metabolism of STZ and precipitate further β-cells DNA damage by strand break^17,18

The concentration of blood glucose was significantly increased in diabetic as compared with STZ as compared to normal control. Administration of APF-NB (200mg/kg and 400mg/kg) significantly reduced the raised blood glucose level in STZ induced diabetic rats and the lowering was almost comparable to glibenclamide 600µg/kg.). Further, this antidiabetic activity of APF-NB was associated with an increase in the serum insulin level revealed that APF- NB may stimulate insulin secretion from regenerated β cells and remaining β-cells (Table 2).^19,20 The blood glucose lowering effect of the APF-NB could be due to the presence of flavonoid and phenolic compounds as reported in the preliminary phytochemical screening and HPTLC of the present study.²¹ These findings were also supported by the previous experimental findings wherein they reported the blood glucose lowering effect of flavonoids. Patients with type 2 diabetes show reduced turn-over of their LDL particles with a reduction of catabolism, leading automatically to increased LDL plasma residence time. Augmented LDL residence time in plasma is likely to promote cholesterol deposition in the arterial wall. Reduced HDL level, in type 2 diabetes, has been shown to be correlated with both hypertriglyceridaemia and obesity. The decrease in HDL cholesterol, noted in patients with type 2 diabetes, is due to increased catabolism of HDL particles.²²

CONCLUSION:

The present study provides methodical indication for anti-diabetic activity of Albizia procera by the evaluation of in vivo models and hence supports the therapeutic usage of barks in traditional medicines for treating diabetes mellitus and its associated complications. This work will be useful for diabetic research workers to isolate the chemical entity from the barks of Albizia procera which is exactly responsible for the antidiabetic activity.

ACKNOWLEDGMENTS:

Authors are thankful to Professor Nagarajaperumal, Karpagam College of Pharmacy, Coimbatore for helping us to carry out the in vivo anti diabetic activity.

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Received on 16.09.2020 Modified on 20.03.2021

Research J. Pharm.and Tech 2022; 15(4):1583-1587.

DOI: 10.52711/0974-360X.2022.00264