Antidiabetic Activity and Toxicity Studies of Ethanolic Extract of Polialthia longifolia Roots

 

D. Benito Johnson*, Akhil Raj, R. Venkatanarayan

Department of Pharmacology, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore

 

ABSTRACT:

Diabetic millets¹ often referred to simply as diabetes (Ancient Greek: diabetes” to pass through [urine]”) is a syndrome of disorder metabolism, usually due to a combination of hereditary and environment causes, resulting in abnormally high blood sugar level (hyperglycemia).Diabetes is described as starvation in the midst of plenty. Because the body will have high amount of glucose level but the cells are incapable of consume it because of osmotic difference Insulin is a hormone produced in the pancreas, which enable body cells to absorb glucose, to turn into energy. If the body cells cannot absorb the glucose, the glucose accumulates in the blood (hyperglycemia), leading to acute metabolic complications including keto acidosis and in the long term contribute to chronic micro-vascular complications

 

 

 

 

 

INTRODUCTION:

Diabetic Mellitus:

Diabetic millets1 often referred to simply as diabetes (Ancient Greek: diabetes to pass through [urine] is a syndrome of disorder metabolism, usually due to a combination of hereditary and environment causes, resulting in abnormally high blood sugar level (hyperglycemia).Diabetes is described as starvation in the midst of plenty. Because the body will have high amount of glucose level but the cells are incapable of consume it because of osmotic difference Insulin is a hormone produced in the pancreas, which enable body cells to absorb glucose, to turn into energy.

 

If the body cells cannot absorb the glucose, the glucose accumulates in the blood (hyperglycemia), leading to acute metabolic complications including keto acidosis and in the long term contribute to chronic micro-vascular complications.

 

Types of Diabetes Mellitus:

The type of diabetes is based on the presumed etiology they are

1. Type 1 diabetes or Insulin dependent diabetes mellitus (IDDM)

2. Type 2 diabetes or Non Insulin dependent diabetes mellitus (NIDDM)

3. Gestational Diabetes (Pregnancy diabetic).

 

1. Type 1 Diabetes:

Insulin-dependent (Type I) diabetes mellitus is a chronic disease characterized by hyperglycemia, impaired metabolism and storage of important nutrients, evidence of autoimmunity, and long-term vascular and neurologic complications.  Insulin secretary function is limited. Cell membrane binding is not primarily involved. The goal of treatment is to relieve symptoms and to achieve blood glucose levels as close to normal as possible without severe hypoglycemia. However, even with education and self-monitoring of the blood glucose level, attaining recommended target values (plasma glucose level less than 8.0 mmol/L before main meals for adults) remains difficult. Therapy with one or two injections per day of mixed short-acting or intermediate-acting insulin preparations is a compromise between convenience and the potential for achieving target plasma glucose levels.

 

2. Type 2 Diabetes:

Type 2 diabetes, once known as adult-onset or noninsulin-dependent diabetes, is a chronic condition that affects the way your body metabolizes sugar (glucose), your body's main source of fuel. With type 2 diabetes, your body either resists the effects of insulin — a hormone that regulates the movement of sugar into your cells — or doesn't produce enough insulin to maintain a normal glucose level. Untreated, type 2 diabetes can be life-threatening. It is the result of failure to produce sufficient insulin and insulin resistance. Elevated blood glucose levels are managed with reduced food intake, increased physical activity, and eventually oral medications or insulin.

 

3. Gestational Diabetes:

This type affects females during pregnancy. Some women have very high levels of glucose in their blood, and their bodies are unable to produce enough insulin to transport all of the glucose into their cells, resulting in progressively rising levels of glucose. Diagnosis of gestational diabetes is made during pregnancy. The majority of gestational diabetes patients can control their diabetes with exercise and diet. Between 10% to 20% of them will need to take some kind of blood-glucose-controlling medications. Undiagnosed or uncontrolled gestational diabetes can raise the risk of complications during childbirth.

 

MATERIALS AND METHOD:

1)       Collection of Plants:

P. Longifolia was produced from the Botany Central council for Research in Ayurvedia and Siddha Govt of India.  The freshly collected sample were thoroughly cleaned and soaked in fresh water repeatedly to separate mud particles sticking on to plant constituents. The plants constituents collected were cut into small bits of about 2-3 in size. After the leaf of the plants are powdered with a mechanical grinder. This powder was subjected to various studies for which the materials and methods which is presented below.

 

2) Authentication of Plant:

The dried whole plant powder of P. longifolia Roots was supplied and authenticated by Chelladurai. V, Research Officer Botany Central Council for Research in Ayurvedia and Siddha Govt. of India.

 

Extraction:

Equal amount of the weighed powder were mixed and placed in the Soxhlet apparatus by using ethanol. From the 500gm of crude powder was extracted with 2.5 liter of ethanol (60 – 80) by continuous hot percolation using Soxhlet apparatus. This can be continued up to 24 hours. After the extraction process filtered and the Soxhlet is removed the obtained residue is stored in the dissector.

 

TOXICITY STUDY:

Acute Toxicity Study:

Selection of Test Animal:

Female adult Wistar rats of 8-12 weeks are selected. Nulliparous and non-pregnant animals were obtained from the centralized animal house of RVS College of pharmaceutical sciences, Sulur and they are acclimatized for holding 1 week prior to dosing.

 

Housing and Feeding Conditions:

Temperature:

As per OECD Guideline-420 the temperature of animal house is maintained at 220C±30C.

 

Humidity:

The relative humidity of animal room maintained at 50-60% preferably not exceeds 70% (OECD guidelines-420, 2001). Otherwise there may be chances of developing lesions such as ring tail and food consumption may be increased.

 

Light:

The sequence of light used is 12 hrs light and 12 hrs dark.

 

Caging:

Polypropylene cages with solid bottom and walls. The lids are made up of stainless steel grill which is capable to hold both feed and water.

 

Feeding Condition and Feed:

Sterile laboratory feed (ad libitum) and water daily. The feed used is brown colored chow diet.

 

Drug Administration:

Animals are fasted prior to dosing (food but not water should be withheld for overnight).After that animals are weighed and the test substance administered. The healthy rats has been taken and divided into 4 different groups. Then the petroleum ether extract was dissolved in 0.6% if sodium carboxyl methyl cellulose on equal. The test substance is administered in a single dose by oral gavages, using a curved and ball tipped stainless steel feeding needle.

Sub Acute Toxicity Study:

Selection of Animals:

Male and Female rats were selected and are acclimatized for 5 days prior to the start of study. The females are nulliparous and non-pregnant. At the commencement of study the weight variation of animals used minimal and not exceed ± 20 % of the mean weight of each sex. Repeated dose oral study was conducted as a preliminary to a long term study preferably animals from the same strain and source were used in both studies.

 

Housing and Feeding Conditions:

The temperature in the experimental animal room was maintained at 220C (±30C). The relative humidity was 50-60% (not exceed 70%) and the lighting sequence was 12 hrs light and 12 hrs dark. For feeding, conventional laboratory diet was used with an unlimited supply of drinking water. Animals were housed in small groups of same sex (NMT 5 animals in each cage)

 

Preparation of Animals:

Healthy young adult animals were randomly assigned to control and treatment groups. Cages were arranged in such a way that possible effects due to cage placement were minimized. The animals were identified uniquely and kept in their cages for five days prior to the start of the study to allow for acclimatization to the laboratory conditions.

 

Dose Administration:

The leaf extracts were administered by oral gavages. Ten animals (5 males and 5 females) were used at each dose level for each extracts. Three test groups and a control group were used for both extracts and the highest dose level was chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter a descending sequence of dose levels selected with a view to demonstrating dosage related response and non-observed-adverse effects at the lowest dose level (NOAEL). The animals were dose with test substance daily 7 days each week for a period of 28 days.

 

Pharmacological Screening:

Animals:

Male Swiss albino rats weighing 150-200g were used for the present work. The animals used for the experiment were maintained under standard laboratory conditions in an animal house of RVS College of Pharmaceutical sciences approved by the committee for the purpose of control and supervision on experiments on animals (Ref. No: IAE1012/C/06/CPCSEA) under 12 h dark/light cycle and controlled temperature 24±20°C. They had free access to food and water ad libitum. The animals were acclimatized to the laboratory for a period of 7 days, before the commencement of experiment.

 

Oral Glucose Tolerance Test:

The ability of an individual rat to handle a standard oral glucose load was evaluated by assessing the blood plasma for glucose level. Study on Oral Glucose Tolerance Test (OGTT) initially, hypoglycemic activity of herbal formulation was carried out in overnight fasted normal rats, which were equally divided into four groups of six rats each. Normal control group received only vehicle (1 ml of water) and test group received the formulation in three different dose level ie low, therapeutic and high dose respectively. Following 30 min post extract administration all the animals were fed with glucose (2 g/kg). Blood samples were collected from tail vein prior to dosing and then at 30, 60, 90 and 120 min after glucose administration. The fasting blood glucose level was analyzed using glucose-oxidase-peroxide reactive strips (Accu-chek, Roche Diabnostics, GmbH, Germany).

 

Antidiabetic Activity:

Induction of Diabetics:

Six adult albino rats weighting 250-300 grams (75-90 days old) were used for inducing diabetes. The animals were injected by Streptozotocin at the dose of 55 mg/kg of the body weight intravenously. Streptozotocin induces diabetes within 3 days by destroying the beta cells. Diabetic animals and non-diabetic control group were kept in metabolic cages individually and separately and under feeding and metabolism control. Glucose in the blood of diabetic rats exceeded that of the non-diabetic control ones. Food consumption was measured in terms of (gr.), water consumption was measured in terms of (ml) and urine volume was measured in terms of (ml) on a daily basis while every 2-4 weeks in 80 days the levels of C-peptide, insulin and glucose in blood serum were also measured, so that chemical diabetes was verified in rats injected with Streptozotocin

 

Assessment of Diabetic:

Diabetic was conformed after 48 hr of streptozotocin injection, the blood samples were collected through retro orbital puncture and plasma glucose level were estimated by enzymatic GOD POD diagnostic kit method. The rat having fasting plasma glucose levels more than 250 mg/dL were selected and used for this study.

 

Evaluation of Extract on Streptozotocin Induced Diabetic rats.

The albino rats on either sex have been selected for the experimental study. The weight of the should be around 170-240 gm. The animals are divided into six groups. Each group has 6 animals. Group 1was kept as normal (normal rat) received only distilled water; group 2 was kept as negative control, Streptozotocin induced and received only water. Group 3 was treated with glibenclamide (10mg/kg) Group 4, 5 and 6 is diabetic induced rat and treated with 100mg/kg,200mg/kg and 400mg/kg b.w of ethanol extract of Polialthia longifolia. (EPLR) EPLR was administered for 21 days at a three different dose levels 100, 200 and 400mg/kg. Dried extract made in aqueous and given orally. The blood was collected by sinus orbital under the light diethyl ether anesthesia. The blood was centrifuged at 3000 rpm for 10 minutes. Body weight glucose was analyzed every week and lipid and lipoprotein profile from serum (TC, TG, HDL, LDL, VLDL.) were analyzed after 28 days.

 

RESULTS:

Soxhlet Extraction of Polialathia longifolia Roots

Table 1- Soxhlet Extraction of Polialathia longifolia Roots

Plant	Part used	Method of Extraction	Solvents	Weight of powder taken (A)	Weight of  product (B)	Percentage yield (W/V)
Polialthia longifolia	ROOT	Continuous Hot percolation by Soxhlet apparatus	Ethanol (60-80°C)	50	2088	5.96

A = Weight of Powder plant material;  B= Weight of extract; Percentage yield = (B/A) x 100

 

Preliminary Phyto Chemical Screening:

EEPLR extracts was subjected various chemical tested as per the standard methods for the identification of the various constituents. The result if this phyto chemical analysis is listed below.

 

Table 2- Preliminary Phyto Chemical Screening

“+” Presence, “-“Absence

Toxicity Studies

Acute Toxicity Studies

Acute toxicity studies on the albino rats show no morality at a dose of 2000mg/kg, during a time period of 14 days. During the study, no noticeable were seen in the rats. This help to predict that it does not contain any type of toxicity and it is full safe. So 200 mg/kg b.w (1/10th and 500mg/kg b.w (1/4th) and 1000mg/kg (1/2th) were selected of that dose for the further study

 

 

Effect of EPLR on Percentage Change in Body Weight

Table 3 - Effect of EPLR on Percentage Change in Body Weight

Sex	Dose	0	7	14	21	28	% Change in body wt
Male	Control	129.4±3.26	139.8±2.37	148.2±2.33	158.8±1.85	169.8±3.15	31.22±0.9
	Lower dose	137.2±2.82	144.4±1.69	154.6±1.63	164±2.07	173.8±2.70	26.6±2.4
	Middle dose	131.2±2.35	135.4±4.31	139.09±0.89	145.5±1.51	146.8±1.59	0.4±2.5
	Higher dose	135.4±4.0	133±2.55	138.8±2.53	143.4±2.42	148.1±1.70	15.3±1.4
Female	Control	123.9±3.2	130±3.92	139.4±3.76	158±5.15	160.2±4.32	29.6±3.81
	Lower dose	125.2±2.92	129±2.43	127.1±4.2	134.1±4.6	140.8±3.26	15.06±3.2
	Middle dose	122.4±2.7	133±2.4	132.8±2.06	138.2±2.18	142.2±1. 24	20.2±1.3
	Higher dose	123.2±2.65	125.6±2.16	128.8±1.28	134.6±1.20	141.6±1.99	18.4±4.2

 

Effect of EPLR on serum biochemical parameter

Table 4 - Effect of EPLR on serum biochemical parameter

Sex	Group	Days	Hb (g/dl)	RBC (x 106mm3)	WBC (x 103 mm3)	Differential WBC count
						N %	L %	E %	M %	B %
Male	Control	0	7.8±2.34	2.67±0.67	7.07±0.31	32.56±7.89	65.8±7.15	0.93±1.3	0	0
		28	7.9±1.00	2.72±0.32	6.87±6.2	32.66±7.09	65.66±7.63	0.67±0.58	0	0
	Lower dose	0	7.8±2.3	2.56±0.65	7.1±8.8	29.93±4.54	67.8±7.23	1.20±1.48	0	0
		28	7.7±2.02	2.42±0.60	7.3±6.3	30.33±4.50	68.66±3.51	1.21±1.00	0	0
	Middle dose	0	7.8±2.3	2.67±0.88	7.06±3.1	29.01±12.54	71±2.72	0.67±2.88	0	0
		28	8.2±1.25	2.78±0.45	6.86±6.2	28.33±10.5	72±10.00	0.67±0.58	0	0
	Higher Dose	0	7.6±2.54	2.64±0.76	8.2±31	32.66±4.43	66.43±11	1.22±1.30	0	0
		28	737±0.57	2.59±0.15	7.4±32	33.35±9.07	65.33±8.50	1.30±0.58	0	0
Female	Control	0	7.9±2.34	2.67±0.67	6.50±2.2	32.56±7.89	64.8±7.15	0.92
		28	7.9±1.00	2.72±0.32	7.00±1.57	31.66±7.09	65.66±7.63	0.67±0.58	1.5±0.57	0.6±0.69
	Lower dose	0	7.7±2.3	2.56±0.65	5.9±2.2	30.96±4.54	66.8±7.26	1.33±1.48	1±1.4	0.4±0.54
		28	7.7±2.02	2.42±0.60	6.01±1.52	30.33±4.50	66.66±3.54	1.26±1.00	0	0.8±0.83
	Middle dose	0	7.8±2.3	2.67±0.88	7.084±2.99	29.01±12.54	72±2.72	0.68±2.88	1±1	0.5±0.8
		28	8.2±1.25	2.72±0.45	6.56±8.6	28.33±10.5	74±10.00	0.96±0.58	0.4±0.5	1.0±1.0
	Higher Dose	0	7.6±2054	2.64±0.746	6.65±1.38	33.66±4.43	67.43±11	1.20±1.30	0.9±0.83	0.2±0.44
		28	7.7±0.57	2.59±0.15	6.85±4.2	31.33±9.07	68.33±8.50	1.17±0.58	0	0.2±0.4

Subacute Toxicity Studies:

Functional observations are necessary to test the neurotoxic effects in the assessment of the toxic effects of ICFE. The functional observational battery is a non-invasive procedure designed to detect gross functional deficits in animals resulting from exposure to chemicals and to better quantify neurotoxic effects detected in other studies. This battery of test is not intended to provide a detailed evaluation of neurotoxicity. It is designed to be used in conjunction with neuropathologic evaluation and/or general toxicity testing. Additional functional is necessary to assess completely the neurotoxic potential of drug.

 

 

 

Effect of EPLR on serum biochemical parameter

Table 5 - Effect of EPLR on serum biochemical parameter

 

Histopatholgical Assessment of Eplr Extracts:

 

Figure 1 - T.S of rat liver showing normal cells in subacute toxicity study of EPLR

 

 

Microscopic appearance:

Section studies from the liver shows normal lobular architecture. Individual hepatocyte, central vein and sinusoids shows p- unremarkable. There is no evidence of inflammation and necrosis.

 

KIDNEY

Figure 2 - T.S of rat kidney showing normal cells in Sub Acute Toxicity Studies of EPLR

 

Microscopic Appearance:

Section studies from the kidney show normal cortex and medulla. The glomeruli, interstitium and blood vessels are unremarkable. There is no evidence of inflammation and necrosis.

 

HEART:

 

Figure 3 - T.S of rat heart showing normal cells in Sub Acute Toxicity Studies of EPLR

 

 

Microscopic appearance:

Section studies from the heart shows normal myocardium with myocytes. There is no evidence of myocytic degeneration or edema.

 

BRAIN:

 

Figure 4 - T.S of rat heart showing normal cells In Sub Acute Toxicity Studies of EPLR

 

Microscopic Appearance:

Section studies from the brain shows normal cerebellum. Brain parenchyma, purkinjic cells and basal ganglion unremarkable. There is no evidence of inflammation and necrosis.

 

ANTIDIABETIC ACTIVITY:

Oral Glucose Tolerance Test:

In the OGTT the EPLR extracts at a dose of 200mg/kg significantly reduced the blood glucose level at 30 minutes after glucose administration. Standard drug Glibenclamide produced activity at all the time interval tested

 

Oral Glucose Tolerance Test of EPLR:

Table 6 - Oral glucose tolerance test of EPLR

Group	Blood Glucose levels (mg/dl)
	0 min	30 min	1st hour	2nd hour	3rd hour	8th hour
Control	84.5±1.14	121.4±1.25	162.91±13.67	139±2.074	111.02±5.805	93.4±1.304
Glibenclamide mg/kg	87.5±10.164	70.3±7.19	84.0±10.271	83.6±13.342	81.2±7.791	80.2±7.328
EPLR 100mg/kg	84.3	110.8	143.12	130.12	128.12	125.22
EPLR 200mg/kg	85.6±5.263	83.6±6.894	83.6±1.924	90.2±2.408	80.4±1.517	84.4±2.302
EPLR 400mg/kg	88.5±1.22	81.5±0.22	79.05±1.22	79.3±2.5	79.5±2.5	80.3±2.1

 

EFFECT OF EPLR ON SERUM GLUCOSE LEVEL

Table 7 - Effect of EPLR on serum glucose level in normal control and STZ induced diabetic rats

S. no	Treatment	Serum glucose level
		Initial	7th day	14th day	21st day	28th day
1	Normal control	90.97±1.47	94.97±1.47	104.3±4.1	102±2.13	89.52±2.16
2	Diabetic control	104.22	259.3±3.51	303.2±5.5	375.1±1.3	405.3±1.26
3	Diabetic+standard	104.12±1.2	267.8±3.15	150.1±1.4	140.2±0.5	129.5±1.0
4	Diabetic+Extract 100mg/kg	85.11±5.5	238.0±2.65	270.5±2.3	299.88±1.1	325.2±2.46
5	Diabetic+Extract 200mg/kg	98.2±1.22	241.9±1.25	240.1±0.7	235.1±1.7	224.2±2.49
6	Diabetic+Extract400mg/kg	94.1±5.5	260.87.8	98.3±1.5	113±1.3	110±3.89

 

Effect of EPLR on Body Weight:

There was gradual increase in body weight in normal control while the diabetic control continues to lose the weight. However treated diabetic group gained 6.25%, 8.24% as compared with the diabetic control and diabetic treated towards normal range. Extract changes in the body weight shows in the tables.

 

Table 8 - Effect of EPLR on body weight in normal control and STZ induced diabetic rats.

Sl. no	Treatment	Body weight
		Initial	7th day	14th day	21st day	28th day
1	Normal control	242.9±1.4	247.8±1.33	254.1±2.333	263.11±8.5	274±1.95
2	Diabetic control	248.9±1.4	221.8±5.5	215.1±2.4	198.1±3.9	189±1.64
3	Diabetic+ Glibenclamide	243.5±1.5	230.2±4.88	245.1±2.3	260.1±1.34	275.8±1.6
4	Diabetic+Extract200mg	243.2±2.5	228.1±4.6	245.1±3.4	260.5±6.7	282.8±2.7
5	Diabetic extract 500mg	250.5±3.2	240.1±1.22	260.11±3.8	280.1±2.3	303.2±2.8
	Diabetic extract 1000mg	241.6±3.8	230.1±4.8	245.9±2.78	254.1±3.44	260.1±1.4

 

Effect of EPLR on Serum Lipid and Lipoprotein Profile in Normal Control and STZ Induced Diabetic Rats

Table 9 - Effect Of EPLR On Serum Lipid And Lipoprotein Profile In Normal Control And STZ Induced Diabetic Rats

Sl. no	Treatment	TC(mg/dl)	TG(mg/dl)	HDL (mg/dl)	LDL(mg/dl)	VLDL(mg/dl)
1	Normal	87.16±4.5	85.62±1.6	42.5±1.32	91.4±1.07	46.5±1.32
2	Diabetic control	155.8±7.4	189.86±4	20.3±1.42	129±1.78	53.3±2.18
3	Diabetic+ Glibenclamide (10mg/kg)	92.3±2.35	101±2.90	45.4±1.48	102±2.56	33.2±1.65
4	Diabetic+ extract (200mg/kg)	135.3±3.9	145.1±2.6	47.3±2.1	110±2.62	41.3±1.56
5	Diabetic+ extract (500mg/kg)	132±3.52	112±3.01	45.3±1.35	102±2.12	33.2±2.06
6	Diabetic+ extract (1000mg)	89.3±1.03	62.3±3.65	3.82±1.37	98.6±1.68	26.7±2.18

 

SUMMARY AND CONCLUSION:

The presented study is an attempt to investigate the effect of petroleum ether extract of EPLR on Streptozotocin induced diabetic in albino rats. The Phytochemical study was screening showed the presence of tannins, carbohydrate, Flavonoids and reducing sugar which is responsible for the anti-diabetic activity. The animals were induced with STZ at a dose of 55mg/kg intraperatoneal and the diabetic animals were treated with EPLR (100, 500, 1000mg/kg) for 28 days orally. The serum glucose, body weight lipid profile, liver glycogen were measured from the pancreas homogenate were measured which showed significant activity. The finding of the presence investigation suggests the EPLR has potential for its evaluation as protective agents against toxicity induced by Streptozotocin. Clinical assessments of EPLR determination of underlying mechanism of the protective effects in interesting topics requiring further study.

 

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Accepted on 25.04.2017           © RJPT All right reserved