Lippia nodiflora is a perennial herb, grows in maritime areas near rivers throughout the sub continent, Africa and other most sub tropical regions. The plant has been reported as vermifuge, antiseptic, antitussive, antipyretic and ant-inflammatory agent and finds uses in the treatment of osteoarticular pain and bronchitis. Antimalarial activity was also reported from the herb.³ Leaves of the plant were reported to possess anti-inflammatory, analgesic and antipyretic activity in rodents and gastroprotective effect were reported.^4,5

In the absence of systematic literature the present study was designed to evaluate the hypoglycemic, hypolipidemic and antioxidant properties of Lippia nodiflora leaves extract in alloxan induced diabetic rats

MATERIALS AND METHODS:

Plant material:

Fresh, mature leaves of Lippia nodiflora were collected from Neyveli, Cuddalore District. The plants were identified and authenticated and a voucher specimen deposited at the Department of Botany, University of Madras, Chennai.

Preparation of Plant extract:

Lippia nodiflora leaves were dried at room temperature and powdered electrical grinder, which was then stored in an airtight container at 5°C until further use. Powdered leaf was delipidated with petroleum Ether (60 - 80°C) for overnight. It was filtered and soxhalation was performed, extracted the residue with 95% Ethanol. Ethanol was evaporated in a rotary evaporator at 40 – 50°C under reduced pressure.

Preliminary phytochemical screening:

The ethanolic extract of Lippia nodiflora leaves were subjected to preliminary phytochemical screening of various plant constituents.^6,7

Experimental Animals:

Male albino Wistar rats (150-180 g) were purchased from TANUVAS, Madavaram, Chennai. The rats were housed in polypropylene cages lined with husk and kept in Animal house, Department of Biochemistry. It was renewed every alternative day. The rats were fed with commercial pellated rats chow (Lipton Ltd., Bangalore, India) and had free access to water. The experimental rats were maintained in a controlled environment (12:12 hours light/dark cycle) and temperature (30 ± 2°C). The experiments were designed and conducted in accordance with the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines for the investigation of experimental pain in conscious rats. The rats were acclimatized for one week before starting the experiments.

Induction of diabetes mellitus:

Rats were induced diabetes by single intraperitonial injection of alloxan monohydrate dissolved in sterile normal saline at a dose 120 mg/Kg, after 18 hours fasting to induce hyperglycemia. After 1 hour alloxan administration, the animals were fed on standard pellets and water ad libitum. Rats were supplied with 5% glucose solution for 48 hours after alloxan injection in order to prevent severe hypoglycaemia. After 1 week time for the development and aggravation of diabetes, the rats with moderate Diabetes having persistant glycosuria and hyperglycemia (Blood glucose range of above 250 mg/dL) were considered as diabetic rats and used for the experiment. The treatment was started on the eighth day after alloxan injection and this was considered as first day of treatment.

Experimental Design:

The rats were grouped into 4 groups, comprising of 6 rats in each group as follows:

Group I : Control rats

Group II : Alloxan induced diabetic rats

Group III : Diabetic rats treated with Lippia nodiflora leaf extract (400 mg/Kg body weight/day) in aqueous solution orally for 30 days.

Group IV : Diabetic rats treated with gliclazide (5mg/kg body weight/day) in aqueous solution orally for 30 days.

Oral Glucose Tolerance Test (OGTT):

OGTT was performed on 28^th day of the experimental period. Prior to an OGTT, all the rats were fasted overnight and then rats were loaded with glucose (2 g/ Kg body weight) in aqueous solution with a feeding syringe. Blood samples were collected from the tail vein just prior to the administration of glucose and at 30, 60, 90 and 120 min after glucose loading. The level of glucose in all blood samples was measured by the method of Trinder (1969).⁸

At the end of the experimental period, the rats were fasted over night, anaesthetized, and sacrificed by cervical decapitation. The blood was collected with and without anticoagulant for plasma and serum separation respectively. Blood glucose level was estimated by the method of glucose oxidase/peroxidase as described by Trinder (1969).⁸ Plasma was separated and used for insulin assay using ELISA kit for rats. Level of glycosylated hemoglobin was estimated according to method of Nayak and Pattabiraman (1981).⁹

Preparation of tissue homogenate:

The pancreatic tissues were excised, rinsed in ice- cold saline. Known amount of the tissues were homogenized in Tris–HCl buffer (100 mM, pH 7.4) at 4° C, in a Potter–Elvehjem homogenizer with a Teflon pestle at 600 rpm for 3 min. The homogenate was then centrifuged at 12,000-×g for 30 min at 4°C. The supernatant was collected as tissue homogenate, which was used to assay various parameters.

The level of lipid peroxides in plasma and pancreas was determined by the method of Ohkawa et al. (1979). ¹⁰Levels of vitamin C, vitamin E, and glutathione (GSH) in plasma were determined by the methods of Omaye et al.(1979), Desai (1984), and Sedlak and Lindsay (1968), respectively.^11-13 Enzymatic antioxidants such as Superoxide dismutase (Misra and Fridovich, 1972), Catalase (Takahara et al., 1960), glutathione peroxidase (Rotruck et al., 1973) in pancreatic tissues were assayed.^14-16

Lipid profile:

Plasma was used for the estimation of lipid profile. Cholesterol content was estimated by the method of Parekh and Jung (1970). ¹⁷ Triglyceride was estimated by the method of Rice (1970).¹⁸ HDL Cholesterol fraction was separated by the precipitation techniques of Burstein and Scholnick (1972) and the cholesterol content was determined by method of Parekh and Jung (1970).^19,17

RESULTS:

Table 1 shows the qualitative analysis of phytochemical in the ethanolic extract of Lippia nodiflora leaves. Phytochemical evaluation revealed the presence of phenols, alkaloids, flavonoids, glycosides, tannins, and terpenoids.

Table 1. Preliminary phytochemical screening

PHYTOCONSTITUENTS	INFERENCE
Phenols	+
Alkaloids	+
Flavonoids	+
Glycosides	+
Saponins	-
Tannins	+
Phytosterol	-
Triterpenoids	+
Anthraquinones	-

Figure 1. Effect of Lippia nodiflora on the blood glucose level in the experimental groups of rats receiving an oral glucose load.

Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows: *compared with control, ^@compared with diabetic rats.

Fig 1 shows the changes in the levels of blood glucose, after oral administration of glucose (3g/Kg) in normal control and experimental group of rats. The data of OGTT revealed that the blood glucose value in normal control rat reach peak at 60 minutes after the oral glucose load and gradually reverted back to near normal levels after 120 minutes. In diabetic control rats, the peak increase in blood glucose concentration was observed after 60 minutes and remained high over the next 60 minutes. Lippia nodiflora leaves extract as well as gliclazide treated group showed showed definite lower peak blood glucose values, and was returned back to near basal level at the end of 120 minutes.

The effect of oral administration of Lippia nodiflora leaves extract on the levels of blood glucose, plasma insulin, glycosylated hemoglobin and urine sugar in the control and experimental groups of rats were depicted in Table 2. The elevated levels of blood glucose, glycosylated hemoglobin in the diabetic group of rats were reverted to near normal level by the administration of Lippia nodiflora leaves extract. Conversely, the decreased level of plasma insulin in diabetic group of rats was elevated by the administration of Lippia nodiflora leaves extract for 30 days. Urine sugar which is present in the diabetic group of rats was absent in Lippia nodiflora leaves extract as well as gliclazide treated diabetic group of rats. The results are comparable with gliclazide, an oral hypoglycemic drug.

Table2. Effect of L.nodiflora leaves extract on the levels of biochemical parameters in the experimental groups of rats.

Groups	Glucose (mg/dl)	Insulin (µU/ml)	Glycosylated hemoglobin (%Hb)	Urine sugar
Control	102.76 ± 12.46	15.21 ± 2.59	6.01 ± 1.49	Nil
Diabetic	290.67 ± 25.14*	5.92 ± 1.21*	14.17 ± 2.95*	+++
Diabetic + L. nodiflora extract	145.99 ± 15.10@	10.97 ± 2.50@	8.56 ± 1.99@	Nil
Diabetic + gliclazide	130.24 ± 16.54@	12.45 ± 1.79@	7.59 ± 2.35@	Nil

Table 3. Effect of L.nodiflora leaves extract on the level of TBARS in plasma and pancreas of experimental groups of rats.

Groups	TBARS
Groups	Plasma	Pancreas
Control	4.26 ± 0.65	38.45 ± 4.59
Diabetic	8.24 ± 1.79*	75.66 ± 9.47*
Diabetic + L. nodiflora extract	5.10 ± 1.65@	50.97 ± 6.19@
Diabetic + gliclazide	4.99 ± 1.52@	52.45 ± 7.34@

Units: mM/100 g in tissues; nM/ml in plasma. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows: *compared with control, @ compared with diabetic rats.

The effect of Lippia nodiflora leaves extract on the plasma levels of non-enzymatic antioxidants such as vitamin C, vitamin E, and reduced glutathione in experimental groups of rats are shown in Table 4. The diminished levels of non-enzymatic antioxidants in the diabetic group of rats were significantly (p<0.05) improved to near normal values by the oral administration of Lippia nodiflora leaves extract as well as gliclazide, after 30 days of treatment.

Table 3 represents the effect of Lippia nodiflora leaves extract on the levels of lipid peroxides in the plasma and pancreas of experimental groups of rats. The levels of lipid peroxides were significantly (p<0.05) elevated in the diabetic group of rats. Upon oral administration of Lippia nodiflora seed extract as well as gliclazide to diabetic group of rats were significantly (p<0.05) reverted to normal levels when compared to control group of rats.

Table 4. Effect of L.nodiflora extract on the levels of nonenzymatic antioxidants of experimental groups of rats.

Groups	Vitamin C	Vitamin E	GSH
Control	1.50 ± 0.18	0.71 ± 0.09	30.45 ± 3.95
Diabetic	0.52 ± 0.07*	0.29 ± 0.05*	14.39 ± 2.56*
Diabetic + L. nodiflora extract	0.99 ± 0.14@	0.56 ± 0.07@	22.67 ± 2.74@
Diabetic + gliclazide	1.03 ± 0.16@	0.62 ± 0.09@	25.52 ± 3.61@

Units: mg/dl. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows: *compared with control, @ compared with diabetic rats

Table 5 depicts the activity of pancreatic enzymatic antioxidants such as superoxide dismutase, catalase and glutathione peroxidase in the experimental groups of rats. The decreased activity of enzymatic antioxidants observed in the diabetic group of rats were significantly (p<0.05) elevated to near normal levels after treatment with Lippia nodiflora leaves extract as well as gliclazide.

Table 5. Effect of L.nodiflora leaves extract on the activity of SOD, Catalase and GPx in pancreas of experimental groups of rats.

Groups	SOD	Catalase	GPx
Control	5.60 ± 1.57	16.25 ± 2.38	6.75 ± 1.21
Diabetic	1.52 ± 0.45*	5.39 ± 1.16*	3.19 ± 0.37*
Diabetic + L. nodiflora extract	3.90 ± 0.95@	12.53 ± 1.79@	4.64 ± 0.59@
Diabetic + Gliclazide	4.02 ± 0.89@	13.24 ± 1.89@	5.01 ± 0.99@

Activity is expressed as: 50% of inhibition of epinephrine autoxidation /min/mg of protein for SOD; µM of hydrogen peroxide decomposed/min/mg of protein for catalase; µM of glutathione oxidized/min/mg of protein for GPx, mg/100 g tissue for GSH. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows: *compared with control,^@compared with diabetic rats.

The levels of cholesterol, triglycerides, HDL- cholesterol and LDL- cholesterol in control and experimental groups of rats are shown in Table 6. The levels of cholesterol, triglycerides, LDL-cholesterol and were significantly increased whereas the HDL-cholesterol was significantly decreased in alloxan-induced diabetic rats. Treatment with c extract as well as gliclazide significantly ameliorated these levels to near normal levels.

Table 6. Effect of L. nodiflora leaves extract on the levels of total cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterol in the plasma of experimental groups of rats.

Groups	Total cholesterol	Triglycerides	LDL	HDL
Control	74.90 ± 10.31	60.19 ± 8.66	35.06 ± 5.21	29.96 ± 2.84
Diabetic	159.27 ± 16.24*	139.72 ± 15.82*	116.51 ± 10.59*	14.49 ± 1.81*
Diabetic + L. nodiflora extract	105.91 ± 14.67@	85.40 ± 10.14@	70.94 ± 6.49@	23.34 ± 2.01@
Diabetic + gliclazide	99.13 ± 12.29@	80.49 ± 9.61@	59.74 ± 6.07@	23.57 ± 2.57@

DISCUSSION:

Alloxan is widely employed to induce diabetes mellitus in experimental animals due to the fact that it causes a massive reduction of the insulin secreting β cells of islets of langerhans, resulting in a decrease in endogenous insulin release, which paves the ways for the decreased utilization of glucose by the peripheral tissue lead to hyperglycemia. Alloxan also increases the oxidative stress which is the possible mechanism of its diabetogenic action.²⁰

The phytochemical analysis of Lippia nodiflora leaves indicated the presence of pharmacologically active ingredients such as phenols, alkaloids, flavonoids, saponins, tannins, terpenoids and glycosides. The phytochemicals identified from traditional medicinal plants are presenting an exciting avenue for the development of novel therapeutic agents especially in combating diabetes and diabetes related complications.

Hyperglycemia is the clinical hallmark of diabetes. The fundamental mechanism underlying hyperglycemia involves the decreased utilization of glucose by peripheral tissues and increased endogenous glucose production. Many reports have shown that the level of blood glucose is elevated in experimentally induced diabetic rats. Oral administration of Lippia nodiflora leaves extract significantly improved the glucose tolerant and this might be due to enhanced glucose utilization by peripheral tissues like skeletal muscles.

Diabetes is known to cause protein glycation, also known as non-enzymatic glycosylation. It has been reported that various proteins, including hemoglobin, albumin, collagen, low-density lipoproteins and fibronectin undergo non-enzymatic glycation in diabetes. During chronic hyperglycemic conditions, the glycosylated form of hemoglobin has an altered affinity for oxygen and this may account for tissues anoxia. The rate of glycosylation is directly proportional to concentration of blood glucose level and with improvement of glycemic control glycosylated hemoglobin also decreases.²¹ The observed normalized level of glycosylated hemoglobin in Lippia nodiflora leaves extract as well as gliclazide treated group of diabetic rats may be due to the maintenance of normal glucose homeostasis.

Oxidative stress is considered to be chiefly associated with many diseases, including cell damage, but diet plays a vital role in human health and in the prevention of certain diseases. Free radicals are formed disproportionately in diabetes by glucose oxidation, non enzymatic glycation of proteins and the subsequent oxidative degradation of glycated proteins. Increased generation of reactive oxygen radicals such as superoxide and hydrogen peroxides leads to decreased activity of antioxidants and cellular defense enzymes in blood and tissues. Inactivation of cellular defense in diabetes can be due to their exhaustion during detoxification of free radicals produced by cell membrane lipid peroxidation.²²

Lipid peroxidation (LPO) is enhanced due to an increased oxidative stress in diabetic condition. Lipid peroxidation products such as MDA are generated under high levels of un-scavenged free radicals and may bring about protein damage and inactivation of membrane bound enzymes and thus, they play an important role in pancreatic damage associated with diabetes. Induction of diabetes in rats uniformly results in an increase in the lipid peroxidation (TBARS), an indirect evidence of intensified free radical production.²³ Increased LPO impairs membrane function by decreasing membrane fluidity and changing the activity of membrane bound enzymes and receptors. Lipid peroxides mediated tissue damage has been observed in the development of both type 1 and type 2 diabetes.²⁴ The increased concentration of lipid peroxides in plasma and pancreatic tissues of diabetic rats indicates detonated production of free radicals. The elevated levels of TBARS in diabetic rats were reduced significantly to near-normal levels upon treatment with Lippia nodiflora.

The enzymatic antioxidants such as SOD, Catalase and glutathione peroxidase are involved in the scavenging of reactive oxygen metabolites that are produced as result of chronic hyperglycemia. In diabetic milieu, the activities of all these enzymatic antioxidants are declined notably because of the detonated production of free radicals arise out of chronic hyperglycemia. Oral administration L.nodiflora leaves extract caused a significant increase in the levels of non-enzymatic antioxidants such as vitamin C, vitamin E, reduced glutathione and enzymatic antioxidants such as SOD, Catalase and glutathione peroxidase suggesting that the L.nodiflora leaves extract possess free radical scavenging and antioxidant activity.

The abnormal high concentrations of plasma lipids in diabetes is mainly due to the increase in the mobilization of free fatty acids from the peripheral depots in the absence (or) deficiency of insulin, since insulin inhibits hormone sensitive lipase on the other hand glucagons, catecholamines and other hormones enhance lipolysis.²⁵ The marked hyperlipemia that characterizes the diabetic state may therefore be regarded as a consequence of the uninhibited actions of lipolytic hormones on the fat depots.²⁶ Our results of increased plasma lipids correlated with the above findings of hyperlipidemia in diabetes. However, oral administration of L.nodiflora leaves extract as well as gliclazide to diabetic rats significantly decreased the levels of cholesterol, triglycerides, LDL and increases HDL cholesterol in plasma are presumably mediated by controlling the lipid metabolism.

CONCLUSION:

The results of the present study clearly indicate that the Lippia nodiflora leaves extract possess hypoglycemic property evidenced from improved OGTT, glycosylated hemoglobin level. Oral administration of Lippia nodiflora leaves extract protects the antioxidant status in the pancreas as well as in plasma which may be due to the attenuation of oxidative stress. In addition, the significant improvement in the plasma lipid profile revealed the hypolipidemic activity of the Lippia nodiflora leaves extract. The phytochemicals present in the leaves extract may account for these pharmacological actions.

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Received on 16.09.2011

Accepted on 30.09.2011

Research J. Pharmacology and Pharmacodynamics. 3(6): Nov.-Dec., 2011, 299-304