Hypoglycemic Activity of Leucas linifolia (Lamiaceae) Spreng.

 

Sneha Anarthe1* and Sanjay Chaudhari2

1Department of Pharmacognosy, Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad, India

2Amrutvahini College of Pharmacy, Sangamner, Tal- Sangamner, Dist- Ahmednagar, Maharashtra, India.

ABSTRACT:

The hypoglycaemic and antihyperglycemic activity of methanolic and ethyl acetate extracts of Leucas linifolia were investigated for in-vitro alpha amylase inhibitory activity and hypoglycaemic activity on normal rats and alloxan induced diabetic rats. The methanolic and ethyl acetate extracts of Leucas linifolia showed good in-vitro alpha amylase inhibitory activity as compare to standard acarbose with IC50 value of 27.13 and 33.61. The methanolic extract (300mg/kg) and ethyl acetate extract (300mg/kg) of Leucas linifolia reduced the normal blood glucose level from 78.2 ± 3.126 to 57 ± 4.266 mg/dl and 82.4 ± 1.208 to 61.2 ± 4.630, 2h after oral administration of the extract (P<0.01). In case of alloxan induced hypoglycaemic activity, the blood glucose levels were measured at 0, 7th 15th and 21st day after the extract treatment. Both methanolic (300mg/kg) and ethyl acetate (300mg/kg) extracts also significantly lowered the blood glucose level in alloxan induced diabetic rat from 311.16 ± 8.356 to 241.0 ± 6.812 mg/dl and 309.5 ± 9.674 to 242.5 ± 9.882 mg/dl, 21 day after oral administration of extract (P<0.001). The antihyperglycemic activity of Leucas linifolia was compared to standard drug glibenclamide (0.25mg/kg), an oral hypoglycaemic activity. Besides total phenolic and total flavonoid contents were also investigated for methanolic and ethyl acetate extracts.

 

KEYWORDS: alpha-amylase inhibitory, hypoglycaemic activity, Leucas linifolia, Lamiaceae.

 

INTRODUCTION:

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia. The management of the blood glucose level is a strategy in the control of diabetes complication1. Inhibitors of carbohydrate hydrolyzing enzymes have been useful as oral hypoglycemic drugs for the control of hyperglycemia especially in patients with type II diabetes mellitus. One therapeutic approach for treating diabetes is to decrease the post-prandial hyperglycaemia, which is done by retarding the absorption of glucose through the inhibition of the carbohydrate hydrolyzing enzymes (alpha-amylase and alpha-glucosidase) in the digestive tract. Inhibitors of these enzymes delay carbohydrate digestion and prolong overall carbohydrate digestion time, causing a reduction in the rate of glucose absorption and consequently blunting the post-prandial plasma glucose rise2. Examples of such inhibitors which are in clinical use are acarbose, miglitol and voglibose3.

 

Plants have long been used for the ethnomedical treatment of diabetes in various systems of medicine. In the recent years research on medicinal plants for the management of diabetes has attracted the interested of scientists.4, 5

 

So present study investigates Leucas linifolia (Family- Lamiaceae) for alpha amylase inhibitory effect and alloxan induced antihyperglycemic effect in rats. Leucas linifolia Spreng (syn. L. lavandulaefolia Rees) is herbaceous annual weed which grows abundantly in fields, pastures and waste lands throughout India. It has a strong flavour and is reputed for its use as sedative, vermifuge, stomachic and dermatosis. This herb is also used in psoriasis, chronic skin eruptions and painful swellings6,7. The antibacterial activity of this plant extract against different micro-organisms has already been reported8

 

Phenylethanoid glycosides were isolated from the aerial parts of Leucas linifolia Linn and they were found to contain antioxidant activity along with the inhibitory activity against xanthine oxidsase enzyme.9 Methanolic extract of aerial parts of Leucas linifolia showed antipyretic activity10 and potential reduction in spontaneous activity and cause a significant decrease in exploratory behavioural pattern by the head dip and Y-maze test. The extract shows a remarkable potentiation of pentobarbitone induced sleeping time in mice.11 Methanolic extract of herb caused a significant reduction of blood glucose levels in streptozotocin induced diabetes12 and has antitussive activity13 as well as wound healing activity.14

 

MATERIAL AND METHOD:

Plant material and preparation of extracts:

The whole Leucas linifolia plant was collected in June 2009 from Ahmednagar district, Maharashtra (India). The plant specimen was authenticated from Botanical Survey of India, Pune (Voucher specimen no. LRM1). Plant materials were dried under shade and coarsely powdered for extraction. The coarsely powdered whole plant (900g) of Leucas linifolia was separately subjected to extraction using ethyl acetate and methanol by maceration for 10 days. The methanolic and ethyl acetate extracts were concentrated by rotary vacuum evaporator under reduced pressure and then dried in open air.

 

In-vitro Alpha- amylase inhibitory activity:15

The assay was carried out following the standard protocol with slight modifications.16 Starch azure (2 mg) was suspended in a tube containing 0.2ml of 0.5 M Tris-Hcl buffer (pH 6.9) containing 0.01 M calcium chloride (substrate). The tube was boiled for 5 min and then pre incubated at 37°C for 5 min. 1ml of 0.1% of dimethyl sulfoxide was used to dissolve 1 mg of dried plant extract in order to obtain concentrations of 9.37, 18.75, 37.5 μg/ml. Then 0.2 ml of plant extract of a particular concentration was put in the tube containing the substrate solution. 0.1 ml of porcine pancreatic amylase in Tris-Hcl buffer (2units/ml) was added to the tube containing the plant extract and substrate solution. The process was carried out at 37°C for 10 min. The reaction was stopped by adding 0.5 ml of 50% acetic acid in each tube. The reaction mixture was then centrifuged at 3000 rpm for 5 min at 4°C. The absorbance of resulting supernatant was measured at 595 nm using UV spectrophotometer. Same procedure was followed for both the plant extracts (methanol and ethyl acetate) to test the alpha amylase inhibitory effects. The experiments were repeated thrice using the same protocol.

 

Animals:

Healthy Albino male rats weighing 160-180 g were used. They were housed under standard environmental conditions of temperature, humidity and light, and provided with standard rodent food and water ad libitum. All the animals were carefully monitored and maintained in accordance with CPCSEA guidelines on control and supervision of experimental animals. The ethical clearance was obtained from the Institutional Animal Ethics Committee before the experiment.

 

Evaluation of extract on normal healthy rats:17

The animals were randomly divided into seven groups of six animals each. Group I served as control and received distilled water. Groups II, III, IV, V, VI and VII received methanolic and ethyl acetate extracts orally at the dose of 150 mg/kg, 300 mg/kg, and 450 mg/kg respectively. At the end of the fasting period taken as zero time (0 h), blood was withdrawn from the tail vein under mild ether anesthesia. Serum was separated by centrifugation and glucose was estimated. Blood glucose levels were determined 30, 60, 120, 150, 180 and 240 minutes following treatment.

 

Evaluation of extract in Alloxan induced diabetes

Induction of diabetes:

Diabetes was induced in male albino rats, aged 4 months (body weight 160-180 g) by intraperitoneal administration of ice-cold aqueous alloxan monohydrate (150 mg/kg body weight) by the method described earlier18. After a fortnight, rats with marked hyperglycemia (fasting blood glucose >250 mg/dl) were selected and used for the study. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in plastic cages.

 

Experimental design:

The rats were divided into nine groups and each group consisted of six rats.

Group I normal untreated rats

Group II diabetic untreated rats

Group III diabetic rats treated with standard Glibenclamide 0.25 mg/kg b.w.

Group IV diabetic rats treated with 150mg/kg b.w. methanolic extract.

Group V diabetic rats treated with 300mg/kg b.w. methanolic extract

Group VI diabetic rats treated with 450mg/kg b.w. methanolic extract

Group VII diabetic rats treated with 150mg/kg b.w. ethyl acetate extract

Group VIII diabetic rats treated with 300mg/kg b.w. ethyl acetate extract

Group IX diabetic rats treated with 450mg/kg b.w. ethyl acetate extract

After an overnight fast Group I and Group II rats were fed distilled water alone and for other groups the plant extract suspended in distilled water were fed to the experimental rats by gastric intubations, using a force feeding needle. Blood samples were collected for the measurement of blood glucose from the tail vein at 0, 7th, 15th and on 21st day after feeding of the plant extract once daily. Blood glucose was measured and the results were compared with those of 1st and 2nd group of rats which were treated with distilled water.

 

Estimation of total phenolics and total flavonoids:

Total phenolic content of methanolic and ethyl acetate extracts of Leucas linifolia  was determined by using Folin-Ciocalteu reagent19. The blue color formed due to the polyphenol content in the extract was measured at 760 nm using a Shimadzu UV-1601 spectrophotometer and the results were expressed as ΅g/mg of gallic acid equivalent. Total flavonoids of methanolic and ethyl acetate extracts of two samples were determined using the method of Liu et al.20 with some modifications. In brief, the extract was diluted with 80% aqueous ethanol (0.9 ml). Aliquots of 0.5 ml of extract were added to test tube containing 0.1 ml of 10% aluminum nitrate, 0.1 ml 1M aqueous potassium acetate and 4.3 ml of 80% ethanol. The reaction tubes were set aside for 40 min at room temperature. At the end of this time, optical density of each sample was determined at 415 nm using a UV spectrophotometer. Total flavonoids content was calculated by interpolation on a standard curve established with a reference standard, quercetin. Quercetin and Folin-Ciocalteu reagent were obtained from Sigma-Aldrich, Germany

 

Statistical analysis:

All the values of body weight and fasting blood sugar were expressed as mean ± standard error of mean (S.E.M.) and analyzed for ANOVA and post hoc Dunnet’s t-test. Differences between groups were considered significant at p<0.05, p<0.01, p<0.001 levels.

RESULT AND DISCUSSION:

Alpha amylase inhibitory activity: Although there are citations of antihyperglycemic and antidiabetic activity of some Leucas species, there are no previous reports, on the activity of the Leucas linifolia on in vitro α-amylase activity. In the present study, methanolic and ethyl acetate extracts of Leucas linifolia was found to possess good a-amylase inhibitory activity with IC50 value of 27.13 and 33.61 as compare to standard as acarbose having IC50 - 9.22 (Table 1).

 

Table 1 Alpha-amylase inhibition activity of Leucas linifolia extracts

Sr. no.

Test extract

Dose (΅g/ml)

Percent inhibition

IC50 (΅g/ml)

1

MEL

9.37

18.75

37.5

13.08 ±0.1832

41.05 ±0.0731

66.23 ±0.2570

27.13

2

EAL

9.37

18.75

37.5

7.046± 0.0606

31.55 ± 0.2810

53.81 ±0.4081

33.61

3

Acarbose

2.5

5

10

4.99 ± 0.05292

28.11 ± 0.0688

53.13 ±0.1386

9.22

Results are expressed as ± SEM (n=3). MEC and EAC represent methanolic and ethyl acetate extracts of Leucas linifolia.

 

Hypoglycemic activity in normal rats:

In the present study methanolic extract and ethyl acetate extracts at 300 mg/kg significantly reduced the normal blood glucose level as compare to 150 mg/kg and 450 mg/kg b.w. dose. The results of acute hypoglycaemic activity showed significant (p < 0.01) increase in plasma glucose level as compare to normal control rats over the 4-h period (Table 2).

 

 

Table 2 Effect of methanolic and ethyl acetate extracts of Leucas linifolia on normal animals

Groups

Treatment

(dose mg/kg)

Fasting

30 min

60 min

120 min

180 min

240 min

Control group

-

81 ± 2.121

81.6 2.337

81.6± 2.227

81.4 ± 2.293

81.8 ± 2.354

82.8 ± 2.653

Group IA

ME-150 mg/kg

80.0 ± 2.121

76.8± 2.177*

73 ± 2.28*

65.2 ± 3.625*

70.6 ± 2.638*

73.2± 2.818*

Group IIA

ME-300 mg/kg

78.2 ± 3.126

75.2 ± 3.216*

67.8 ± 2.311*

57 ± 4.266*

68.4 ± 2.205*

71.8± 1.855*

Group IIIA

ME-450 mg/kg

81.6± 1.327

78.4± 1.208*

74.6± 1.288*

59.8 ± 1.020*

63 ± 1.844*

71.4± 0.871*

Group IB

EA-150 mg/kg

80.6 ± 1.720

77.8 ± 1.562*

74 ± 1.414*

69.8 ± 1.281*

71 ± 1.140*

75.4±  1.600*

Group IIB

EA-300 mg/kg

82.4 ± 1.208

78.8 ± 1.159*

75± 1.265*

61.2 ± 4.630*

74.8± 1.158*

78.4 ± 1.288*

Group-IIIB

EA-450 mg/kg

81.6± 2.015

77.8± 2.245*

73.4± 2.205*

64.4± 2.502*

71.2± 3.382*

75.4± 2.522*

Results are expressed as ± SEM (n=6), Data processed by one way ANOVA followed by Dunnett’s test,*P< 0.01, significant when compared to normal group.

 

Table 3 Effect of methanolic and ethyl acetate extracts of Leucas linifolia on diabetic animals

Group

Treatment

Blood glucose concentration in mg/dl

0 day

7 day

15 day

21 day

I

Control (Normal saline)

81.5 ± 2.262

81.5 ± 2.202

80.3 ± 2.092

80.33 ± 2.028

II

Diabetic control

307.66 ± 10.108

310 ± 10.529*

311.5 ±10.468 *

312.5 ± 10.868*

III

Glibenclamide (0.25mg/kg)

312.33 ± 9.559

276.16 ± 10.387*a

245.83 ± 11.368*a

199.66± 10.398*a

IV

MeOH-100mg/kg

303 ± 9.191

293.33 ± 9.069*a

280.33 ± 2.0292*a

270.33 ± 8.831*a

V

MeOH-200mg/kg

311.16 ± 8.356

284.5 ± 6.737*a

254.33 ± 6.787*a

241.0 ± 6.812*a

VI

MeOH-400mg/kg

305.0 ± 14.257

278 ± 14.257*a

250.5 ± 13.968*a

242.33 ±12.632*a

VII

EA-100mg/kg

309 ± 7.104

301.83 ± 6.374*a

291.0 ± 5.621*a

280.00 ± 5.209*a

VIII

EA-200mg/kg

309.5 ± 9.674

284.33 ± 9.559*a

264.33 ± 9.559*a

242.5 ± 9.882*a

IX

EA-400mg/kg

310.15 ± 9.061

274.166± 9.061*a

257.16 ± 9.061*a

234.83 ± 8.252*a

Results are expressed as ± SEM (n=6), *P< 0.001, significant when compared to control group. aP<0.001 significant when compared to diabetic control.

 

Table 4 Total phenolic and flavonoids content of methanolic and ethyl acetate extract of Leucas linifolia

Sr. no.

Crude drug samples

Total phenolic content (΅g/mg)

Total flavonoids content (΅g/mg)

1

MEL

79.65 ±0.3384

7.56 ±0.4807

2

EAL

54.88 ±0.0669

9.23 ±0.3844

Results are expressed as ± SEM (n=3) and compared with standard as gallic acid for Total phenolic content and Quercetin for total flavonoids content.

 

Table 5 Changes in body weight of animals during the activity

Days

Normal group

Diabetic control

Test group-IA

Test group-IIA

Test group-IIIA

Test group- IB

Test group-IIB

Test group-IIIB

0

169.8 ± 3.680

172.2 ± 3.499

167.8 ± 2.518

171.2 ± 2.922

175.8 ± 2.922

171.4 ± 3.140

174.8 ± 1.960

171.4 ± 2.272

21

171.4 ± 3.44b

116.6 ± 3.782*

120.6 ± 4.98*

129.5 ±5.958*a

124.7 ± 6.979*

123.6 ± 3.362*

126.4 ± 6.025*

137.8 ± 3.499*b

Results are expressed as ± SEM (n=6).  * p<0.01 significant when compared to normal group. a - p<0.05, b - p<0.01.

 

 

Alloxan induced diabetic rats:

The methanolic and ethyl acetate extracts of Leucas linifolia also have shown significant (p<0.001) antihyperglycemic activity (Table 3). But methanolic extract showed good antihyperglycemic activity as compare to ethyl acetate extract. The phytochemical screening of Leucas linifolia revealed the presence of flavonoids, phenolic compounds, sterols/triterpenoids, alkaloids, tannins. Flavonoids, sterols/triterpenoids, alkaloids and other phenolics are known to be bioactive principles helpful in diabetes21-24. The flavonoids are known to regenerate the damaged beta cells in the alloxan diabetic rats25 and phenolics are found to be effective antihyperglycemic agents26. In the present study, 7.56 ΅g/mg of flavonoids and 79.65 ΅g/mg phenolic compounds were found to be present in the methanolic extract of Leucas linifolia (Table 4) so it may be one of the reason that methanolic extract shows good hypoglycemic and antihyperglycemic activity.

 

Changes in initial and final body weight in control and experimental groups are shown in Table 5. Significant weight loss was observed in diabetic rats compared to control non-diabetic rats. Treatment with Leucas linifolia extracts or glibenclamide improved the body weight as compared to normal control rats.

 

CONCLUSION:

In conclusion, the present study reveals the potential of Leucas linifolia for inhibition of carbohydrate hydrolyzing enzyme and regulating carbohydrate metabolism in Insulin dependence diabetes mellitus.

 

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Received on 01.12.2010

Accepted on 25.12.2010     

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 3(1): Jan. - Feb. 2011, 34-37