Evaluation of glucose utilization capacity of bioactivity-guided fractions of Barleria prionitis Linn and Hyptis suaveolens (L.) Poit in isolated rat hemidiaphragm.

INTRODUCTION: Diabetes mellitus (DM) is a chronic disease characterized by high blood glucose levels due to absolute or relative circulating insulin levels. Plants represent a major potential source of drugs for treating diabetes. The study of plants having antidiabetic activity may give a new approach in the treatment of DM. AIM: This study was aimed to investigate the glucose utilization capacity of bioactivity-guided fractions of Barleria prionitis and Hyptis suaveolens.
MATERIALS AND METHODS: The dried coarse powdered plant material was extracted in aqueous ethanol by cold maceration; further, ethanolic extracts were fractionated using solvents of varying polarity and were investigated in isolated rat hemidiaphragm using 0.1% and 0.2% concentrations of plant extracts.
RESULTS: The tested fractions of both plants showed significant and dose-dependent increased glucose uptake capacity and was found to be maximum with petroleum ether and aqueous ethanolic extracts of both plants; 0.2% concentration of both plant extracts is superior in activity when compared to 0.1% of the test extracts. H. suaveolens has produced more glucose utilization capacity when compared to B. prionitis.
CONCLUSION: The activity of H. suaveolens is comparable to standard insulin (P < 0.01). Both the plant materials have some extra pancreatic mechanism like glucose uptake by peripheral tissue.

Introduction

Diabetes mellitus is a group of metabolic disorders and not a single disease sharing a common underlying feature of hyperglycemia.[1] The antihyperglycemic agents are categorized into oral hypoglycemic agents and insulin. The oral hypoglycemic agents are effective in type 2 diabetes only while insulin is used for all types of people with diabetes. In several studies, treatment with traditional medicine in the form of plant extract has been reported to give remarkably good results. Available ethnobotanical information reports about 800 plants which may possess antidiabetic potential. However, most of these plant materials are not scientifically evaluated and are incompletely characterized.

Barleria prionitis Linn.belongs to the family Acanthaceae (BPA) which is a prickly shrub 1.5 m high with simple opposite decussate leaves. The flower is yellow and sessile. The plant is cultivated as hedge plant and is distributed throughout the warmer parts of India.[2] The major constituents found are acetylbarlerin and barlerin, [34] and other chemical constituents present are 6-O-acetylshanzhiside methyl ester, [5] 6-O-cis-p-coumaroyl-8-O-acetylshanzhiside methyl ester its transisomers, shanzhiside methyl ester α-amyrin, verbascoside, [6] β-sitosterol, [7] and stigmasterol-3-O-D-glucoside.[5] The plant is known to have anti-inflammatory, expectorant, analgesic, diuretic, antirheumatic, [89] and antidiabetic properties.[10] The plant extract is also known to possess hepatoprotective activity, [11] in respiratory infections[6] and tuberculosis.[12]

Hyptis suaveolens Poit.belonging to the family Lamiaceae (HSL) is an annual and aromatic plant found in open and wasteland at low and medium altitude in bushes. Traditionally, various parts of this plant are used in different ailments such as respiratory tract infections, cold, pain, inflammation, fever, skin diseases, and in diabetes[1314] and is also used for antifungal, [15] antibacterial, [16] and anticonvulsant activities.[17] Phytochemical constituents present in the leaves of H. suaveolens are hentriacontane, hentriacontanone, lupeol, and its acetate and friedelin.[18] The aerial parts are also reported to contain antiplasmodial diterpenoids[19] and triterpenoid heptadienic acid.[20]

In traditional medicine and locally, both plants have claimed to be useful in diabetes;[101314] till for studies on the hypoglycemic effects of B. prionitis have not so far been scientifically evaluated, and a limited report available on the antihyperglycemic activity of H. suaveolens with an unknown mode of action that too not investigated scientifically was found. Hence, the present investigation was carried out on isolated rat hemidiaphragm to ascertain whether the fractions of both plants have any direct insulin-like activity or any effect which can augment the effect of insulin so that further studies could be taken up to isolate the concerned active principle if any. 0.1% and 0.2% concentrations of both plant extracts were investigated in the present study.

Materials and Methods

Plant material

The aerial parts of B. prionitis were collected in the month of September from Attapur, Hyderabad, Telangana, and aerial parts of H. suaveolens were collected in the month of October from Rajendra Nagar, Hyderabad. A voucher specimen of B. prionitis (AUB-BPA-2013) and H. suaveolens (AUB-HSL-2013) is being maintained in the Department of Phytochemistry and Pharmacognosy. Identification and authentication of the plants were done by scientist and taxonomist of the Botanical Survey of India, Hyderabad, Telangana. The aerial parts of both the plants were cut, air-dried, and grounded into powder for extraction.

Preparation of ethanolic extract

Extraction of the dried aerial part powder of B. prionitis (1.4 kg) and H. suaveolens (1.3 kg) was performed with 80% aqueous ethyl alcohol by maceration process for 5 days. The percentage yield of crude aqueous ethanolic extract of B. prionitis was 10.71 and that of H. suaveolens was 9.23.

Fractionation of extract

The concentrated aqueous ethanolic extracts were suspended in 500 ml of water. The solvents, petroleum ether (4x500 ml), chloroform (4x500 ml), ethyl acetate (4x500 ml) and n-butyl alcohol (4x500 ml) were used for the fractionation of the concentrated extracts of both the plants. The percentage yields of B. prionitis were 0.46, 0.65, 0.29, 0.64, and 6.86, whereas for H. suaveolens were 0.56, 0.59, 0.2, 0.55, and 5.74 with petroleum ether, chloroform, ethyl acetate, n-butanol, and leftover aqueous extract respectively.

Animals

The protocol of the animal experimentation was approved by the Institutional Animal Ethics Committee (GPRCP/IAEC/02/12/3/PCG/AE-06-Rats-M/F-250). Animals used in the experiment were the Wistar rats of either sex, maintained under standard conditions of temperature, relative humidity, and dark: light cycles with free access to standard diet (Hindustan Unilever, India) and water.

Acute toxicity studies

Five animals were used for the acute toxicity studies and performed according to OECD 425. A composition of 2000 mg/kg was used as test dose; the first animal was given a test dose and observed continuously for a period of 2 h, intermittently for 6 h, and then 24 h for lethality and abnormal behavioral changes. If the animal survived with the given test dose, then the other four animals were also administered with the test dose sequentially. All the animals under study survived with the test dose of 2000 mg/kg. Hence, LD50 of ethanolic extract of both the plants was >2000 mg/kg.[21]

Effect on glucose uptake by isolated rat hemidiaphragm

Glucose uptake by rat hemidiaphragm was estimated by method described by Walaas and Walaas[22] and Chattopadhyay et al.[23] Thirteen sets containing six numbers of graduated test tubes (n = 6) for each group of B. prionitis [Table 1] were taken as follows:

Table 1

Effect of Barleria prionitis (0.1%) on glucose utilization by isolated rat hemidiaphragm

Group 1: 2 ml of tyrode solution with 2% glucose

Group 2: 2 ml of tyrode solution with 2% glucose and regular insulin (Nova Nordisk) 0.62 ml of 0.4 U/ml solution (or 6.2 μl of 40 U/ml)

Group 3–13: 2 ml of tyrode solution with 2% glucose + 1.38 ml of aqueous ethanolic, petroleum ether, chloroform, ethyl acetate, butanol, and left-over aqueous fractions of B. prionitis (0.1%) with and without insulin [Table 1].

Effect of 0.2% B. prionitis plant extracts’ concentration was investigated as per the scheme illustrated in Table 2.

Table 2

Effect of Barleria prionitis (0.2%) on glucose utilization by isolated rat hemidiaphragm

Similarly, H. suaveolens extracts (0.1% and 0.2%) were also tested for glucose uptake by rat hemidiaphragm. The scheme of evaluation is represented in Tables 3 and 4.

Table 3

Effect of Hyptis suaveolens (0.1%) on glucose utilization by isolated rat hemidiaphragm

Table 4

Effect of Hyptis suaveolens (0.2%) on glucose utilization by isolated rat hemidiaphragm

The volumes of all test tubes were made up to 4 ml with distilled water. Overnight fasted albino rats were sacrificed by cervical dislocation. The diaphragms were dissected out quickly with minimal trauma and divided into two halves. Two diaphragms from the same animal were not used for the same set of experiment. Six numbers of diaphragms were used for each group. The hemidiaphragms were placed in test tubes and incubated for 30 min at 37°C at an atmosphere of 100% oxygen with shaking at 140 cycles/min. Glucose uptake per gram of tissues was calculated as the difference between the initial and final glucose content in the incubated medium.

Statistical analysis

All values were expressed as mean ± standard error of mean. Results were analyzed statistically by analysis of variance followed by Dunnett’s test. P < 0.05 was considered statistically significant.

Results

The B. prionitis and H. suaveolens plant extracts were tested at 0.1% and 0.2% concentrations to evaluate the glucose uptake by isolated rat diaphragm. Tables 1 and 2 summarize the result of glucose uptake by isolated rat hemidiaphragm of B. prionitis. Tables 3 and 4 illustrate the results of H. suaveolens. Insulin served as positive control. The difference between the initial and final glucose content is considered as the amount of glucose uptake per gram of tissue.[24] The glucose uptake by rat hemidiaphragm was significantly more in the test groups compared to control. The results showed that fractions of both the plants have in vitro glucose utilization capacity and the uptake of glucose is more when the test substance is tested along with insulin. On comparing the results of glucose utilization, it was observed that the fractions of H. suaveolens possess more significant potency compared to B. prionitis test fractions.

Among all the fractions of H. suaveolens petroleum ether, aqueous ethanol and chloroform fractions produced pronounced effect of glucose uptake and the uptake of glucose was further increased when tested along with insulin. Butanol and ethyl acetate fractions had low glucose utilization capacity. The uptake of glucose with respect to 0.2% of petroleum ether fraction was 75.51 ± 0.91, and when tested this fraction along with insulin, the glucose uptake was 94.69 ± 0.73. These results suggest that the action of extracts is to synergistically enhance the effect of insulin and vice versa. However, the uptake of glucose with respect to 0.1% concentration of petroleum ether and aqueous ethanol was similar.

B. prionitis test fractions have produced significant in vitro glucose utilization. The degree of utilization of glucose was comparatively low with respect to H. suaveolens fractions. The aqueous ethanol extract and petroleum ether fraction of B. prionitis produced a significant potency when compared to other fractions. The glucose utilization was not influenced much when the extracts of B. prionitis were tested along with insulin. Ethyl acetate and butanol fractions had significant low utilization capacity. The left-over aqueous fractions of both plants had no glucose utilization capacity.

Discussion

The estimation of glucose content in rat hemidiaphragm technique is a commonly employed and reliable method for in vitro study of peripheral uptake of glucose. Diaphragm is a dynamic tissue which utilizes a large amount of glucose compared to other tissues.[2223] This model is reliable and sensitive to evaluate the peripheral utilization of glucose. Both plant extracts produced significant and dose-dependent glucose utilization when tested alone and tested along with insulin.

The petroleum ether and aqueous ethanol fractions of both the plants enhance the uptake of glucose by isolated rat hemidiaphragm when compared to the other test fractions. During the course of study, it was observed that the fractions of H. suaveolens had produced more glucose utilization capacity when compared to B. prionitis fractions. The effect of glucose utilization was more when tested in the presence of insulin. It is well known that insulin promotes glucose uptake by peripheral cells and tissues.[24] In the present experiment, the extracts of H. suaveolens along with insulin further increased the glucose utilization capacity by hemidiaphragm.

Conclusion

It can be concluded that aqueous ethanolic and petroleum ether extracts of B. prionitis and H. suaveolens reduce blood glucose levels by increasing the peripheral glucose utilization. However, further studies are needed to substantiate these observations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.