Lectins from seeds of Abrus precatorius: Evaluation of Antidiabetic and Antihyperlipidemic Potential in Diabetic Rats

 

Sampada S. Sawant*, Vishal R. Randive, Savita R. Kulkarni

Bombay College of Pharmacy, Kalina, Santacruz (West), Mumbai – 400 098.

*Corresponding Author E-mail: sampada.bcp@gmail.com

 

ABSTRACT:

There is an increasing patient demand to use natural products for diabetes treatment due to the side effects associated with the use of insulin and oral hypoglycemic agents. This work highlights the potential of Abrus precatorius seeds for their antidiabetic activity. The aim of this study was to evaluate the antidiabetic, antihyperlipidemic and in vivo toxicity of the lectins purified from the seeds of Abrus precatorius (Fabaceae), locally known as Gunja. The present work involves purification of lectins from the seeds of A. precatorius (ABA), followed by characterization. The in vivo acute toxicity study of ABA was carried out as per the OECD 425 guidelines. The antidiabetic potential was evaluated in alloxan monohydrate (AXN) induced diabetic rats at two dose levels (150 mg/kg and 200 mg/kg) body weight, daily for 14 days. Various parameters such as body weight, food consumption, serum glucose levels, serum cholesterol levels and liver glycogen levels were determined post ABA treatment. Acute toxicity study revealed ABA to be non-toxic even at a high dose of 2000 mg/kg body weight (bw). Increased body weight, food consumption and decreased serum glucose levels were observed in diabetic rats treated with the ABA compared to the diabetic control rats. Diabetic rats treated with the ABA showed altered lipid profiles and liver glycogen levels which were found to be reversed to near normal levels than the diabetic control rats. These findings highlight the efficacy of A. precatorius seed lectins as potential antidiabetic and anti hyperlipidemic agent that can be used as adjunct therapy for diabetes treatment.

 

KEY WORDS: Abrus precatorius, Lectins, Antidiabetic, Antihyperlipidemic, Alloxan.

 


 

INTRODUCTION:

Diabetes is a chronic metabolic disorder that occurs either due to pancreas not producing enough insulin or when the body cannot effectively use the insulin it produces. Insulin is a hormone that regulates blood sugar1. Hyperglycaemia, or raised blood sugar, is a common effect of uncontrolled diabetes and over time leads to serious damage to various organ systems, especially the nerves and blood vessels.

 

Diabetes mellitus is associated with several micro-and macrovascular complications resulting into significant morbidity and mortality. It is reported to be amongst the leading causes of death worldwide. In 2014, 9% of adults were reported of diabetes. In 2013 diabetes was the direct cause of 1.5 million deaths. More than 80% of diabetes deaths occur in low and middle-income countries2. WHO has projected diabetes to be the 7th leading cause of death by 20303. Diabetes is characterized by dysfunction of carbohydrate, protein and fat due to insufficient production of insulin or insufficient response of the produced insulin. Hyperglycaemia is associated with long term damage or failures of vital organs like eyes, kidneys, liver, heart and blood vessels with abnormal changes in lipid and protein metabolism. Several therapies are currently available for the treatment of diabetes but suffer with several drawbacks such as liver toxicity, gastrointestinal disturbances, irregular weight gain, hypoglycaemia and high cost to include a few. Hence there is an increasing demand by patients to use natural products with antidiabetic activity due to side effects associated with the use of insulin and oral hypoglycemic agents4. Traditional medicinal plants, on the other hand, provide an alternative option for the control and management of diabetes. The WHO has listed approximately 21,000 plants for use of medicinal purposes with around 150 species being used on a commercial scale. Several plants from the Fabaceae, Asteraceae, Lamiaceae, Liliaceae, Poaceae and Euphorbiaceae family have been reported for antidiabetic potential5.

 

Abrus precatorius (Fabaceae) is a perennial shrub (locally known as gumchi/gunja) and the roots and the seeds (rosary beads) have been traditionally used for several medical implications. The roots of A. precatorius are reported to possess tonic, emetic and diuretic properties and are used in preparations for gonorrhoea, jaundice and haemoglobinuric bile and alcoholic extract of roots is known to possess anti-estrogenic activity6.  The seeds of A. precatorius have been reported for traditional use in the treatment of inflammatory disorders like arthritis7, ulcer8 and skin disorders9, trypanocidal activity10, moluscicidal11 and antiallergic treatment in asthma12.  Methanolic extract of seeds is reported to have sperm antimotility activity in males and abortifacient activity in females 13. Plant polyphenolic compounds, such as flavonoids are described as scavengers of reactive oxygen species. The seeds are known to have antitumour, immunomodulating, antiplatelate, insecticidal, antidiabetic, antibacterial and antidiarrhoeal properties.  In ayurveda the plant is considered beneficial for the hair14.

 

The main constituent present in the seeds are isoflavonoids, flavonoids, proteins, alkaloids, carbohydrates and triterpinoids15. The seed proteins of Abrus are essentially lectins/agglutinins. Lectins are protein or glycoprotein substances, of non-immunoglobulin nature, capable of specific recognition of and reversible binding to, carbohydrate moieties of complex glycoconjugates without altering the covalent structure of any of the recognized glycosyl ligands. Lectins bind to sugar moieties in cell walls or membranes and thereby change the physiology of the membrane to cause agglutination, mitosis, or other biochemical changes in the cell. The Abrus precatorius agglutinin (ABA) is the least toxic lectin among five type 2 ribosome inactivating proteins (RIP) isolated from the seeds of Abrus. Some plant lectins such as wheat germ agglutinins (WGA) and concanavalin A (con A) have shown to have in vitro insulin mimicking action in fat cells16, as well as lectins from Urtica pilulifera, UPSL (U. pilulifera seed lectin) showed antidiabetic activity 17. The lectins from A. precatorius have been studied for their glycoprotein recognition processes18, immunomodulatory activity19,20. Monago et al, 2005, have reported chloroform-methanol extract to have antidiabetic potential. However no specific component responsible for the antidiabetic effect has been     identified 21. Though several activities of A. precatorius seed components including the seed lectins, have been reported no detailed scientific study on its antidiabetic potential and effect on the lipid levels has been established. Hence our aim was to study the antidiabetic and antihyperlipidemic effect of A. precatorius lectin on AXN induced diabetic rats.

 

MATERIALS AND METHODS:

Chemicals:

AXN and all other chemicals were purchased from S.D Fine chemicals. Pioglitazone was obtained as a kind gift fom Macloeds Pharma Pvt. Ltd., Mumbai Glucose estimation kit was purchased from Erba diagnostics and Total cholesterol estimation kit was obtained from Accurexbiomedica Ltd.

 

Plant Material Identification:

Abrus precatorius seeds were collected from Rajiv Gandhi National Park, Mumbai, Maharashtra. The seeds were authenticated by the taxonomist at the Botanical Survey of India (BSI), Pune. The seeds were cleaned, shade dried and powdered coarsely.

 

Preparation of Plant Material:

Extraction was done by cold maceration process22. Dry seeds of A. precatorius were decorticated and powdered. The powder was passed through the # 20 mesh sieve. This powder (100 g) was soaked in 750 ml of 5% acetic acid for 12 hours at 4˚C with the ratio of powder: solvent as 1:7.5. The marc was separated by centrifugation at 4000 r/min for 30min in Remi Cooling centrifuge at 4°C. The solid pellet was discarded and supernatant was used for further processing. Supernatant was subjected to 30% ammonium sulphate precipitation and centrifuged at 4000 rotations/min (r/min) for 15 mins in Remi cooling centrifuge at 4°C. Supernatant obtained here was subsequently subjected to 90% ammonium sulphate precipitation and was again centrifuged at 4000 r/min for 20min at 4°C. The supernatant was discarded and pellet was dissolved in 80ml water. This colloidal solution was heated to 60°C for 30min, with occasional stirring in a constant temperature bath. This was then centrifuged at 4000 r/min and supernatant was then dialyzed against 10mM Tris-HCl buffer (pH 7.4) to free it of traces of salt. The dialyzed extract was centrifuged to remove the precipitated impurities. The pellet was discarded and the supernatant was further purified by passing through Sephadex G-100 column (1x25cm), previously equilibrated with Phosphate buffer saline, pH 7.4 (PBS). The dialyzed extract was passed through the column at the rate of 15ml/hr. The column was run for 24hrs using PBS solution. The solution of 0.4 M lactose in PBS was used to displace A. precatorius agglutinin (ABA) from Sephadex bed. Fractions of 5ml were collected and monitored by absorbance at 280nm for lectin content using Folin Lowry method of protein estimation23. The fractions showing hemagglutination activity were pooled and further dried in vacuum. Then it was stored in the refrigerator (2- 8°C) till further use.

 

Quantitative and Qualitative evaluation of plant lectins:

Qualitative estimation:

Presence of proteins and carbohydrates in purified ABA was determined using the standard tests as per the protocols reported in literature previously 24.

 

Quantitative Estimation of Protein:

Protein content of crude, dialyzed and purified extract was determined by Folin Lowry method23. Graph of absorbance v/s concentration was plotted and unknown protein concentration was calculated by interpolating the absorbance values on standard curve (Graph not shown here).

 

Determination of Hemagglutination unit (H.U), Specific activity and Fold purity of ABA:

Determination of Hemagglutination unit (H.U), Specific activity and Fold purity was done by the hemagglutination assay using freshly collected rat RBCs using method described previously25. A 96 well micro titre plate was used for hemagglutination test. Initially 100 µl saline solution was added to the 48 wells of the plate. 100 µl ABA solution (1 mg/ml) was added to the first well to make 1:2 dilutions and mixed well. From this first well 100 µl mixture was pipetted out and transferred to the next well to make 1:4 dilutions and continued till 11th well to make further dilutions. From 11th well, 100 µl of mixture was transferred to the 13th well and continued till the 23rd well to make further dilutions.

 

From 23rd well 100 µl of mixture transferred to the 25th well and continued till the 35th well. From 35th well 100 µl of mixture transferred to the 37th well and continued till the 47th well. Finally 100µl mixture from 47th well was discarded. Wells 12th, 24th, 36th and 48th were kept as control containing 100µl saline without lectin extract. Normal Rat erythrocytes were washed 3 times with Alsever’s solution and used as a 3% w/v suspension. Erythrocyte suspension (25 µl) was added to all the 48 wells including control wells (well 12th, 24th, 36th, and 48th). This prepared plate was incubated for 30 minutes at room temperature. Titre i.e. the highest dilution that showed hemagglutination was noted down. From these results titre, specific activity and fold purity of lectin solution was calculated. One hemagglutination unit (H.U.) is defined as the reciprocal of highest dilution causing visible agglutination. The specific activity was expressed as hemagglutination unit per mg and the minimum dose as the minimum amount of protein still promoting a visible agglutination. Titre, specific activity and fold purity for crude, dialyzed and purified lectin extract was calculated by applying the same procedure.

 

Effect of ABA on different blood group erythrocytes26:

A series of two-fold dilutions of ABA solution (1 mg/ml), as that prepared for hemagglutination assay, was prepared in a 96 well micro titre plate (in triplicate for three blood groups). 50 µl of erythrocyte suspension (3% w/v in Alsever’s solution) of human blood groups A+, B+ and O+ was added to each of the series respectively. The highest dilution showing visible agglutination was noted

 

Effect of sugar on ABA27:

Sugar solutions of increasing concentrations were made. 50 µl of ABA (1 mg/ml), was added to each of the above dilutions. After 15 minutes of incubation at room temperature, 50 µl of 3% rat erythrocyte suspension was added to each well. Minimum concentration of sugar solution inhibiting agglutination was noted. The sugars used for the test were as follows: lactose, sucrose, fructose, dextrose, arabinose, mannose and galactose.

 

 

Effect of EDTA and divalent cations on ABA26:

Two fold serial dilutions of ABA (1 mg/ml) were prepared in 0.2M PBS alone and 0.2M PBS containing 5mM EDTA (in triplicate). 50 µl of 3% rat erythrocyte suspension was added to each dilution well. To the 1st series of dilutions containing 5mM EDTA, 50 µl of 10mM MgSO4 was added to each dilution and to the 2nd series, 50 µl of 10mM BaCl2 was added to each dilution in order to evaluate their capacity to restore hemagglutination.

 

Effect of pH on lectins26:

Two fold serial dilutions of ABA (1 mg/ml) were prepared in Citrate phosphate buffer (pH 3.6), Phosphate buffered saline (pH 7.4), Borate buffer (pH 9.0), Glycine-NaOH buffer (pH 11.3). Equal volume (50 µl) of 3% rat erythrocyte suspension was added to each dilution well. The highest dilution showing visible agglutination was noted.

 

Experimental Animals:

For acute toxicity study, Swiss albino female mice (25 – 30 g) were used. Healthy female rats of Wistar strain, (100 – 150 g), were used to evaluate the antidiabetic potential. All the study animals were quarantined under the standard laboratory conditions. Animals were fed with commercially available Amrut rat and mice feed, manufactured by M/s. Nav Maharashtra chaken Oil Mills Limited, Pune and were allowed to drink water ad libitum. The experiments were conducted in accordance with the protocol duly approved by the institutional animal ethics committee of the Bombay College of Pharmacy, Mumbai.

 

Acute oral toxicity:

Acute oral toxicity study was conducted in accordance to the OECD guidelines no. 42528.The sighting study was started at the dose of 175 mg/kg of animal weight by using three animals. Toxicity study was performed in mice that had been fasted for 3-4 hours prior to study (deprived of food for 3-4 hrs but allowed free access to water). Following the period of fasting the animals were weighed and ABA was administered orally with feeding cannula. After ABA administration, food was withheld for another 1-2 hours. Treated animals were observed critically for first 4 hours and thereafter for 24 hours and daily for 14 days.

 

This procedure was followed for all the animals used in the toxicity study. Since no mortality was observed for the dose of 175 mg /kgbw, the next higher dose i.e.550 mg/ kg was administered and followed the same procedures mentioned earlier. Since no mortality was observed for 550 mg/kg bw of dose administered then the next higher dose,2000 mg / kg bw was administered and observed carefully as mentioned earlier. All the study animals were carefully observed for clinical signs, symptoms, and conditions associated with pain, sufferings and death. Additional observations included change in skin, fur, eyes, and behaviour pattern of animals. Animals were also observed for tremors, convulsion, salivation, diarrhoea, sleeps and coma. Body weights of individual animals were determined shortly before ABA administration and at least weekly thereafter. Food consumption was determined every day for 14 days by considering food given and food left on the lid of cage. All the study animals were observed for morbidity/mortality every day for 14 days.

 

Induction of experimental diabetes:

Rats were made diabetic by a single intraperitoneal injection of AXN (130 mg/kg bw) 29. AXN was weighed individually for each animal according to the body weight and solubilized in 0.2ml of normal saline just prior to injection. Six days after AXN injection, rats with plasma glucose levels of >250 mg/dl were included in the study. Treatment with ABA was started one week after AXN injection. The doses were selected based on the minimum concentration that promoted visible hemagglutination and one higher dose.

 

Antidiabetic activity assessment:

Diabetic animals were assigned into following groups (n=6).

Normal control: Normal rats received saline solution

Diabetic control: Diabetic rats received vehicle (placebo sodium CMC suspension)

PIO: Diabetic rats received Pioglitazone suspension (10 mg/kg bw)

ABA - 150: Diabetic rats received ABA lectin(150 mg/kg bw)

ABA - 200: Diabetic rats received ABA lectin (200 mg/kg bw)

 

Experiment was performed on the rats that had been fasted overnight (deprived of food for at least 12 hrs but allowed free access to water).

 

ABA and Pioglitazone were suspended in PBS (pH 7.4) using sodium CMC and was administered orally once daily at 10.00 h for 14 days. The effect of vehicle, ABA and Pioglitazone on serum glucose and serum cholesterol levels was determined in fasting rats on 0, 7th and 14th day. On 14th day animals were sacrificed and liver of animals were isolated for the determination of glycogen content. Body weight of the animals was taken on day 0, 7th and 14th day.

 

Biochemical Parameters:

Blood samples were collected from the retro orbital plexus into the centrifuge tubes and allowed to clot for 30 mins at room temperature. Blood samples were centrifuged at 4000 r/min for 20 mins. Serum was separated and stored at -20 °C, until analysis was performed.

 

Glucose estimation study:

Serum was analyzed for glucose using glucose oxidase- peroxidase (GOD-POD) method, spectrophotometrically as per the standard procedure given in the Erba glucose diagnostic kit, using UV spectrophotometer (Jasco V-530) at 505nm30.

 

Cholesterol estimation study:

Serum was analyzed for cholesterol, using cholesterol oxidase-peroxidase (CHOD–POD) method, spectrophotometrically as per the standard procedure given in the Accurexbiomedica cholesterol diagnostic kit using UV spectrophotometer (Jasco V-530) at 540nm31.

 

Calculations:

The glucose or cholesterol levels were calculated using the following equation:

                                                      (A) of sample 

Cholesterol or Glucose (mg/dl) = ----------------- X 100

                                                      (A) of standard

 

Liver Glycogen content study:

Glycogen content of the isolated liver was determined spectrophotometrically as per the standard procedure described in previous reports31.

 

Digestion of glycogen from tissues:

About 0.1 g of liver was weighed and minced. 0.2 ml of 30% KOH was added (2 ml of KOH to be added per g of tissue). Tubes were heated in boiling water bath for 15-20 minutes until clear solution was formed. Tubes were cooled and 0.24 ml of 95% ethanol was added to precipitate glycogen (1.2 ml of ethanol to be added per ml of glycogen solution). Tubes were centrifuged at 5000 r/min for 10 minutes. The supernatant was discarded and tubes were kept on boiling water bath to remove any remaining ethanol. The precipitated glycogen was dissolved in 1 ml of distilled water.

 

Assay of glycogen:

One ml of unknown glycogen solution /standard solution /water was taken along with50 µl of 80 % phenol and 2.5 ml of concentrated sulphuric acid. The mixture was shaken after each addition. (Concentrated sulphuric acid should be added slowly). The test tubes were allowed to attain room temperature and absorbance was measured at 490 nm (Eliza micro plate reader, Bio-rad 550).

 

Statistical analysis:

The data of serum glucose concentrations, serum cholesterol levels and body weight was expressed as mean ± standard deviation and analyzed by one-way ANOVA and Tukey’s multiple comparison tests. p< 0.05 was considered to be statistically significant. The data of glycogen content of the liver was expressed as mean ± standard deviation and analyzed by paired t-test. p< 0.05 was considered to be statistically significant.

 

RESULTS:

Plant Material Identification:

The seeds were authenticated by the taxonomist at the Botanical Survey of India (BSI), Pune. The authentication certificate number was BSI/SS2/2008.

 

Quantitative and Qualitative evaluation of plant lectins:

The first step of extraction yielded 6.21 % w/w of crude protein expressed in terms of BSA, calculated in terms of dried seed powder weight. The yield of protein extract after dialysis was found to be 1.80 % w/w. The yield of purified protein (ABA) after separation using affinity chromatography was found to be 0.77 % w/w in terms of BSA, with respect to protein content when compared to weight of dry seed powder. The characterization for the presence of lectin in the extract was done using various chemical tests for proteins and carbohydrates. The positive observations of Biuret, Ninhydrin, Xanthoproteic and Barfoed’s tests for proteins signify the presence of proteins in ABA. The positive observations for Molisch, Iodine, Benedict’s tests for carbohydrates signify the presence of aldohexoses as the sugar part. Based on these observations it can be concluded that the purified lectin ABA, is a glycoprotein. The characterization of ABA by hem agglutination showed step-by-step increase in hem agglutination titre, as the lectin was purified further. The hemagglutination titre, specific activity and fold purity results are given in table 1. Molecular weight of ABA was found to be134k Daltonsas determined by SDS PAGE analysis (Results not shown here).


 

 

Table no. 1. Total Protein content (mg) and Hemagglutination Units (H.U) of ABA obtained at different steps of extraction and purification

Fraction

Vol.(ml)

Protein content in terms of BSA (mg/dl)

Total protein (mg)

H.U

Specific activity

Fold purity

Crude extract

460

13.5

6210

32

2.37

1

-6.21

Dialyzed

extract

78

23.19

1808

128

5.51

2.32

-1.8

Purified

60

12.86

771

512

39.81

16.79

Lectin (ABA)

-0.77

 


 

Lectins show different agglutination titres on different human blood group erythrocytes. ABA showed hemagglutination titre of 512, 64 and 32 for human blood group A+, B+ and O+ erythrocytes respectively. This specificity towards blood grouping can be further explored as a diagnostic tool and for targeted treatment of hematological disorders by exploring ABA as a stealthing agent. Sugars did not inhibit the agglutination even at 500mM concentration except lactose at 200mM and galactose at 300mM concentration. ABA can thus be classified as a lactose and galactose binding lectin. EDTA completely inhibited the hemagglutination activity of ABA whereas Mg+2 and Ba+2 ions restored the activity, when incubated in the presence of ABA. Mg+2  restored the activity to 256 H.U whereas Ba+2 restored the activity to 128 H.U. The divalent cations Mg+2 and Ba+2 are thus proved to be essential for ABA activity These results are in conjunction with the properties of ABA, as reported by Panneerselvam et al, 200022.ABA showed no agglutination in the presence of Citrate phosphate buffer (pH 3.6) and Glycine-NaOH (pH 11.3) buffer. Activity was reduced to 2 H.U in Borate buffer (pH 9.0) whereas Phosphate buffer (pH 7.4) showed activity of 512 H.U for ABA. pH of the medium highly influenced the ABA activity and can have a profound effect during ABA formulation for clinical use.

 

Acute oral toxicity study:

Three animals were used for individual dose level 28. As no preliminary information was available, the dose of 175 mg/kg was used as a starting dose for the preliminary toxicity studies28. No mortality was observed for the dose of 175 mg/kg. Hence subsequent higher dose (progression factor 3.2) i.e.550 mg/kg and 2000 mg/kg was used. No mortality was observed for 550 mg/kg and 2000 mg/kg for ABA extract. The animal weight was found to be steadily increased when weighed at weekly intervals (Table no.2).

 

Table no. 2: Effect on body weight (g) in ABA treated animals studied for acute toxicity

Group/

Body Weight (g)

Treatment (mg/kg)

mean ± S.D (n=3)

 

Day 0

Day 7

Day 14

Control

26.2±2.54

27.8±3.34

29.3±3.71

175

28.3±2.13

30.4±2.12

33.6±2.24

550

29.8±2.32

32.6±2.41

34.1±2.8

2000

29.6±2.69

33.8±2.56

34.9 ± 2.94

 

Organs of sacrificed animals (including control) were isolated and weighed. Organ weight of treatment animals were compared with control animals. No significant changes were observed in organ weights of ABA treated animals as compared with that of control animals (Table no. 3).


 

 

Table no.3: Effect on body organ weights (g) in ABA treated animals studied for acute toxicity. Data represented as mean ± S.D (n = 3)

Group/

treatment (mg/kg)

Heart

Brain

Liver

Lungs

Kidneys

Spleen

Control

0.14±0.01

0.44±0.02

2.19±0.01

0.19±0.02

041± 0.02

0.25± 0.01

175

0.16±0.03

0.44±0.09

2.26±0.31

0.25±0.01

0.47± 0.01

0.24± 0.09

550

0.19±0.02

0.47±0.03

2.14±0.54

0.26±0.08

0.46± 0.08

0.25± 0.08

2000

0.18±0.08

0.46±0.06

2.52±0.25

0.27±0.06

0.49± 0.08

0.27± 0.01

 

 

 


No signs of toxicity (clinical signs, symptoms, and conditions associated with pain, sufferings) or death were noted. No significant changes in skin, fur, eyes, and behavior pattern of animals were observed. Animals showed no signs of tremors, convulsion, salivation, diarrhoea, sleepiness or coma. As the high doses of ABA showed no signs of toxicity, it was further evaluated for its antidiabetic and antihyperlipidemic potential.

 

Antidiabetic study:

AXN was used to induce Type I diabetes mellitus in female wistar rats. AXN is selectively toxic to the pancreatic beta cells as it preferentially accumulates in the beta cells as glucose analogues 32. Further, the massive increase in cytosolic calcium concentration ultimately causes rapid destruction of beta cells of pancreatic islets. In diabetic condition, elevated blood glucose, reduction in body weight and polyuria are commonly observed. In our study, the AXN induced diabetic rats showed elevated blood glucose levels, increased cholesterol levels, decrease in body weight and polyuria. Reduction in body weight was due to the catabolism of fats and proteins. The screening of the anti-diabetic activity of ABA was done in AXN induced diabetic rats (AXN-diabetic rats). The dose of 130mg/kg of AXN was used to induce diabetes. Six days after the injection, the treatment was initiated and given for 14 days. Rats treated with AXN showed diabetic symptoms of hyperglycemia, polyuria, polyphagia, polydypsia and weight loss. In present study 75% of the rats treated with AXN became diabetic. Fasting serum glucose levels above 250mg/dl indicated diabetic state. During the prolonged study, various physical parameters such as body weight, food intake, water intake and polyuria were also found to be normalized.

 

Table no. 4: Effect on food intake (g) in ABA treated diabetic animals, post 14 days of treatment

Treatment group

Food intake

Normal Control

4.2 ± 1.9

Diabetic Control

1.29 ± 3.58a

PIO

3.65 ± 3.56b

ABA- 150

2.62 ± 3.44b

ABA - 200

3.38 ± 2.78b

Data expressed as mean ± s.d (n = 6). aStatistically significant w.r.t normal control, bStatistically significant w.r.t diabetic control. (p< 0.05).

 

The decrease in body weights were diminished by the ABA treatment, hence this effect may be useful for the diabetic patients. Food consumption was found to be significantly decreased in case of AXN diabetic rats. The food consumption of the diabetic control group was reduced by 69% when compared to normal group on 14th day, whereas the food consumption of the ABA- 150, ABA-200 treated and Pioglitazone treated group was increased by 103%, 162% and 182% respectively when compared to diabetic control group on 14th day. This shows a significant increase in food consumption of the ABA treated and Pioglitazone treated groups as compared to diabetic control. The observations are recorded in Table.no.4.

 

Decrease in body weight is characteristic of the rats injected with AXN. Loss of body weight in diabetic rats could be due to derangement in protein metabolism such as decreased protein synthesis and increased breakdown33. The effect of ABA suspension on the body weight was evaluated on day 0, day 7 and day14 of treatment. The observations are recorded in Table no.5.


 

Table no.5: Effect on body weight (g) in ABA treated diabetic rats. Data represented as mean ± s.d (n = 3)

Treatment group

DAY 0

DAY 7

DAY 14

Normal Control

157.00 ± 5.36

168.50 ± 4.75

170.84 ± 4.50

Diabetic Control

101.16 ± 3.25a

92.78 ± 5.71b

80.28 ± 4.28d

PIO

110.19 ± 8.75a

140.00 ± 5.86c

146.54 ± 5.63e

ABA - 150

107.66 ± 2.06a

126.00 ± 2.73c

137.00 ± 3.23e

SBA - 200

109.00 ± 7.08a

132.86 ± 4.65c

138.00 ± 8.98e

Data expressed as mean ± s.d. (n = 6): aStatistically significant w.r.t normal control, bStatistically significant w.r.t normal control on day 7, cStatistically significant w.r.t. diabetic control on day 7, dStatistically significant w.r.t normal control on day 14, eStatistically significant w.r.t diabetic control on day 14. (p < 0.05)

 

 


From the observations it is evident that untreated diabetic rats exhibited lower body weight as compared to normal control. Progressive decrease in body weight of untreated animals was observed. The body weight of diabetic control group was decreased by 28% and 34% respectively on 7th and 14th day respectively, when compared to the normal control group. The body weight of animals treated with ABA – 150 suspension increased by 17% and 31% on 7th and 14th day respectively. The body weight of ABA - 200 suspension treated group increased by 20% and 32% on 7th and 14th day respectively, when compared to diabetic control group. Whereas body weight of Pioglitazone treated group was increased by 24% and 36% on 7th and 14th day respectively, when compared to diabetic control group. A significant change in body weight was observed in lectin treated rats on the 14th day of the study. The results are given in Table no.5. The enzymatic GOD-POD method was used for estimation of serum glucose levels. The concentration of the standard provided with the kit was 100mg/dl. Repeated once daily oral administration of ABA – 150, ABA- 200and Pioglitazone (10mg/kg) produced significant decrease in serum glucose levels on day 7 and day14 as compared to diabetic control, which demonstrates the antidiabetic activity of the ABA suspension. The serum glucose level of diabetic control group was increased by 137% and 140% on 7th and 14th day respectively, when compared to the normal control group. The serum glucose level of ABA-150 treated group was decreased by 15% and 40% on 7th and 14th day respectively, when compared to diabetic control group. The serum glucose level of ABA - 200 treated group decreased by 13% and 43% on 7th and 14th day respectively, when compared to diabetic control group. Whereas the serum glucose level of Pioglitazone treated group was decreased by 17% and 49% on 7th and 14th day respectively, when compared to diabetic control group (Results given in Table no.6).


 

Table no.6: Effect on serum glucose levels (mg/dl) in ABA treated diabetic rats.

Treatment group

DAY 0

DAY 7

DAY 14

Normal Control

115.82 ± 2.58

120.88 ± 1.25

117.83 ± 2.45

Diabetic Control

265.53 ± 5.45a

287.14 ± 5.40b

282.33 ± 6.28d

PIO

260.41 ± 3.58a

239.02 ± 5.68c

144.41 ± 5.89e

ABA - 150

278.38 ± 6.00a

244.19 ± 3.93c

169.21 ± 5.85e

ABA - 200

266.91 ± 3.29a

251.55 ± 4.87c

162.63 ± 4.54e

Data expressed as mean ± s.d. (n = 6): aStatistically significant w.r.t normal control, bStatistically significant w.r.t normal control on day 7, cStatistically significant w.r.t. diabetic control on day 7, dStatistically significant w.r.t normal control on day 14, eStatistically significant w.r.t diabetic control on day 14. (p < 0.05)


The results of this investigation indicate that the lectins from the seeds of A. precatorius have hypoglycemic effect on AXN-induced diabetic rats. Though the serum glucose levels were not completely normalized at the end of first week, low levels indicate gradual normalization of metabolic disturbance as diabetes gets controlled in ABA suspension and Pioglitazone treated diabetic animals. At the end of second week, however, the serum glucose levels reach below the diabetic level (< 250 mg/dl). ABA treatment, significantly decreased the serum glucose level at both the doses (P<0.05). When ABA was compared with standard (Pioglitazine), ABA showed equivalent antidiabetic activity in normalizing various parameters. Increase in body weight and decrease in blood glucose might be due to improving the glycemic control mechanisms and insulin secretions from remnant pancreatic cells in diabetic animals. The possible mechanisms of action are due to insulin mimicking activity of the lectins and improvement of glycogenesis process34.

 

Cholesterol Estimation Study:

The most common lipid abnormalities in diabetes are hypertriglyceridemia and hypercholesterolemia35. The abnormal high concentrations of serum lipids in diabetic animals are mainly due to an increased mobilization of free fatty acids from the peripheral fat depots36. The serum cholesterol level at various time points was estimated using a commercial kit (Cholesterol estimation kit, Accurex fine chemicals). The enzymatic method referred as CHOD-POD method. The concentration of the standard provided with the kit was 100mg/dl. The observations are recorded in table no.7.


 

 

 

Table no.7: Effect on serum cholesterol levels (mg/dl) in ABA treated diabetic rats.

Treatment group

DAY 0

DAY 7

DAY 14

Normal Control

40.04 ± 5.28

46.48 ± 4.75

50.20 ± 4.93

Diabetic Control

69.85 ± 3.82a

81.54 ± 9.28b

100.89 ± 9.08d

PIO

69.19 ± 5.66a

62.65 ± 8.62c

62.14 ± 9.00e

ABA - 150

69.55 ± 6.47a

68.52 ± 2.86c

67.24 ± 7.60e

ABA - 200

69.14 ± 8.69a

66.54 ± 6.40c

64.57 ± 11.91e

Data expressed as mean ± s.d. (n = 6): aStatistically significant w.r.t normal control, bStatistically significant w.r.t normal control on day 7, cStatistically significant w.r.t. diabetic control on day 7, dStatistically significant w.r.t normal control on day 14, eStatistically significant w.r.t diabetic control on day 14. (p < 0.05)

 

 

 


Repeated once daily oral administration of ABA- 150, ABA- 200 suspension and Pioglitazone (10mg/kg) produced significant decrease in serum cholesterol levels on day 7 and day 14 as compared to diabetic control, which demonstrates the cholesterol lowering activity of the ABA suspension. The serum cholesterol level of diabetic control group was increased by 75% and 79% on 7th and 14th day respectively, when compared to the normal control group. The serum cholesterol level of ABA- 150 treated group decreased by 16% and 33% on 7th and 14th day respectively, when compared to diabetic control group. The serum cholesterol level of ABA-200 treated group decreased by 19% and 36% on 7th and 14th day respectively, when compared to diabetic control group; whereas the serum cholesterol level of Pioglitazone treated group decreased by 23% and 38% on 7th and 14th day respectively, when compared to diabetic control group. Though the serum cholesterol levels were not completely normalized, a serious symptom of high cholesterol gets controlled in ABA suspension and Pioglitazone treated diabetic animals.  In the present study, we observed significantly increased levels of serum cholesterol. ABA administration also reduced the serum cholesterol levels at both the dose levels, the effect being comparable to the standard pioglitazone treatment. The above action can prove to be useful in preventing diabetic complications like the coronary heart diseases and atherosclerosis37.

 

Liver Glycogen content study:

Glycogen is primary intracellular storable form of glucose and its levels in various tissues like skeletal muscle, heart muscle and liver reflects the insulin activity as insulin promotes intracellular glycogen deposition by stimulating glycogen synthatase and inhibiting glycogen phophorylase. Diabetic rats showed marked reduction in the liver glycogen levels. The glycogen content of the diabetic control group was reduced by 75% when compared to normal group on 14th day, whereas the glycogen content of the ABA-150, ABA – 200 and Pioglitazone treated group was reduced by 21%, 26% and 15% respectively when compared to normal control group on 14th day.This shows a significant difference in the glycogen content of the ABA suspension treated and Pioglitazone treated groups as compared to diabetic control. The observations are recorded in Table.no.8.

 

Table no.8: Effect on liver glycogen content (mg/g of tissue) in ABA treated diabetic rats

Treatment group

Glycogen content of liver

Normal Control

35.94 ± 0.76

Diabetic Control

9.03 ± 1.11a

PIO 

30.43 ± 0.49b

ABA - 150

28.35 ± 3.32b

ABA - 200

26.55 ± 2.46b

Data expressed as mean ± s.d (n = 6). aStatistically significant w.r.t normal control, bStatistically significant w.r.t diabetic control. (p< 0.05).

 

However ABA treated animals showed significant increase in the glycogen levels, reaching almost the normal levels, as also in the case of standard drug treatment. This may be due to the insulin mimicking activity of the lectins. Similar activity has been reported in case of lectins from Agaricus bisporus and Agaricus campestris stimulate insulin and glucagon release from isolated rat islets in the presence of 2 mM glucose34. It confirms that ABA functions on the protection of vital tissues (pancreas, liver and heart), thereby reducing the diabetes abnormalities in experimental animals.

 

CONCLUSION:

Non-insulin dependent diabetes is a much rapidly prevalent type of diabetes, leading to more than 90% of all the diabetes cases leading to severe socioeconomic consequences especially in the developing countries. The use of ethnobotanicals has a long folkloric history for the treatment of blood glucose lowering abnormalities. Therefore, the search for more effective and safer antidiabetic/ hypoglycaemic agents has continued to be an important area of active research. Seed extracts of A. precatorius have been reported for antidiabetic potential, however no scientific study showing the exact component responsible for glucose lowering effect has been established. In our study, we have extracted seed lectin (ABA) and purified it using affinity chromatography method. ABA was found to be non toxic at high dose of 2000 mg/kg, and hence was further explored for its antidiabetic potential. ABA, reduced the serum glucose levels significantly, probably by the insulin mimicking property of the lectins. ABA has also proven to be effective in regulating the biochemical indices associated with diabetes mellitus such as cholesterol and restoration of liver function. Further studies are in progress to elucidate exact mechanism(s) of ABA activity. Attempts are in progress in our lab to formulate the lectins as nano drug delivery systems to achieve targeted delivery using the carbohydrate specificity, blood group specificity of the lectins to further reduce the dose required and enhance patient compliance.

 

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Received on 14.03.2017       Accepted on 12.05.2017     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2017; 7(2):71-80.

DOI:  10.5958/2231-5691.2017.00013.2