International Journal of Pharma and Bio Sciences ISSN … · 2016-01-18 · Original Research Article Pharmaceutics International Journal of Pharma and Bio Sciences ISSN 0975-6299

Int J Pharm Bio Sci 2016 Jan; 7(1): (P) 185 - 198

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Original Research Article Pharmaceutics

International Journal of Pharma and Bio Sciences ISSN

0975-6299

PHYTOCHEMICAL PROFILING AND EVALUATION OF PHARMACOLOGICAL

ACTIVITIES OF TRIBULUS TERRESTRIS (LINN.)SEED

1VINODHINI S AND

2 DEVI RAJESWARI V*

1 School of BioScience and Technology, VIT University, Vellore-632014.

2Division of Biomolecules and Genetic, SBST, VIT University, Vellore-632014.

ABSTRACT

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycaemia generally associated with oxidative stress. The objective of this study is to screen the phytochemical constituents of the seeds of the medicinal plant Tribulus terrestris and to obtain preliminary in vitro data about its antioxidant, antidiabetic and anti-inflammatory activity effects. The in vitro antioxidant activity was assessed for T.terrestris seeds; extracts with different solvents such as methanol, ethyl acetate and chloroform were investigated. Antioxidant activity was measured with three methods, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, Hydrogen peroxidation and Total antioxidant activity. Each extract exhibits a dose-dependent free radical scavenging action against DPPH, hydrogen peroxidation and total antioxidant, but exhibits diverse antioxidant activities. To determine the antidiabetic activity, each extracts were analysed the assays of glucose uptake, α-amylase and glucose diffusion and anti-inflammatory activity was performed. The results show that methanolic extract have an effective activities when compared to ethyl acetate and chloroform extracts. Hence, methanolic extract of T.terrestris possess higher antioxidant, antidiabetic and anti-inflammatory activity, while compared to ethyl acetate and chloroform extract. The compounds were separated by thin layer chromatography. Intervention studies are essential to provide confirmation for a safe and effective use of the identified medicinal plants in the treatment. The present study shows that the methanolic extract of T.terrestris act as a potent antioxidant, antidiabetic activity and anti-inflammatory activity. KEYWORDS: Tribulus terrestris, TLC,DPPH, phytochemicals.

*Corresponding author

DEVI RAJESWARI V Division of Biomolecules and Genetic, SBST, VIT University, Vellore-632014.



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1. INTRODUCTION

Diabetes mellitus is a metabolic disorder, its affecting protein, fat and carbohydrate metabolism an increased risk of complications from vascular disease

1. A

worldwide review reported in every year 10% of the populations are affected by the diabetes mellitus and is considered as leading causes of death

2. Diabetes

mellitus is the fourth (or) fifth leading disease, it cause of death mainly in developed countries and most important evidence that it outbreak in globally

3-4.

Diabetes mellitus is one of most common disorder and non-communicable disease through all over universal

5.

Global increase in the commonness of diabetes mellitus and mostly a leading non-communicable disease related to life styles and the resulting pour in obesity. Diabetes is a lifelong progressive disease; it is a chronic metabolic disorder due to the relative deficiency of insulin secretion and varying in insulin resistance and it is characterized by high circulating glucose. In next 25 years, diabetes mellitus to become one the most main killers in worldwide

6, number of people with diabetes

takes forever increasing part of national and international human health care resources. Around 230 million people in worldwide have been affected by diabetes mellitus and approximately 366 million people are to get diabetes mellitus and expected by 2030

7.

Historically, Medicinal plants such as herbs have been used for centuries in daily life

8. Herbal plants have

been used in folk medicine to treat the diseases throughout the world

9-10. Medicinal plants are a good

source of producing large quantity of phyto constituents in mostly capable with precise selectivity

10. While in the

middle of the 19th century, different bioactive compounds are isolated and characterized from medicinal plants

11. In 2008 according to the World

Health Organization (WHO), traditional plants are assuming more than 80% of the populations are giving greater importance on herbal medicine

12 for their prime

health care needs of folks and communities in the developing countries

13. Traditional medicines are

derived from medicinal plants, plants have a rich source of compounds that are importance to the health of human beings, rich source of novel drugs which contain in the forms of traditional medicine

11, Applications of

the plant parts used in modern medicine like phytochemical compounds are most important to the medicinal benefits of the plant

14. Plants parts may use

properly and scientifically and it reach to the worldwide

10. Natural bioactive constituents of plants are

Alkaloids, Cardiac glycosides, Steroids, Saponins, Tannins, and Flavonoids. It is estimated that more than 250,000 to 500,000 species of higher plants on globe level, but some plants are relatively small percentage (5-15%) has been analytically investigated for the presence of natural bioactive compounds of photochemicals

12. Natural bioactive compounds were

found in plants parts, phtochemicals which act as a defence system against disease or protect against disease

12. Plants are the good source of natural

antioxidants; natural antioxidants which contain phytochemical antioxidants are the secondary

metabolites. Ascorbic acid, flavonoids, tocopherols, folic acid, cinnamic acids, benzoic acids, tocotrienols, carotenoids etc., these are some important antioxidants produced by the plant

15. Antioxidants substances that

are present in low concentrations, when compared to an oxidisable substrate significantly delays oxidation or prevent oxidation

16. The oxidative damage caused by

certain oxygen species are nucleic acids, proteins and, lipids. Since trigger from different chronic diseases, such as coronary heart disease, ageing an atherosclerosis, cancer etc,

17 and the antioxidants

substances which are free radicals scavenging play an important role to prevent the free radical-induced diseases. At present, mainly antioxidants are synthetically manufactured belong to the group of synthetic antioxidants. The main disadvantage with the synthetic antioxidants it occurs the side effects when taken in vivo. Severe governmental rules regarding the safety of the food have necessitated the search for alternatives as food preservatives

18. Some

synthetic antioxidants are toxic and carcinogenic to animal a model which is occurred by the butylated hydroxyanisole (BHA) and butylated hydroxyl toluene (BHT) require to be replaced between natural antioxidants

19. Natural antioxidants are more ideal as

food additives, not only in free radical scavenging properties and also presents in the natural products; these are healthy and safer than synthetic ones India is capable with rich flora and fauna of various origins. Tribulus terrestris Linn. (gokshura) is a herb which belongs to Zygophyllaceae family

20. It is adapted to

moderate region and is there common in areas like dry soils. In India, it is commonly known as gokharu and other vernacular names are in (Tamil) nerinjil, (English) caltrops fruit, (kannada) neggilamullu, (Malayalam) nerinjil, (Bengali) gokhri etc., Tribulus terrestris having a nutritive values and it is native to southern Africa, Europe and tropical Asia

21. Tribulus terrestris have

medicinal values known for it’s application in the folk medicine, it used in parts of the world. Tribulus terrestris parts are fruit and root (Caltrop fruit) it contains some pharmacological constituents such metabolites are flavonoids, alkaloids, phytosteroids and glycosides

22-23 and other reported pharmacogolical

activities were included such as antihypertensive, antioxidant, antidiabetic, anthelmintic, aphrodisiac, anticancer, antifungal, antiurolithiatic and antimolluscicidal activity

24. The present study was

undertaken to investigate the Tribulus terrestris three different extracts for its antioxidant and antidiabetic activities and phytochemical screening.

2. MATERIALS AND METHODS

2.1. Plant sample Plant material consisting mature seeds of Tribulus terrestris were collected from Malecherry forest. It was authenticated at Plant Anoatomy Research Centre in West Tambaram, Chennai, India. Its authentication number is (PARC/ 2015/3175).The plant material was cleaned, shade dried and powdered for extraction (Figure 1and Table 1).



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Figure 1 Tribulus terrestris seeds

Table 1 Classification of the plant material

2.2. Extraction process Direct extraction with three different solvents chloroform, ethyl acetate and methanol were used following the method of (Eloff, (1998) in this method, finally ground plant material (10 gram) was extracted with 100ml of chloroform, ethyl acetate and methanol in conical flask in shaking condition. The extract was

decanted into pre weighed glass vials. The process was repeated three times with the same material using fresh solvent. The solvent was removed by placing the extracts in front of a steam of air. The combined extract was concentrated by condensation. The extracted residues were weighed and re-dissolved in different solvents to yield 10mg/ml solution (Figure 2).

Figure 2

Solvent extraction of T. terrestris

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Rosidae

Order: Sapindales

Family: Zygophyllaceae

Genus: Tribulus

Species: Tribulus terrestris



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2.3. Phytochemical screening analysis Qualitative and quantitative Phytochemical screening analysis of the T. terrestris extract was performed to identify the bioactive constituents; this study was carried out by using standard procedures, plant extracts were detected by the following tests. 2.3.1. Qualitative analysis 1. Detection of Alkaloids 5mg of powder was taken and added with a few ml of dilute Hcl and filtrate the sample. The alkaloid reagent was carefully added to the test solution. Wagner’s test 2ml of filtrate, two or three drops of Wagner’s reagent were added by the side of the sample tube. A reddish- brown precipitate indicates presence of alkaloids

25.

2. Detection of Carbohydrates 2 mg of extract is dissolved in 2 ml of water and filtrate the sample. The sample solution was subjected to the following tests. Benedict’s test 1 ml of filtrate added a 0.5 ml of Benedict’s reagent and mixed well. The sample mixture was kept in a boiling water bath for 2 minutes. A characteristic coloured precipitate indicates the presence of carbohydrates

26.

3. Detection of Glycosides Test for cardiac glycoside: To 2 ml of extract filtrate, 1 ml of glacial acetic acid, 1 ml ferric chloride and added a few drops of conc. sulphuric acid was added. Brown colour indicated the presence of glycosides

27.

4. Detection of Saponins 2mg of extract was dissolved with 2ml of distilled water. The suspension is shaken well for few minutes. A layer of foam indicates the presence of saponins

28.

5. Detection of Proteins and Amino acids 2 mg of extract is diluted in 2ml of distilled water then filtered the sample extract using whatmann No.1 filter paper and then filtrate the extract tests used for proteins and amino acids. Millions test To 2ml of filtrate, few drops of millions reagent was added, if protein was present in the sample it showed white precipitate indicates the presence of protein

26.

6. Test for tannins Lead acetate test The extract 2 mg is dissolved in 1ml of distilled water and few ml of 10% lead acetate solution is added to the sample mixture. A bulky white precipitate indicates the presence of tannins compound

26.

7. Detection of Phenolic compounds Lead acetate test The extract 2 mg is dissolved in 1ml of distilled water and few ml of 10% lead acetate solution is added to the sample mixture. A bulky white precipitate indicates the presence of phenolic compounds

26.

8. Detection of Steroids Salkowski test 0.2 mg of the extract of sample was mixed with 2 ml of chloroform and 1ml ofconc. H2SO4 was carefully added to form a layer. A reddish brown colour in the interface indicates the presence of steroid

29.

9.Detection of Flavonoids Sodium Hydroxide Test 2 mg of extract was dissolved in 2ml of distilled water; 2ml of 10% sodium hydroxide solution was added to the test sample and to give yellow colour, few drops of dilute hydrochloric acid was added yellow colour change into colourless indicates the presence of flavonoids

30.

2.3.2. Quantitative analysis 1. Determination of total phenolic content Total phenol content was determined by the Folin- Ciocalteau method of Mc Donald et al., (2001). 0.5ml of three different extract were taken and 0.1ml (0.5N) folin- ciocalteu’s reagent was mixed and the mixture was incubated at room temperature for 15mins. Then, 2.5ml sodium carbonate solution was added and further incubated for 30min at room temperature and the absorbance was measured at 765nm. These data were used to estimate the total phenolic content using a standard calibration curve obtained from various diluted concentrations of gallic Gallic acid was used as a positive control

31.

2. Determination of total flavonoids content Total flavonoids were determined by Aluminium Chloride Colorimetric, chang et al., 2002 method was followed. Briefly, 0.5ml of plant extract was mixed with 1.5ml of ethanol, 0.1ml of 10% aluminium chloride, 0.1ml of 1M potassium acetate and 2.8ml of distilled water and it was kept at room temperature for 30mins. The absorbance of the reaction mixture was measured at 415nm. The results were expressed as milligrams quercetin equivalents (QE) per gram of extract (mg QE/g extract). The quercetin is used as a standard curve in different concentrations

32.

2.4. Antioxidant Activity 2.4.1. Total Antioxidant Activity Total antioxidant activities of crude methanol, ethyl acetate and chloroform extracts were obtained from Tribulus terrestris used to determine. 1mg/ml of sample stock solution were diluted to five different concentrations of 25, 50, 75,100, 200 µg/ml were used for the assay. Briefly, 1.23g Ammonium of Molybdate [4mM] , 7.45ml of Sulphuric acid (0.6mM) and 0.99g of Sodium sulphate (28mm) were mixed in 250 ml of distilled water and represented as Total antioxidant capacity TAC. Different concentrations of the samples were mixed with 1ml of TAC solution. Reaction mixture was incubated at room temperature for 15 minutes. Ascorbic acid was used as standard. Absorbance of sample mixtures was measured at 695nm. Total antioxidant activity was expressed as the number of equivalents of ascorbic acid in milligram per gram of extract. Absorbance values are calculated by using the formula

33.



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Where, A0 – Absorbance of control; A1 – Absorbance of sample, Ascorbic acid was used as positive controls. The experiment was repeated in triplicate.

2.4.2. DPPH (2, 2- diphenyl-1-picrylhydrazyl) Radical Scavenging Activity DPPH scavenging effects of methanol, ethyl acetate and chloroform extracts obtained from Tribulus terrestris were determined. Sample stock solutions were taken into five different concentrations of 25, 50, 75,100, 200 µg/ml were used for the assay. Briefly, 2.0 ml of 0.16mM DPPH solution (in methanol) was added to the test tube containing 2.0 ml aliquot of sample. The mixture was vortexes for 1 minute and

kept at room temperature for 30 minutes in the dark. After incubation the absorbance of all the sample solutions were measured at 517nm; it showed decrease in the absorbance of test mixture (due to quenching of DPPH free radicals). The scavenging effect percentage was calculated by using the formula. Sample blank and control samples were performed according to the method

34.Scavenging effect of DPPH radical was

calculated using the following equation

Where, a sample is the Absorbance of DPPH solution & test sample, A sample blank is the absorbance of the sample only without DPPH solution). Ascorbic acid was used as positive controls. The experiment was repeated in triplicate.

2.4.3. Hydrogen Peroxide (H2O2) Radical Scavenging Activity Tribulus terrestris extracts on H2O2 by spectrophotometric method. 40mM H2O2 was prepared in phosphate buffer (pH-7.4) and the H2O2 concentration was determined. Extract and ascorbic acid was dissolved in distilled water, sample stock

solutions were taken into five different concentrations of 25, 50, 75,100, 200 µg/ml were used for the assay. 0.6 ml of 40mM H2O2 solution was added to extract, after 10 minutes incubation, these solution was absorbed at 230nm.Blank solution contain phosphate buffer without H2O2

35.

The percentage of H2O2 scavenging to aqueous extract was calculated using the following formula

Where, A0 – Absorbance of control; A1 – Absorbance of sample, Ascorbic acid was used as positive controls. The experiment was repeated in triplicate

2.5. Antidiabetic Activity 2.5.1. Glucose uptake by yeast cells Yeast cells were prepared according to the method of Cirillo, (1962). Commercial baker’s yeast was washed by repeated centrifugation (3,000rpm for 5 minutes) in distilled water until the supernatant fluids were clear and a 10% (v/v) suspension was prepared in distilled water. Various concentrations of extracts were added to 1ml of glucose solution (5, 10 and 25mM) and incubated together for 10 min at 37 °C. Reaction was started by adding 100µl of yeast suspension, vortex and further incubated at 37 °C for 60min. After 60 min, the tubes were centrifuged 2,500 rpm for 5 minutes and glucose was estimated by DNS method in the supernatant. The percentage increase in glucose uptake by yeast cells was calculated using the following formula Increase in glucose uptake (%) = Abs sample– Abs control× 100/Abs sample Where, Abs control is the absorbance of the control reaction (containing all reagents except the test sample) and Abs sample is the absorbance of the test sample. All the experiments were carried out in triplicates

36.

2.5.2. Effects of various extracts on inhibition of glucose diffusion Glucose diffusion test was done by Edwards et al., 1987 method was followed. Dialysis tube (6cm x15mm) into which 1ml of 50g/litre plant extracts in 1% CMC and 1ml of 0.15M sodium chloride containing 0.22M D-glucose was added. The dialysis tube was sealed at

each end placed in a 50ml centrifuge tube containing 45ml of 0.15M sodium chloride. The tubes were placed on an orbital shaker and kept at room temperature. The movement of glucose into the external solution was monitored at set time intervals

37.

Glucose estimation Add 1ml of DNS reagent to 1ml of glucose sample in a lightly capped test tube. (To avoid the loss of liquid due to evaporation, cover the test tube with a pick of paraffin film if a plain test tube is used).Heat the mixture at 90ºc for 5 -15minutes to develop the red brownish colour.400µl of a 40% potassium sodium tartrate (Rochelle salt) solution of stabilize the colour . After cooling to room temperature in a cold water bath record the absorbance with a spectrophotometer

38.

2.5.3. α-Amylase inhibition assay The α-amylase inhibitory activity was determined by an assay modified from the Worthington Enzyme Manual (Worthington, 1993). A total of 500 µl of sample extracts and 500 µl of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing α-amylase solution (1ml mg/ml) were incubated at 25°C for 10 minutes. After pre incubation, 500 µl of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added to each tube at timed intervals. The reaction was stopped with 1.0 ml of dinitrosalicylic (DNS) acid colour reagent. The test tubes were incubated in a boiling water bath for 5



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minutes and cooled to room temperature. The reaction mixture was then diluted after adding 5 -15 ml of distilled water, and the absorbance was measured at

540 nm using the UV/Visible spectrophotometer. The readings were compared with the controls, containing buffer instead of sample extract

39.

Where, Abs control is the absorbance of the control reaction (containing all reagents except the test sample), and Abs sample is the absorbance of the test sample. All the experiments were carried out in triplicates.

2.6. In-vitro anti-inflammatory activity This study is followed by Varadarasu et al., 2007

40

using human red blood cell membrane stabilization method. The blood was collected from healthy human volunteer. Alsever solution contains (2% dextrose, 0.8% sodium citrate, 0.5% citric acid and 0.42% NaCl) mixed with the equal volume of blood sample and centrifuged at 3,000 rpm. The packed cells were washed with isolaline and a 10% suspension was made. Different concentrations of extracts were prepared (200 to 1000µg/ml) using distilled water. Samples were mixed with 1ml of phosphate buffer, 2ml hyposaline and 0.5ml of HRBC suspension was added. It was incubated at

37°c for 30min and centrifuged at 3,000 rpm for 20min. The haemoglobin content of supernatant solution was measured at 560 nm. 2.7. Thin Layer Chromatography The thin layer chromatography was done to determine the number of compounds present in the plant sample Tribulus terrestris methanol extract. The sample was placed in the pre coated TLC plate as a spot and was allowed to run with various solvent ratios are (0.5:9.5). The solvents used were methanol:chloroform. Then the separation of compounds was viewed under a UV illuminator. Rf factor values were calculated.

3. RESULTS AND DISCUSSION

Diabetes mellitus an enduring progressive disease is a chronic metabolic disorder due to the relative deficiency of insulin secretion. Diabetes mellitus is the main causes of premature deaths in globally. It affects mostly the developing countries like India

39, 41. Insulin

plays a key role in the control of glucose homeostasis. The carbohydrate; fat and protein metabolism are affected by lack of insulin

42. Many complications of

diabetics are prone due to the nature of disease 43

. Diabetes mellitus has reached epidemic proportion among the challenging unresolved health problems of the 21st century

44. Natural source of medicinal plants

have been utilized in ayurvedic medicines, since 2000 years. Traditional Indian literatures such as Atharva Veda, Rig Veda, Yajur Veda, Sushrut Samhita and Charak Samhita have reported the use of medicinal plants to cure human diseases in ancient times

45. At

present, scientists are focussed on the bio-active compounds from the natural sources to develop newer drugs. In earlier period, a variety of medicinal plants have been reported to possess different medicinal properties viz, antimicrobial activity

46, antioxidant

activity 47

, antidiabetic activity, anticancer activity, anti-inflammatory activity

48, haemolytic activity etc.,

46.

3.1. Phytochemical screening analysis However, the preliminary pharmacological investigation is an important process to perform before or after the isolation of the bioactive molecule.

Phytochemicals are the non-nutritional plant compounds that are produced by the plants in self-defence to protect them from pest, microbes and environmental stress factors

49. In earlier period,

medicinal plants have been reported to contain different phytochemical compounds with an extensive range of activities, which may help in the development of new drugs. 3.1.1. Qualitative test Preliminary phytochemical screening results reveals that the seed extract of Tribulus terrestris showed many types of chemical constituents. Among different solvents used for extraction in a series, methanolic and ethyl acetate extract showed positive results for many numbers of chemical compounds. It contains Steroids, Saponins, Carbohydrates, Tannins, flavonoids and phenolic compounds. Chloroform extract showed positive results, presence compounds are Steroids, Saponins, and Carbohydrates, alkaloids, tannins, flavonoids and phenolic compounds. The summery results for the preliminary phytochemical screening of different solvent extracts of Tribulus terrestris are shown in Table 2. The present finding of Phytochemical screening of Tribulus terrestris extracts confirmed the presence of several bioactive compounds like alkaloids, tannins, phenols and flavones which could be responsible for the versatile medicinal properties of this plant.



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Table 2 Phytochemical screening analysis in Qualitative test

3.1.2. Quantitative Analysis 1. Total phenolic content Tribulus terrestris extracts were tested for total phenolic content; the reaction was based on the Folin-Ciocalteu reagent used to identify the phenolic content. The potential antioxidant assay was used to measure the inhibit oxidation reagent. Gallic acid act as a positive control, these values were compared with the standard of Gallic acid it shows in Table 3. The linear regression

equation was found to be y = 0.114x + 0.028 while the correlation was found to be 0.9995. The amount of Phenolic content present in the extract in terms mg GAE/g of methanol extract was found to be 85.9 in ethyl acetate it shows 58.12 and in chloroform it showed 32.01 by using the above linear regression equation.

Table 3

Estimation of total phenolic content

Conc. of Gallic acid in µg/ml

Absorbance at 765nm

Conc. of extract in µg/ml

Amount of total Phenolic content in terms mg GAE/g of extract

Methanol extract Ethylacetate extract Chloroform extract

2 0.208± 0.021

4 0.418±0.117

6 0.657±0.213 100 85.9 58.12 32.01

8 0.896±0.04

10 1.114±0.01

2. Total flavonoids content An antioxidant effect of Tribulus terrestris extracts the compounds such as flavonoids; flavonoid is responsible for the functional group of hydroxyl and effects on considerable health and human nutrition. Quercetin act as a positive control, aqueous extract value was compared with the standard of quercetin it

Shows in Table. 4. The linear regression equation was found to be y = 0.0298x + 0.0305 while the correlation was found to be 0.9995. The amount of Flavonoids content present in the Tribulus terrestris extracts in terms mg Quercetin equivalent/g of extract was found in table 4 it showed the values of three different extracts by using the above linear regression equation.

Table 4 Estimation of total flavonoids content

Conc. Of Quercetin in µg/ml

Absorbance at 415nm

Conc. of extract in µg/ml

Amount of total Flavonoids content in terms mg Quercetin /g of extract

Methanol extract Ethylacetate extract Chloroform extract t

20 0.43± 0.01

40 0.751±0.04

60 1.31±0.09 100 129.35 112.23 94.56

80 1.69±0.03

100 2.412±0.03

3.2. Antioxidant activity 3.2.1. Total antioxidant activity Total antioxidant activity is expressed as the number of equivalents of ascorbic acid in milligram per gram of extract (Figure 3). Ascorbic acid was taken in different concentration viz; 25-200µg/ml. Graph was plotted using the O.D value of the extract and Ascorbic acid taken as a standard it showed (0.523±0.217) - (1.365±0.197). The total antioxidant activity of the

Tribulus terrestris was determined. The antioxidant activity was exhibited by the chloroform extract (0.117±0.348)-(0.275±0.634) it showed partial activity, ethyl acetate shows the lowest activity was recorded (0.133±0.328) - (0.467±0.651). Similarly findings also showed that the maximum total antioxidant activity was shown by the methanol extract (0.321±0.203)-(1.051±0.214) compared to the Ascorbic acids.

S.no Phytochemic Qualitative analysis Results

Methanol extract Ethylacetate extract Chloroform extract

1. Alkaloids Wagner’s test - - +

2. Carbohydrates Benedict,s test + + +

3. proteins and aminoacid

Lead acetate test - - -

4. Glycoside Borntrager’s test - + -

5. Saponins Foam test ++ + +

6. Steroids Salkowski test +++ ++ ++

7. Phenols Lead acetate test +++ ++ +++

8. Tannins Neutral ferric chloride test +++ + ++

9. Flavanoids Sodium hydroxide test +++ ++ +++



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Figure 3 Total antioxidant activity of different solvent extracts from Tribulus terrestris

3.2.2. DPPH (2, 2- diphenyl-1-picrylhydrazyl) Radical Scavenging Activity DPPH have been used extensively as a free radical to evaluate reducing substance, the three different extracts were preformed with DPPH free radical scavenging assay. Tribulus terrestris extracts to evaluate the DPPH scavenging activity against the free radicals. DPPH solution was freshly prepared to use. The deep purple colour disappeared and changes into yellow colour once an antioxidant is present in the sample. The antioxidant molecules can be quenching between DPPH free radicals, with providing a hydrogen atoms or electron contribution, possibly through a free-

radical attack on the DPPH molecule and then convert to colourless product

50. This result is decreased in a

maximum absorbance at 518nm. The antioxidant activity increases with an increase in the concentration (Figure 4). The maximum scavenging effect was shown by the methanol extract of (27.5±0.634)-(62.75±0.235% inhibition) and chloroform extract showed (13.14±0.345)-(54.03±0.231) % inhibition minimum scavenging activity of test sample. The lowest inhibition was shown by chloroform extract showed (11.21±0.623)-(44.35±0.12) partial scavenging activity values are compared with ascorbic acid as a standard.

Figure 4

DPPH radical scavenging activity of different solvent extracts from Tribulus terrestris

3.2.3.Hydrogen Peroxide (H2O2) Radical Scavenging Activity Hydrogen peroxide is a weak oxidizing agent that inactivates a few enzymes directly, usually by oxidation of essential groups. Hydrogen peroxide itself is not very reactive, but sometimes it can be toxic to cells, because of rise in the hydroxyl radicals in the cells. It can probably react with and Fe

2+possibly Cu

2+ ions to form

hydroxyl radicals and this may be the origin of many of

its oxide effects51

. The H2O2 Radical scavenging assay was performed with the different extracts of the samples. The maximum scavenging activity was shown on the methanol extract (8.23±0.254)-(41.01±0.685). The lowest inhibition was shown on ethyl acetate (3.21 ±0.129)-(31.02±0.498), chloroform extract showed (16.34±0.31)-(38.43±0.287) partial scavenging activity values are compared with ascorbic acid as a standard it shown (Figure 5).



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Figure 5 H2O2 radical scavenging activity of different solvent extracts from Tribulus terrestris

3.3. Evaluation of antidiabetic potential of Tribulus terrestris 3.3.1. Glucose uptake by yeast cells assay Glucose transport occurs across the yeast cell membrane through facilitated diffusion down the concentration gradient and it takes place only when there is removal of intracellular glucose

52. In the case

of human small intestine, intracellular glucose uptake is accomplished by passive diffusion and transport is via a glucose transporter. The effect of plant extracts on the transport of glucose across yeast cell membrane is considered as a vital measure for screening the antidiabetic efficacy of medicinal plants

53. The ability of

the plant extracts to increase glucose uptake by yeast cells is considered as their potential to regulate glucose transport across cell membrane which is in turn a measure of their efficacy to regulate glucose levels. It is reported that in yeast cells, Tribulus terrestris glucose transport is extremely complex and it is generally

agreed that glucose is transported in yeast is by a facilitated diffusion process. The specific carriers that transport solutes down the concentration gradient

54.

The rate of glucose transport across cell membrane in yeast cells system is presented. The amount of remaining glucose in the medium after a particular time serves as an indicator of the glucose uptake by the yeast cells. The rate of uptake of glucose into yeast cells was linear in all the 3 glucose concentrations. The results obtained for the glucose uptake in 5mM, 10mM and 25mM concentration assay are presented. The glucose uptake assay was performed for methanol extract was efficient in enhancing the glucose uptake by yeast cells .From 13-50 % (concentration ranging from 250-1000µg) (Figure 6). While, the ethyl acetate extract and chloroform extracts were found (Figure 7 and 8) to increase glucose uptake only to 17% to 15% respectively.

Figure 6

Effect of methanol extract of Tribulus terrestris on glucose uptake by yeast cells



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Figure 7 Effect of Ethyl acetate extract of Tribulus terrestris on glucose uptake by yeast cells

Figure 8 Effect of chloroform extract of Tribulus terrestris on glucose uptake by yeast cells

3.3.2. α-Amylase inhibition assay Among various commercial antidiabetic drugs, some are claimed to act through inhibiting the digestion of complex carbohydrates in the gastrointestinal tract

55.

Pancreatic α-amylase is a key enzyme which catalyses the initial step of starch hydrolysis producing a mixture of smaller oligosaccharides such as maltose, maltotriose and a number of α-(1-6) and α-(1-4) oligoglucans. These small saccharides are further acted upon by α-glucosidases releasing glucose which is absorbed into the blood stream. Thus, a correlation is said to exist between post-prandial glucose levels and

the activity of Human Pancreatic α-amylase (HPA) in the small intestine

56. Hence, regulating the activity of

pancreatic α-amylase in small intestine plays a pivotal role in the treatment of diabetes. α –amylase inhibition assay was performed for the three different extracts of Tribulus terrestris. There was a dose-dependent increase in percentage inhibitory activity against α-amylase enzyme. At a concentration of 250 to1000 µg/ml of methanol extract showed a percentage inhibition (31.47±0.424 to 80.34±0.576 µg/ml) and in ethyl acetate it showed (25.67±0.634 to 61.71±0.753 µg/ml) and chloroform showed (19.23 ±0.66 to 57.32±0.797µg/ml) (Figure 9).

Figure 9

Effect of Tribulus terrestris extracts on α-amylase inhibition



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3.3.3. Glucose diffusion assay Antihyperglycemic activities of most effective plants were partly explained by the ability of the phytoconstituents to increase glucose transport and metabolism in muscle and to stimulate insulin secretion. In the present study, research has been carried out to evaluate the potential of selected extract to additionally retard the diffusion and movement of glucose in the intestinal tract

37. The effect of Tribulus terrestris

seeds as antidiabetic agents has been studied; all extracts showed varying effect on glucose utilization. These extracts caused a significant decrease in glucose concentration during the experiment. The effects of Tribulus terrestris seed extracts on glucose

diffusion inhibition were summarized in (Fig. 10). At the end of 5 hrs, glucose movement of control (without plant extract) in the external solution had reached a plateau with a mean glucose concentration. It was evident from the figure that the methanol, ethyl acetate and chloroform extracts were found to be potent inhibitors of glucose diffusion. The methanol extract was found to be more potent than other extracts shows the lowest mean glucose concentration of methanol (104±0.275)-(967±0.923), ethyl acetate (83±0.456)-(819± 0.826) and chloroform (60±0.756)-(795± 0.217) extract of Tribulus terrestris signifies inhibitory potential of glucose diffusion at the end of 5 hrs.

Figure 10

Effect of Tribulus terrestris extracts on Glucose diffusion

3.4. In-vitro anti-inflammatory activity This study shows that the extracts exhibited membrane stabilization effect by inhibiting hypotonicity induced lysis of erythrocyte membrane. Erythrocyte membrane is analogous to the lysosomal membrane

57; different

extracts may stabilize lysosomal membrane, which is important in limiting response to preventing the release

of lysosomal constituents of neutrophils such as enzymes which may cause further tissue damage and inflammation upon extra celluar release

58. Extracts

showed (Table 5) significant in vitro anti- inflammatory activity. This study provides a confirmation for the Tribulus terrestris seeds in folk treatment as anti-inflammatory agent.

Table 5

In-vitro anti-inflammatory activity

Concentration µg/ml

% inhibition

Methanol Ethylacetate Chloroform

200 26.21± 0.021 19.47±0.141 12.37±0.175

400 31.02±0.012 23.01±0.07 17.65±0.264

600 47.43±0.032 29.21±0.214 21.57±0.321

800 53.09±0.12 32.37±0.17 28.64±0.274

1000 61.52±0.04 44.05±0.36 34.52±0.184

3.5. Thin layer chromatography The TLC analysis of methanol extract of Tribulus terrestris revealed the presence of 4 compounds with Rf values 0.96, 0.8, 0.64 and 0.56 (Figure 11).



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Figure 11 Thin layer chromatogram of methanol extract of Tribulus terrestris

Under UV illuminator Under iodine camber

4. CONCLUSION

According to all tested methods, the antioxidant activity of T. terrestris was compared with that of Ascorbic acid, which is a well known potent antioxidant. In general, the present study reports that extracts can be a good source of antioxidant and the ability of various extracts of T. terrestris to inhibit glucose diffusion, α-amylase activity and to enhance glucose uptake by yeast cells using in vitro models. The data suggested that the plant

may be useful in effective management of Type 2 Diabetes mellitus. Further studies are required to elucidate whether in vitro effects represent therapeutic potential in In vivo models for improving glycemic control in type 2 diabetic. However, further study is needed to isolate the compounds responsible for its promising anti-diabetic activity; this study adds credence to the traditional use of T. terrestris to treat diabetes.

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International Journal of Pharma and Bio Sciences ISSN … · 2016-01-18 · Original Research Article Pharmaceutics International Journal of Pharma and Bio Sciences ISSN 0975-6299