phytochemical screening and biological ativities of ...

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Shivaji Bole et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 2, Issue 6, 5363-5376.

Research Article

ISSN 2278 – 4357

PHYTOCHEMICAL SCREENING AND BIOLOGICAL ATIVITIES OF IMPATIENS BALSAMINA. L SEEDS Shivaji Bole*1, Shivakumara1, Seitajit Singh Wahengbam1, Naveen kumar Rana1, Swarnava Kundu1, Siddharth Dubey1, Vedamurthy AB1 Dept. of Biotechnology, The Oxford College of Science, 19th Main, 17th B cross, HSR Layout, Bangalore, India.

Article Received on 26 August 2013, Revised on 27 Sept 2013, Accepted on 05 November 2013

ABSTRACT In vitro antioxidant, antidiabetic, anti-inflammatory activity and phytochemical evaluation of solvent extracts of seed of Impatiens Balsamina. L. was analyzed. Phytochemical screening of the ethanolic extracts of seeds revealed the presence of alkaloids, flavanoids,

*Correspondence for

terpenoids, tannins and saponins. Antioxidant activities of these extracts were evaluated by in vitro antioxidant assay models like

Author:

DPPH, reducing power assay and phospho-molybdenum method. *Shivaji B Bole Associate Professor, PG Dept of Biotechnology,

antidiabetic activity was evaluated by amylase inhibition assay method and anti-inflammatory activity was determined by induced protein

The Oxford College of Science,

denaturation method. The Results obtain in this study showed that the

Bangalore. India.

seeds of Impatiens Balsamina. L has antioxidant, antidiabetic and anti-

[email protected]

inflammatory activity which provide a basis for traditional use of medicinal plant. IC50 value was calculated for both antioxidant and

anti-inflammatory activity and was found to be 320µG/mL. and 210 µG/mL. respectively KEY WORDS: Antioxidant, Antidiabetic, Anti-inflammatory, BSA Denaturation. INTRODUCTION In India, drugs of herbal origin have been used in traditional systems of medicines such as Unani and Ayurveda since ancient times. The drugs are derived either from the whole plant or from different organs, like leaves, stem, bark, root, flower, seed, etc. Some drugs are prepared from excretory plant product such as gum, resins and latex. In many of the developing countries the use of plant drugs is increasing because modern life saving drugs is beyond the www.wjpps.com

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reach of three quarters of the third world’s population. With the rapid depletion of forests, impairing the availability of raw drugs, Ayurveda, like other systems of herbal medicines has reached a very critical phase. About 50% of the tropical forests, the treasure house of plant and animal diversity have already been destroyed. Green plants synthesize and preserve a variety of biochemical products, many of which are extractable and used as chemical feed stocks or as raw material for various scientific investigations. Many secondary metabolites of plant are commercially important and find use in a number of pharmaceutical compounds [1]. In some cases, the crude extract of medicinal plants may be used as medicaments. On the other hand, the isolation and identification of the active principles and elucidation of the mechanism of action of a drug is of paramount importance. The scientific study of traditional medicines, derivation of drugs through bioprospecting and systematic conservation of the concerned medicinal plants are thus of great importance. Impatiens balsamina L is an annual plant belongs to family Balsaminaceae commonly called as Balsam, Jewelweed. The average height of the plant is about 1 to 2.5 feet. It is a very variable species particularly in the size of leaves and flower these plants tend to grow in wet shady soils along stream banks. Leaves are of 2 to 4 inches, simple, pinnate venation, lanceolate shaped, serrate margin and green in color. Flowers are of different colors like white: pink: salmon: purple: lavender. The genus has been widely distributed throughout the northern hemisphere and native of India and parts of mainland South East Asia [2.3].

Oxygen is a highly reactive atom that is capable of becoming part of potentially damaging molecules commonly called “free radicals.” Free radicals are capable of attacking the healthy cells of the body, causing them to lose their structure and function. Cell damage caused by free

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radicals appears to be a major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular disease, cataracts, immune system decline, and brain dysfunction. Overall, free radicals have been implicated in the pathogenesis of at least 50 diseases [4-9] fortunately; free radical formation is controlled naturally by various beneficial compounds known as antioxidants. Compounds with reducing power indicate that they are electron donors and can reduce the oxidized intermediates of lipid peroxidation process, so that they can act as primary and secondary antioxidants. In reducing power assay, the antioxidant compounds convert the oxidation form of iron from ferric chloride (Fe+3) to ferrous (Fe+2). The reducing power increased with increasing amount of the extract. The phosphor molybdenum assay is based on the reduction of Mo (VI) to Mo (V) by the sample analyze and subsequent formation of a green phosphate Mo (V) complex at acidic pH. Increase in the absorbance shows higher reducing property. DPPH assay is based on the reduction of DPPH in presence of methanol due to the hydrogen– donating antioxidant leads to the formation of the non radical form of DPPH. This transformation results in a color change from purple to yellow, which is measured spectrophotometrically. DPPH radicals react with suitable reducing agent, the electrons become paired off and the solution loses color stoichiometrically depending on the number of electrons taken up. Decreasing of the DPPH solution absorbance indicates an increase of the DPPH radical-scavenging activity. The result of the different tests carried on the plant material is compared with standard result of ascorbic acid [10-11]. Diabetes is a defect in the body’s ability to convert glucose (sugar) to energy. Carbohydrates, when digested, change to glucose. In order for glucose to be transferred from the blood into the cells, the hormone - insulin is needed. Insulin is produced by the beta cells in the pancreas (the organ that produces insulin).In individuals with diabetes, this process is impaired. Diabetes develops when the pancreas fails to produce sufficient quantities of insulin Type 1 diabetes or the insulin produced is defective and cannot move glucose into the cells. In Type 2 diabetes. Either insulin is not produced in sufficient quantities or the insulin produced is defective and cannot move the glucose into the cells. In vitro screening of anti-diabetic drug is carried out by estimating the levels of PPARγ and the two principle enzymes which involve in the carbohydrate digestion and glucose absorption process. Various methods are reported for antidiabetic activity. In the work that is described here, inhibition of α-Amylase assay method has been adopted for our study [12-13]. www.wjpps.com

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Alpha amylase is an enzyme that hydrolyses alpha-bonds of large alpha linked polysaccharide such as glycogen and starch to yield glucose and maltose. Alpha amylase inhibitors bind to alpha-bond of polysaccharide and prevent break down of polysaccharide in mono and disaccharide. Due to suppression of the activity of enzymes amylase would delay the degradation of starch and oligosaccharides, which would in turn cause a decrease in the absorption of glucose. INFLAMMATION Inflammation is a complex process which involves many steps. The region of inflammation is associated with various physical changes which are visible to the unaided eye. However, these visual representations are associated with a lot of chemical changes occurring within the tissue. Denatured proteins produced at the site of inflammation are also known to act as chemotactic factors that can prolong inflammatory process. A candidate that can inhibit denaturation of protein may be a suitable anti-inflammatory agent. Bovine serum albumin denaturation assay is a non specific In-Vitro model to evaluate anti-inflammatory agents [1415]. MATERIALS AND METHODS COLLECTION OF SAMPLE The seeds of the plant Impatiens balsamina.L were collected from Nursery of Lalbhag Botanical garden, Bangalore. The plant was authenticated by Botanist Bangalore University Bangalore. Karnataka, India. CHEMICALS AND REAGENTS All the chemicals and reagents used were of analytical grade and are purchased from Lancaster Research Lab, Chennai, India and Himedia Lab, Mumbai, India. PREPARATION OF EXTRACT Ethanolic extract: The seeds of the plant collected were dried under low sunlight for 7-8 days and then grinded to a fine powders in a grinder. The powdered plant material (15g) was subjected to maceration using ethanol for 4 days, then filtered with muslin cloth and evaporated to dryness. Extract was kept in desiccator.

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PHYTOCHEMICAL ANALYSIS The seed extract was used for preliminary screening of phytochemicals such as alkaloids (Wagner’s and Meyer’s tests), saponins (foam test), tannins (gelatin test), and flavonoids (Alkaline reagent and Lead acetate tests), the screening was done as per the standard method. Test for alkaloids 

Dragendroff’s test: 2mg of the test extract and 5ml of distilled water was added, 2M hydrochloric acid was added until an acid reaction occurs. To this 1ml of Dragendrof’s reagent was added. Formation of orange red precipitate indicated the presence of alkaloids.



Hager’s test: 2mg of the test extract was taken in a test tube, a few drops of Hager’s reagent was added. Formation of yellow precipitate confirmed the presence of alkaloids.



Wagner’s test: 2mg of extract was acidified with 1.5% v/v of hydrochloric acid and a few drops of Wagner’s reagent were added. A yellow or brown precipitate indicated the presence of alkaloids.



Mayer’s test: few drops of Mayer’s reagent, 2mg of extract were added formation of white or pale yellow precipitation indicated the presence of alkaloids.

Test for flavanoids 

Ferric chloride test: test solution with few drops of ferric chloride solution shows intense green color.



Zinc hydrochloride acid reduction test: test solution with zinc dust and few drops of hydrochloric acid shows magenta red color



Lead acetate solution test: test solution with few drops of lead acetate (10%) solution gives yellow precipitate.

Test for terpenoids: taken 0.5 gm of plant extract in a 2ml of chloroform. Added concentrated sulphuric acid carefully to form a layer. Observe for the presence of reddish brown color interface to show positive result for the presence of terpenoids. Test for saponins 

Foam test: to the extract solution, a drop of sodium bicarbonate solution was added. The test tube was shaken vigorously and left for 3 minutes. The formation of honeycomb like froth indicates the presence of saponins.

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Test for tanins 

Ferric chloride test: 1-2ml of the extract, few drops of 5%w/v FeCl3 solution was added. A green color indicated the presence of gallotanins; while brown color indicates the Presence of pseudotanins.



Gelatin test: test solution when treated with gelatin solution gives white precipitate

ANTIOXIDANT ACTIVITY Reducing Power Assay (Iron (III) to iron (II) reduction) 2.5 mL of extract solution of different concentrations (100 to 500 µg) was mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 mL, 0.1%). This was incubated at 500 C for 20 min. After the incubation, 2.5 mL of 10% trichloroacetic acid was added.2.5 mL of the reaction mixture was mixed with distilled water (2.5mL) and ferric chloride (0.5 mL, 0.1%). The solution absorbance was measured at 700 nM. Increasing absorbance of the reaction mixture indicates increasing reducing power. The same procedure was applied for ascorbic acid which acts as the standard. Increase in the absorbance indicates increase in reducing power. PHOSPHO MOLYBDENUM ASSAY The antioxidant activity of all the extract was determined by the phospho‐molybdenum Method as described by Prieto et al [16]. 0.3 ml of extract of different concentrations (100 to 500µg) was mixed with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodiumphosphate and 4 mM ammonium molybdate). The reaction mixture was incubated at 950C for 90 min and cooled to room temperature. Finally, absorbance was measured at 695 nm using a spectrophotometer against blank. Distilled water (0.3 ml) in place of extract was used as the blank. The total antioxidant capacity was expressed as the number of equivalents of Ascorbic acid. DPPH FREE RADICAL SCAVENGING ASSAY Plant extract of different concentration ranging from 100-500µg was taken in test tubes and made the volume to 1 ml with methanol. 4ml of DPPH solution was added to all the tubes. The reaction mixture was kept 30 min at room temperature. Measure the Optical density at 517 nm against Blank containing 5 ml of methanol, and control is containing 1 ml methanol and 4 ml of DPPH. The % of inhibition is calculated by using formula

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% of Inhibition Where, Ac is absorbance of control As is absorbance of sample. ANTIDIABETIC ACTIVITY INHIBITION OF ALPHA AMYLASE ENZYME STANDARD MALTOSE CURVE: 0.2-1ml of standard maltose (1mg/ml) was taken into different test tubes. Made the volume to 1.0 ml in each case with distilled water. Added 1.0 ml of DNS reagent to the each tube & then placed all the tubes in boiling water bath for 15 minutes. Added 8.0 ml of distilled water in each test tube & Mix the contents of the tube thoroughly. Then read the absorbance of the solution in calorimeter at 570 nM against blank solution. ALPHA AMYLASE INHIBITION ASSAY 100-500µl (100-500µg) of extract was taken into different test tubes. Made the volume to 0.5ml with phosphate buffer of pH 6.9, Blank was measured by taking 1 ml of phosphate buffer. Control was measured by taking 0.5ml of phosphate buffer. The solution was then treated with 0.5ml of alpha amylase (0.5mg/ml). The solution was incubated at 25oC for 10 minutes. Added 0.5ml of 1% starch solution in 0.02 M sodium phosphate buffer of pH 6.9 to all the tubes, and then incubate at 25oC for 10 minutes. The reaction was stopped by adding 1.0 ml of DNS and the reaction mixture was kept in boiling water bath for 5 minutes, cooled to room temperature. The solution was mixed with 8 ml distilled water. Read the absorbance of the solution in calorimeter at 570 nM against blank solution. Amount of maltose produced is calculated using standard maltose curve, and Enzyme activity is calculated by using formula Enzyme activity =

ANTI INFLAMMATORY ACTIVITY BSA DENATURATION ASSAY 0.5ml of test solution having different concentrations (100 to500µg) of extract was taken with 1.5ml of bovine serum albumin (BSA). The mixture was incubated at 27±1oC for 15minutes. Denaturation was induced by keeping the reaction mixture at 600C in a water bath for 10 minutes. After cooling the turbidity was measured spectrophotometrically at 660 nm.

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Percentage inhibition of denaturation was calculated from control where no drug was added. Percentage of anti‐inflammatory activity of the extract is given by, % AI = (Ac ‐ At) x 100 Ac

Where Ac is the absorbance of the control At is the absorbance of the test.

RESULTS AND DISCUSSION PHYTOCHEMICAL ANALYSIS In this study In vitro antioxidant activity, antidiabetic, and anti-inflammatory activity of seed extracts of Impatiens Balsamina. L was evaluated by different In vitro screening methods. Preliminary phytochemical screening of ethanolic extract of the dried seeds revealed the presence of secondary metabolites like alkaloids, tannins, and flavonoids. The results are tabulated in Table 1. The results of the free radical scavenging activity of extract tested by reducing power assay method, phosphor-molybdenum method and DPPH method, the results are summarized in table 2-3 and fig 1-2. The phosphor-molybdenum method is quantitative since the total antioxidant capacity is express as ascorbic acid equivalents. The percentage of antioxidant activity was found 71.43%. In reducing power assay extracts of Impatiens Balsamina. L seemed to have quite significant antioxidant properties compared to ascorbic acid. The results are summarized in table 2 and fig 1. In DPPH assay extracts of Impatiens Balsamina. L shows the minimum (24.98%) free radical scavenging activity compared to standard (43.8%). The results are compared with standard ascorbic acid. The results are summarized in table 3 and fig-2. In vitro anti-inflammatory was estimated by inhibiting protein denaturation. The inhibitory effect on protein denaturation by extracts of Impatiens Balsamina. L is shown in table 5 and fig- 4. From the results revealed that extracts of Impatiens Balsamina. L showed significant activity of protein denaturation (79.24%), which is more activity than standard sodium diclofenac (67.07%). In vitro antidiabetic activity was done by alpha amylase inhibition assay method, the inhibitory effect on amylase by extracts of Impatiens Balsamina. L was shown in table 4 and fig-3.

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Results shows that extracts of Impatiens Balsamina. L has Antidiabetic activity which is compared with acarbose. Table 1: Screening of secondary metabolites. Test for alkaloids; Dragendroff”s test:

++

Hager”s test:

++

Wagner”s test:

++

Mayer”s test:

++

Test for flavanoids; Ferric chloride test:

++

Zinc test:

++

Lead acetate solution test:

++

Test for terpenoids:

++

Test for saponins: Foam test:

++

Test for tanins:

++

Gelatin test:

++

ANTIOXIDANT ACTIVITY Table 2: Reducing power assay and Phospho molybdenum assay:

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Test (µg/ml)

% of Antioxidant activity

100

7.29

(Phosphomolybdenm assay) 53.8

200

16.37

59.6

300

25.90

68.91

400

42.77

70.96

500

74.33

71.43

(Reducing Power assay)

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Figure 1. Reducing power assay and Phospho- molybdenum assay Table 3: DPPH free radical scavenging assay Extract (µg/ml)

% of Inhibition Extract Ascorbic acid

100

1.59

3.4

200

7.53

12.0

300

10.04

21.65

400

17.15

34.4

500

24.98

43.8

Figure 2. DPPH free radical Scavenging activity

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ANTIDIABETIC ACTIVITY Table 4: Inhibition of alpha amylase enzyme Extract Extract (µg/ml)

Amt. of Maltose liberated (µg)

Amylase activity (µmol /ml/min)

30 70 120 170 210

0.017 0.040 0.070 0.099 0.12

100 200 300 400 500

Standard Acarbose Amt. of Amylase Maltose activity(µmol/ml/min) liberated (µg) 35 0.020 130 0.076 270 0.157 400 0.233 440 0.257

Figure 3. Alpha amylase inhibition assay ANTI INFLAMMATORY ACTIVITY Table 5: BSA Denaturation assay

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% of Inhibition Std (Sodium diclofenac)

Extract (µg/ml)

Test

100

16.2

25.04

200

32.2

36.33

300

56.41

46.52

400

65.05

58.16

500

79.24

67.07

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Figure 4. BSA Denaturation activity CONCLUSION Impatiens balsemina.L seeds were collected, extracted and screened for phytochemical analysis. phytochemical analysis of the extract revealed the presence of alkaloid, flavonoid, terpenoid and tannins. The extract of Impatiens balsemina.L was also subjected to antioxidant, anti-inflammatory and antidiabetic activity. From research investigations we observed that the increase in the % of Antioxidant activity. Maximum activity was seen in 500µg/ml concentration. IC50 320 µg/ml was found in DPPH free radical scavenging activity compared to the standard Ascorbic acid with IC50 310µg. Extract was also shown very good anti-inflammatory with IC50 value of Standard and extract is found to be 196 µg/ml and 210 µg respectively. And finally results are infirmed that extract was also shown antidiabetic activity. ACKNOWLEDGEMENT Authors are thankful to the Oxford College of Science for providing laboratory facilities to carry out this research work. REFERENCE 1. Hassawi D, Kharma A. Antimicrobial activity of some medicinal plants against Candida albicans. Jl. Bio. Sci, 2006; 6(1): 109-114. 2. Bu-Li Su, Rong Zeng, Jin-Yin Chen, Chu-Ying Chen, Juan-Hua Guo, and Chang-Gan Huang, Antioxidant and Antimicrobial Properties of Various Solvent Extracts from Impatiens balsamina L. Stems. Jl. Food Sci, 2012; 77(6): C614-619

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