International Journal of Life science and Pharma Reviews (IJLPR)  
   
 
International Journal of Life science and Pharma Research (IJLPR)
Life Science
Volume 10 Issue 4, October - November 2019    Pages:36-40
Phytochemical Screening and Evaluation of Antidiabetic Activity of the Marine Microalgae: Nannochloropsis Sp.

Deepa P. K, Subramanian A and Manjusha W.A.
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DOI: http://dx.doi.org/10.22376/ijpbs/lpr.2020.10.4.L36-40
Abstract:
Diabetes mellitus is a metabolic disorder and is one of the major challenges on health of people all over the world. The phytochemicals of various marine algae are found to possess antioxidant, anti-inflammatory and anti-diabetic properties. Microalgae are photosynthetic organisms that produce oxygen and are capable of producing beneficial secondary metabolites. The bioactive metabolites like pigments, phenolics, amino acids, polyunsaturated fatty acids etc. produced by them have a positive effect on human health. In the present investigation, the marine microalgae Nannochloropsis sp. was screened for phytochemicals qualitatively and quantitatively. The evaluation of antidiabetic activity was determined against the enzymes such as alpha amylase and alpha glucosidase. The extracts were prepared in five different solvents such as petroleum ether, aqueous, isopropanol, methanol and ethyl acetate. The qualitative analysis for phytochemicals showed the presence of alkaloids, phenols, flavonoids, carbohydrates, proteins, cardiac glycosides, sterols, coumarin and tannins. The maximum total phenolic, flavonoid and sterol content was present in ethyl acetate extract of Nannochloropsis sp. and the values are found to be 42.17 ± 0.12mg GAE/g, 60.23 ± 0.09 mg QE/g, 55.34 ± 0.07 mg cholesterol/g of extract respectively. The ethyl acetate extract showed highest inhibitory effect of 78.52 ± 0.56 % (IC50 121.96 μg/ml) on α-amylase and 80.42 ± 0.13 % (IC 50 178.53μg/ml) on α-glucosidase enzyme at a concentration of at 1mg/ml which implies the microalgae possess anti-diabetic property. The results indicate that the phytochemicals present in the microalgae contribute the antidiabetic activity. Hence Nannochloropsis sp. can be applied in pharmaceuticals for the development of antidiabetic drugs which have the benefits over side effects by the use of synthetic drugs. (These 3 lines are in different font style- Change it)
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1.    INTRODUCTION

Diabetes mellitus is one of the most prevalent diseases among the world. It is due to lack of insulin secretion (relative and absolute) and insulin resistance, which upsets fat, protein and carbohydrate metabolism and genetic factor may be one of the causes. One of the major symptoms is hyperglycemia and major complications include hypertension, blindness, and kidney failure and so on. It is a metabolic disorder which has induced substantial morbidity and mortality due to micro vascular (neuropathy, retinopathy and nephropathy) and macrovascular (stroke, heart attack and peripheral vascular disease) complication.1 Microalgae are eukaryotic photosynthetic organisms that account for approximately 40 % of the global photosynthesis and play a key role in aquatic ecosystems.2 Diverse varieties of functional metabolites are produced by them. They are rich sources of phenols, flavonoids and pigments which have several roles in health ailments.  Nowadays researchers show interest on algae because of its ability to produce several beneficial natural products used as anti-inflammatory, antimicrobial, antimalarial, antiproliferative, and anticancer agents.3 Several microalgae are reported to have antidiabetic property.4 Hence the present study was aimed to screen the phytochemicals and to evaluate the antidiabetic property of marine microalgae, Nannochloropsis sp.
 
2.    MATERIALS AND METHODS

10 gms of dried microalgae cells were extracted in 100ml of different organic solvents specifically ethyl acetate, methanol, aqueous, isopropanol and petroleum ether for 7 days at room temperature. The solution was filtered through Whatman No.1 filter paper. The crude extract was kept in a sealed container and stored in a refrigerator for further studies.

2.1    Phytochemical screening 

The Nannochloropsis sp. extracts of petroleum ether (PN), Aqueous (AN), Isopropanol (IN), Methanol (MN), and Ethyl acetate (EN) were subjected to phytochemical analysis for the presence of bioactive constituents. 5

2.2    Quantitative analysis of Phytochemicals

2.2.1    Estimation of total phenol content 

Various solvent extract (0.5g) was extracted in 10 ml of 80% ethanol. Supernatant was evaporated to dryness and the extract was dissolved in 5 ml water. Different aliquots 0.1-1 ml was taken and the final volume was made to 3 ml with water. Folin’s reagent of 0.5ml was added followed by 2ml of 20% Na2CO3.(2 and 3 to be written subscript) Tubes were vortexed and kept in boiling water for one minute. The tubes were cooled and measured the absorbance at 650nm against blank. The standard curve of gallic acid was obtained using the same procedure and the results were expressed as gallic acid equivalent GAE/g dry weight of microalgae . 6

2.2.2    Estimation of total flavonoid content    

Total flavonoid content was determined by aluminium chloride colorimetric method was used with some modifications. Various solvent extract (1ml) was added to 3 ml of methanol followed by 0.2 ml of 10% aluminium chloride, 0.2 ml of 1 M potassium acetate and 5.6 ml of distilled water and was incubated at room temperature for 30 minutes. The absorbance was measured at 420 nm. Quercetin was used as standard (1 mg/ml). Flavonoid contents were determined from the standard curve and were expressed as quercetin equivalent (mg/g of extracted compound). 7

2.2.3    Estimation of steroid 

A solution of 2.5 g (ml) of various solvent extract was prepared in 50 ml of distilled water after vigorous shaking for 1 hour. From that, 2ml of extract solution was washed with 3 ml of   0.1 M NaOH (pH 9) and with 2 ml of chloroform was added and 3 ml of ice-cold acetic anhydride followed by adding two drops of concentrated H2SO4 cautiously. Cholesterol was taken as blank. The absorbance was measured spectrophotometrically at 420 nm. 8

2.3    Antidiabetic Activity 

2.3.1    Inhibition of alpha amylase enzyme 

A starch solution of 0.1% w/v was prepared by stirring 0.1 g of potato starch in 100 ml of 16 mM of sodium acetate buffer. The enzyme solution was prepared by mixing 27.5 mg of alpha amylase in 100 ml of distilled water. The colorimetric reagent was prepared by mixing sodium potassium tartrate solution and 3, 5 di nitro salicylic acid solution (96 mM). The various concentrations of the algal extract (100 to 1000 μg/mL) were added to 1 ml of starch solution and left for 10 min. Further, the reaction was initiated by the addition of the enzyme solution and allowed to react for 10 min under alkaline condition at 25ºC. Finally, the reaction was terminated by adding 1 mL of calorimetric reagent and then incubated in a boiling water bath for 5 min, cooled to room temperature. The reaction mixture was then diluted after adding 10 ml distilled water and absorbance was measured at 540 nm. Control represent 100% enzyme activity and were conducted in a similar way by replacing algal extract with DMSO. A similar experiment was conducted with the standard drug Acarbose and the experiments were conducted in triplicate. 9 

2.3.2    Inhibition of alpha-glucosidase enzyme 

To various concentrations of algal extract, 1ml of starch substrate (2% w/v maltose or sucrose) and 1 ml with 0.2 M Tris buffer pH 8.0 were added and incubated it at 37ºC for 5 minutes.  Then 1 ml of alpha glucosidase enzyme (IU/ml) was added followed by incubation for 40 minutes at 37ºC. The reaction was terminated by the addition of 2 ml of 6N HCl. The intensity of colour was measured at 540 nm. Control experiment was done by replacing the extract with DMSO. 10 Percentage of inhibition was calculated by using the following formulae, 


3.    STATISTICAL ANALYSIS

The data were subjected to two way and one-way ANOVA using statistics software package (SPSS, ver.22) to analyze the statistical significance. The data were analyzed and expressed as means ± SD. The IC50 values were calculated from linear regression analysis.

4.     RESULTS AND DISCUSSION

4.1    Phytochemical screening

Various solvent extracts of Nannochloropsis sp. were screened for phytochemicals and are shown in table 1.
 

Table 1: Phytochemical screening of various solvent extracts of Nannochloropsis sp.

Test

PN

AN

IN

MN

EN

Test for alkaloids

(Mayer’s test)

-

-

-

++

+

Test for phenol

(Ferric chloride test)

-

-

+

-

+++

Test for flavonoids (Shinoda test)

-

-

-

+

++

Test for carbohydrates (Benedict’s test)

-

-

-

+++

++

Test for protein

(Xanthoproteic test)

+

-

+++

+

++

Test for sterols

(Liebermann Burchard’s test)

-

-

-

+

++

Test for cardiac glycosides

(Keller–Kiliani test)

-

-

+++

-

++

Test for coumarin

-

-

-

++

+

Test for tannin

-

-

+

+++

+

 +++: highly present, ++: moderately present: +: trace amount *Abbreviation to be given for PN, AN, IN, MN and EN 
for this tabular column) is given below the materials and methods: phytochemical screening

 4.2    Quantitative analysis of phytochemicals

The qualitative analysis of phytochemicals showed the presence of most of the analyzed phytochemicals. Of them total phenol, flavonoid and sterols were quantified. The total phenolic content of EN and IN was found to be 42.17 ± 0.12mg GAE/g and 11.64 ± 0.06 mg GAE/ g of extract respectively. The flavonoid content was found to be 60.23 ± 0.09 mg QE/ g in EN and 14.13 ± 0.21 mg QE/ g of extract in MN. The Sterol content was found to be 55.34 ± 0.07 mg cholesterol/g and 12.31 ± 0.12 mg cholesterol/g of extract in EN and MN accordingly. All experiments were performed in triplicates and the results were expressed as mean ± SD. These phytochemicals are well known sources of antioxidants. Many studies proved that flavonoids and phenols are capable of scavenging activity. Various phenolic compounds were reported to inhibit in vitro α-glucosidase activity and thereby diabetes mellitus can be controlled. Flavonoids are a family of polyphenolic compounds which are found to possess antihyperglycemic activity.11 

4.3    Anti-diabetic activity

Diabetes is defined as a state imbalance in homeostasis of carbohydrate and lipid metabolism due to improper regulation by pancreatic hormone, leading to hyperglycemia. The regulation of enzymes such as alpha amylase and alpha glucosidase is one of the major tasks involved in the treatment of Diabetes.

4.3.1    Inhibition of alpha amylase activity

α-amylase is one of the key enzymes that breaks down starch to simpler sugars and increase the absorption rate of glucose. The highest level of inhibition was exhibited by EN of about 78.52 ± 0.56 % at 1000 μg/ml and the IC50 was found to be 121.96 μg/ml. The inhibitory effect of acarbose was found to be 98.10 ± 0.10 % with an IC50 of 103.69 μg/ml. The inhibition level was found to be 3.89 ± .08 to 78.52 ± 0.56 % on varying extract at different concentrations. The dose dependent activity of various extract against alpha amylase is shown in figure 1. Variance among various levels of extract was P<0.01 is statistically significant.  Maintenance of blood glucose level near normal during fasting as well as postprandial state is one of the major goals involved in the treatment of diabetes mellitus.12 These α-amylase inhibitors inhibit the action of α-amylase enzyme leading to a reduction in starch hydrolysis which shows beneficial effects on glycemic index control in diabetic patients.13The inhibition of these enzyme is very important to regulate the release of glucose during digestion. Sangeetha et al found methanolic extract of Nannochloropsis oculata is a potent inhibitor of alpha amylase with the IC50 of 762.613 μg/ml.14 The EN, in the present study exhibited better result with a significant inhibitory effect. 
 

Fig 1.Inhibition of alpha amylase

 4.3.2    Inhibition of alpha-glucosidase activity 

In the present study, EN exhibited a significant inhibitory action on α-glucosidase enzyme followed by MN, IS, AN and PN. The EN, at the concentration of 1000 μg/ml exhibited α-glucosidase inhibitory activity of 80.42 ± 0.13 % with an IC50 of 178.53 μg/ml when compared to the remaining extracts. The inhibition level was found to be 12.02 ± 0.09 to 80.42 ± 0.13%. The standard drug acarbose showed an inhibition of 97.81 ± 0.04 % and the IC50 was found to be 103.69 μg/mL at the highest concentration. The dose dependent activity of various extract against alpha glucosidase is shown in figure 2. Variance among various levels of extract was found to be statistically significant (P<0.01). Alpha glucosidase breaks down starch and disaccharides to glucose.15 Inhibition of the enzyme is the one of the strategies to avoid hyperglycemia. The Nannochloropsis sp. present study showed the higher α- glucosidase inhibition when compared to the study reported by Sangeetha et al, in which methanolic extract of Nannochloropsis oculata showed an IC50 of 613.72 µg/ml.14
 

Fig 2. Inhibition of alpha-glucosidase

 5.     CONCLUSION

The marine green microalgae, Nannochloropsis sp. was found to contain various phytochemicals. The major compounds with bioactive potential such as phenols, flavonoids and sterols were present in significant quantity. The ethyl acetate extract of the microalgae possesses effective inhibitory activity on alpha amylase and alpha glucosidase enzyme which implies that the extract has antidiabetic effect. The phytochemicals present in the microalgae delivers the antidiabetic property and hence the Nannochloropsis sp. can be explored in pharmaceutical industry for the development of antidiabetic drug. 

6.    AUTHORS CONTRIBUTION STATEMENT

Professor Dr. A Subramanian gives the guidelines for the research study and Deepa. P.K. carried out the research work. Dr. W.A. Manjusha evaluated the results and supported in manuscript preparation.

7.    CONFLICT OF INTEREST

Conflict of interest declared none.

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Keywords: marine microalgae, Nannochloropsis sp., phytochemicals, antidiabetic, bioactive compounds
 
 
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