The Novel Source Of Natural Products

Published Date: 02 Nov 2017

Ipsita Mishra, Rachita Gupta & K Suthindhiran

SCHOOL OF BIOSCIENCE AND TECHNOLOGY, VIT UNIVERSITY, VELLORE

TAMIL NADU – 632004, INDIA

[email protected]

Abstract

Seaweeds are the novel source of natural products. Marine seaweeds have many antimicrobial, anticancer, anti-inflammatory and antidiabetic activities. In the present studies, preliminary phytochemical analysis were concluded to determine the presence of alkaloid, carbohydrate, amino acids, proteins, tannins, phenolic compound, oils & Fats and flavonoids in Cladophora rupestris & Ulva linza. In the DPPH scavenging assay, Cladophora rupestris & Ulva linza showed higher inhibition percentage of 78% (±0.22) and & 71% (±0.24) at 5 mg/ml in compared to positive control BHT which showed 63% (±0.29). The IC50 values of methanol extract of Cladophora rupestris & Ulva linza was found to be 1.20 & 2.20 in mg/ml respectively. The further tests such as Minimum inhibitory concentration (MIC) test to determine their growth in minimum concentration against three human & three shrimp pathogenic bacteria. At higher concentration of 1000 µg/ml the growth is inhibited in compared to the minimum concentration of 250 µg /ml. The MIC50 values of Cladophora rupestris on tested pathogens was found to be 357.5, 425, 657.5, 475, 400, 375 in µg/ml respectively. The MIC50 values of Ulva linza on tested pathogens was found to be 312.5, 325, 325, 480, 350, 287.5 in µg /ml respectively. The well diffusion method as done to check the antimicrobial property against the tested pathogens and their zone of inhibition was compared with the zone of inhibition of standard antibiotic Amphicillin.

Key Words: Macro algae, Algal extract, Antioxidant, Antimicrobial, MIC

Introduction:

Antibiotic treatment of bacterial diseases in fish culture has been applied for many years. The occurrence of antibiotic resistant bacteria associated with fish disease is a worldwide problem in aquaculture, which has received considerable attention in the last years and continues to increase due to the absence of a more effective and sager use of antibiotics. The prevention and treatment of these infectious diseases by applying products from marine organisms appears as a possible alternative. Marine organisms are a rich source of structurally novel and biologically active metabolites. There are numerous reports concerning the inhibiting activities from macro algae against human pathogens, fungi and yeasts, but only few contain data about effects against fish pathogens. The aim of the present study is to investigate the antimicrobial activity of seaweed associated marine bacteria against three human and three shrimp pathogenic bacteria. Several macro algae (Ulva, Undaria, Ascophyllum, Porphyra, Sargassum )are widely used in fish diets and there are number of studies into their effects that have been reviewed by (Nakagawa and Montgomary 2007). The chemical composition of macro algae varies with species , physiological status and environmental conditions and they are rich in nonstarch polysaccharides ,vitamins and minerals seaweeds are used in animal food for their mineral contents or for the functional properties of their polysaccharides. Seaweeds are rarely promoted for the nutritional value for their protein (Fleurence ,1999).For most seaweeds aspartic and glutamic acid constitute together a large part of the amino acid fraction . In brown algae these two amino acids can represent between 22 and 44 %of the total amino acid and in green seaweeds up to 26 to 32% , and in red algae 14 to 19% of the total amino acid (Fleurence ,1999).The fatty acid and pigment composition of seaweeds also differ between groups ;brown seaweeds are a better potential source of EPA and DHA than green one (Ackman ,1981). Brown seaweeds generally contain more vitamin C than red and green seaweeds . Marine organisms are rich source of biologically active metabolites (Abou- Elela et al., 2009). Macro algae contains some active compounds that can improve animal resistance against bacterial and viral diseases. Some seaweed species can be co-cultivated with shrimps , resulting in a sustainable alternative to reduce the need for artificial feed .

Materials and Methods:

Sample Collection: The algae was collected on January 2013 between 12-2 pm on the rock surface of the sea shore of Kovallam beach, Chennai (120 47’3" N 800 15’11"E,350 c). After collection sample was kept in the cold room at 70c until washing and drying. Taxonomic identification the species was performed with the aid of the standard literature and determination keys.

Extract Preperation: The seaweeds were rinsed with distilled water and washed 4-5 times for the complete removal of sand and other particles attached to the seaweed. After washing seaweeds were kept for drying. The dried seaweeds were crushed by an electrical grinder and the obtained powder were stored at 70c until the extraction step. The powder (5g) was extracted with methanol for 4 hours at 650c using soxlet apparatus. The obtained extracts were concentrated under vaccum using a rotator evaporator. Residues were then diluted in 2 ml pure methanol. The extracts were kept open for the proper evaporation of the solvent and the slurry remains at the bottom (Sreenivas Rao & Parekh, 1981). The slurry were then lyophilised and kept for further experiments.

Preliminary Phytochemical Analysis : The preliminary phytochemical analysis of the algal extracts was determined. The following tests were performed on the extracts to determine the presence of various phytoconstituents.

Detection of Alkaloids: 5 mg of extract was stirred with few ml of dilute hydrochloric acid and filtered. The filtrate was tested with various alkaloid reagents (Evans., 1997)

Mayer’s Test: Few ml of filtrate, two drops of Mayer’s reagent were added side by side of the test tube. A white or creamy precipitate indicates the test as positive (Evans., 1997)

Wagner’s Test: Few ml of filtrate, few drops of Wagner’s reagent were added by the side of the test tube. A reddish-brown precipitate confirms the test as positive (Wagner., 1993)

Detection of Carbohydrate: 5 mg of extract were dissolved in 100 ml of distilled water and filtered. The filtrate were used for various tests (Ramakrishnan et al., 1994)

Molish’s Test: 1 ml of filtrate, two drops of alcoholic solution of α-naphthol were added, mixture is shaked well and add 0.5 ml of concentrated sulphuric acid is added slowly along the sides of the test tube and allowed to stand. Violet ring indicates the presence of carbohydrates.

Fehling’s Test: 1 ml of filtrate is boiled with 1 ml of Fehling’s solution. Red precipitate indicates the presence of carbohydrates.

Detection of Amino acids and Proteins: 10 mg of extract was dissolved in 1 ml of distilled water and filter through Whatmann filter paper and the filtrate is subjected to tests for amino acids and proteins (Fisher., 1968; Ruthmann., 1970)

Ninhydrin Test: Two drops of ninhydrin solution (1 mg ninhydrin in 20 ml acetone) were added to two ml of aqueous filtrate. Purple colour indicates the presence of amino acids.

Biuret Test: 2 ml of filtrate is mixed with 1 drop of 2% copper sulphate solution. Add 1 ml of 95% ethanol, followed by potassium hydroxide pellets. Pink colour indicates the presence of proteins (Gahan., 1984)

Detection of Phenolic compounds: 10 mg of extract was dissolved in 1 ml of distilled water and filtered.

Ferric chloride Test: Few drops of filtrate were mixed with 5% ferric chloride solution. Dark green colour indicates the presence of phenolic compounds (Mace., 1963).

Lead acetate Test: 1 ml of filtrate, add 3 ml of 10% lead acetate solution were added. A white precipitate indicates the presence of phenolic compound.

Detection of Oils & Fats: Few drops of 0.5 N alcoholic potassium hydroxide solution were added to 3 mg of extract with a drop of phenolphthalein. The mixture is heated on waterbath for 2 hours. Formation of soap indicates the presence of oils & fats (Kokate., 1999).

Detection of Flavonoids: 2 mg of extract is mixed 10 ml of ethyl acetate. Boiled in water bath and cooled at room temperature. The layers are separated and the red colour of ammonia was noted (Francis., 2007).

Detection of Saponins: 10 mg of extract is mixed with distilled water and made upto 10 ml. The mixture was shaken for 15 minutes. A layer of foam indicates the presence of saponins (Kokate., 1999).

Detection of Phytosterols: 5 mg of extract is dissolved in 1 ml of acetic anhydride. Add 1-2 drops of concentrated hydrochloric acid are added slowly along the sides of the test tube. A change of colours showed the presence of phytosterols (Finar., 1986).

Detection of Glycosides: 5 ml of 50% hydrochloric acid is mixed with 1 mg of extract. The mixture was heated for 5 minutes in waterbath. Add 5 ml of Fehling’s solution and boiled for 10 minutes in waterbath. A brick red precipitate indicates the presence of glycosides (Francis., 2007).

Detection of Tannins: 2 drops of 5% ferric chloride solution was added to 1 mg of extract. Dark green indicates the presence of Tannins (Francis., 2007).

Antioxidant Assay: The antioxidant activity of the seaweed were studied by their ability to scavenging the free radicles using 2,2-Diphenyl-1-Picryl hydroxyl (DPPH) reducing power (Jayashri et al., 2008).

Preperation of Positive Control: The synthetic antioxidant Butylated Hydroxy Toluene (BHT) was used positive control at a concentration of 2 mg/2 ml (Jayashri et al., 2008)

Preperation of Test Extracts: 1 mg of extract was dissolved in 1 ml of methanol. Mix the solution and make different concentration viz. 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml & 5 mg/ml.

Preperation of DPPH (2,2-Diphenyl-1-Picryl Hydrazyl): 11.8 mg of DPPH was dissolved in 100 ml of methanol (0.3 mM concentration). The content was kept in dark condition because DPPH is light sensitive (Jayashri et al., 2008)

DPPH Free Radical Scavenging Assay: The free radical scavenging activity of seaweed extract was measured by DPPH proposed by Hatano et al.,1988. Percentage inhibition or DPPH scavenging activity was calculated by following expression.

Percentage of scavenging = [(A0 – A1)/ A0] X 100

Where, A0 = Absorbance

A1 = Absorbance of sample

The samples were kept in the dark for 30 minutes and the optical density was measured at 517 nm where as positive control = 250 µl BHT + 250 µl DPPH + 2.5 ml methanol, control= 250 µl DPPH+ 2.5 ml methanol and test sample = 250 µl sample +250 µl DPPH +2.5 ml methanol.

Antimicrobial Activity: Three shrimp bacterial pathogens and three human bacterial pathogens were collected from VIT University laboratory, Tamil Nadu. Thus the antimicrobial activity of the crude extract of Cladophora rupestris & Ulva linza were determined by measuring the zone of inhibition in the agar well diffusion method. The results were compared with the zone of inhibition of standard antibiotic Amphicillin.

Minimum Inhibitory Concentration (MIC): Minimum inhibitory concentration of all the pathogens were determined by inoculating the algal extracts of various concentration viz. 1000 µg/ml, 750 µg/ml, 500 µg/ml and 250 µg/ml along with the pathogens and kept for incubation for 48 hours. The optical density was measured at 540 nm.

Results and Discussion

Preliminary Phytochemical Analysis: The preliminary Phytochemical Analysis of both the marine algae contain Alkaloid, Carbohydrate, Amino acids & proteins, Phenolics, Oils & fats, Flavonoids, Saponins, Phytosterols, Glycosides and Tannins, but in Cladophora rupestris & Ulva Linza Saponins and Glycosides were absent.

Table 1: Preliminary Phytochemical analysis of Cladophora rupestris and U .Linza

SL. NO

TESTS

Cladophora rupestris

Ulva Linza

1

ALKALOIDS

+

+

2

CARBOHYDRATE

+

+

3

AMINO ACID & PROTEIN

-

+

4

PHENOLIC COMPOUND

+

+

5

OILS & FATS

+

+

6

FLAVONOIDS

+

+

7

SAPONINS

-

_

8

PHYTOSTEROLS

+

_

9

GLYCOSIDES

-

_

10

TANNINS

+

+

Antioxidant Assay

DPPH Scavening Assay: In the present DPPH study of seaweeds extracts, the concentrations of the algal samples are 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml & 5 mg/ml respectively. Scavenging capacity increased with the increased concentration. DPPH assay showed significant difference when compared with the positive control. DPPH assay show highest inhibition of 78% (0.22) in Cladophora rupestris & 72% (0.24) in Ulva linza in 5 mg/ml respectively.

Figure : Inhibition percentage of crude of methanol extracts n scavenging of DPPH

DPPH is a relatively a free radical. DPPH reacts with suitable reducing agents, the electrons paired off and the solution losses its colour depend upon the number of electrons taken off. The assay provides the result on reaction with the test samples. In Cladophora rupestris the highest inhibition percentage is 78% (±0.22) and in Ulva linza the highest inhibition percentage is 72% (±0.24) in compared to positive control which had a inhibition of 63% (±0.29). The IC50 values of Cladophora rupestris & Ulva linza was found to be 1.20 & 2.20 in mg/ml respectively.

Well diffusion method: The antimicrobial activity of methanol extract of both the macroalgae is determined with three human pathogenic bacterial strains viz. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and three shrimp pathogenic bacterial strains viz Vibrio harveyii, Vibrio parahaemolyticus ,Vibrio alginolyticus and the zone of inhibition were compared with the zone of inhibition of the standard antibiotic Amphicillin.

Pathogenic Bacteria

Zone of inhibition (mm)

250 µg/ml

500 µg/ml

1000 µg/ml

Ampicillin

Escherichia coli

8.3

13.9

15.9

17.3

Pseudomonas aeruginosa

9.1

12.5

15.3

16.9

Staphylococcus aureus

10.1

13.3

16.3

18.2

Vibrio harveyii

11.3

13.4

15.1

17.5

Vibrio parahaemolyticus

9.8

10.4

11.3

15.3

Vibrio alginolyticus

7.8

10.9

11.7

16.2

Table 2: Antimicrobial activity of Cladophora rupestris against human pathogenic bacteria and shrimp pathogenic bacteria

Pathogenic Bacteria

Zone of inhibition (mm)

250 µg/ml

500 µg/ml

1000 µg/ml

Ampicillin

Escherichia coli

13.9

15.9

17.3

Pseudomonas aeruginosa

9.1

12.5

15.3

16.9

Staphylococcus aureus

10.1

13.3

16.3

18.2

Vibrio harveyii

11.3

13.4

15.1

17.5

Vibrio parahaemolyticus

9.8

10.4

11.3

15.3

Vibrio alginolyticus

7.8

10.9

11.7

16.2

Table 3: Antimicrobial activity of Ulva linza against human pathogenic bacteria and shrimp pathogenic bacteria

In the above Table 1&2 the methanol extract of Cladophora rupestris showed good antimicrobial activity in human and shrimp pathogenic bacteria. The diameter of the zone of inhibition was larger in higher concentration (1000 µg/ml) in compared to lower concentration (250 µg /ml).In table 2 Vibrio harveyii shows more zone of inhibition than Escherichia coli in 250 µg/ml.

Minimum Inhibition Concentration (MIC) Test: The minimum inhibitory concentration (MIC) test of three human pathogenic bacterial strains and three shrimp pathogenic bacterial strains with the crude methanol extract of both the algae to determine the growth at minimum concentration. At minimum concentration of 250 µg/ml showed better growth in compared to the highest concentration of 1000 µg/ml. Bacterial growth inhibited by increasing the concentration.

Figure 3: Minimum Inhibition Concentration of Cladophora rupestris against the tested pathogens

Figure : Minimum Inhibition Concentration of Ulva linza against the tested pathogens

The higher the concentration of the algal extract the less will be the growth of all the pathogenic bacteria which means at higher concentration the algal extract inhibit the growth of the pathogenic bacteria. In the above figure at 250 µg/ ml and 700 µg/ ml th growth of pathogens less inhibited than 1000 µg/ml. All the pathogens are inhibited at 1000 µg/ml. The MIC50 value of methanol extract was found for E.coli(375.5), P.auregenosa(425), S.aureus(357.5) V.harveyii (475), V.parahaemolyticus (400) & V.alginolyticus (375) in µg/ml respectively for Cladophora rupestris .The MIC50 value of methanol extract for Ulva linza was found for E.coli (312.5), P.auregenosa (375), S.aureus (325), V.harveyii (450), V.parahaemolyticus (350) & V.alginolyticus (286.5) in µg/ml respectively

Conclusion

Marine seaweed are the novel source of natural products. In our present studies we can concluded that marine algae Cladophora rupestris had showed good inhibitory results in antioxidant and antidiabetic inhibition assays. Therefore it can be used in dietry supplements and in therapeutic use for the treatment of diabetes.

Acknowledgement

We thank VIT University for providing necessary facilities to carry out our research work.