Chapter 7

PHYTOCHEMICAL STUDY ANDANTIOXIDANT ACTIVITY OF

Euglena tuba

PHYTOCHEMICAL STUDY AND ANTIOXIDANT ACTIVITY OF

Euglena tuba

7.1 Introduction

In recent years, there has been an upsurge of interests toward the field of free radical

biochemistry. Free radicals - reactive oxygen species (ROS) and reactive nitrogen species

(RNS) are generated in our body by various endogenous pathway, exposure to different

physiochemical conditions or pathological states. A balance between free radicals and

antioxidants is therefore essential for proper physiological function. Excess free radicals

beyond the body's ability to regulate them lead to a condition known as oxidative stress.

Free radicals thus adversely alter lipids, proteins, and DNA and trigger a number of

human diseases. External application of antioxidants can provide from this oxidative

stress. Synthetic antioxidants such as butylated hydroxytoluene and butylated

hydroxyanisole have recently been reported to be dangerous for human health. Search for

effective, nontoxic natural compounds with antioxidant activity has been intensified in

recent years (Lobo et al., 2010).

Under normal conditions, free radicals are produced through biological activities which

are scavenged by various enzymes and antioxidants present in the organisms. When the

critical balance between free radical generation and antioxidant defences is unfavourable,

this may lead to some biological basis of several acute diseases. Tissue injury after

ischemia, chronic conditions such as artheriosclerosis and cancer may be prevalent under

these circumstances (Rock et al., 1996). Various antioxidants ( tocopherols, ascorbic

-carotene etc.) in modifying the risk for conditions which may result from

oxidative stress has stimulated research efforts about their bio-availability and synthesis

in the cells (Kamat et al., 1997 ;Palozza and Krinsky 1992). Tocopherols (vitamin E) are

usually fat-soluble and its derivatives are widespread in nature. Their occurrence range

from photosynthetic green leaves to non-photosynthetic seedling tissues of higher plants

and algae (Green 1958 ;Kruk & Strzalka 1995). Among microorganisms, tocopherols

are only available in unicellular algae like Dunaliella (Abalde et al., 1991), Chlorella,

Chlamydomonas ( all Chlorophyceae ), Ochromonas ( Chrysophyceae ) ( Taketomi et al.,

1983 ) and Euglena ( Euglenophyceae ) ( Hughes and Tove 1982).These organisms have

attracted immense attention for their significant antioxidant properties. Interestingly ,

Euglena, in particular, was screened by Takeyama et al. (1997) who reported for their

simultaneous production of more than a single antio -carotene,

vitamins C and vitamin E , rendering it a promising dietary supplement. The use of

natural antioxidants in food, cosmetic, and therapeutic industry are fast emerging as

promising alternative for synthetic antioxidants in respect of low cost, highly compatible

with dietary intake and almost zero side effects in the human body. Many antioxidant

compounds, naturally occurring in plant sources have been identified as free radical or

active oxygen scavengers. Antioxidant potential of a wide variety of vegetables like

potato, spinach, tomatoes, and legumes have been documented. Several reports addressed

antioxidant potential of fruits. Rather strong antioxidants properties were observed in

berries, cherries, citrus, prunes, and olives. Green and black teas containing upto 30% of

the phenolic compounds(dry weight) also have been demonstrated for antioxidant

properties (Lobo, 2010; Docampo, 1995; Corpas, 2001).

During our bloom exploration study in Barak valley ( Assam ), we have come across a

species of Euglena identified as Euglena tuba contributing a substantial amount of

biomass to the aquatic bodies of the region. The alga exhibit a green colouration to the

aquatic bodies in the morning which gradually changes to orange to deep brick red

during noon time and revert to green colour in the afternoon to evening time ( vide infra:

Chapter 5 ). Local fishermen opine the alga to be one of the important components of the

pond ecosystem and many indigenous fishes feeds on this alga. This alga, being non-

toxic, do not appear to pose any threat to the health of the ecosystem in general and

organisms consuming this alga in particular thus presenting a interesting case for

exploring the value added products from this alga.

Keeping in mind the algal biomass formation, its availability in plenty and in light of

other species of Euglena documented for their potential antioxidant compounds, the

work discussed in this Chapter involves screening of the phytochemicals and assessment

of the total antioxidant property of this indigenous algal species from this region of

Southern Assam.

Material and Methods

The Euglena algal sample was collected from the Cachar district of Assam, India and

authenticated as described in Chapter 3. The fresh algal sample was dried at room

temperature for 7 days, finely powdered and used for extraction. The powder (100 g) was

mixed with 1000 ml methanol:water (7:3) at 37°C for overnight using shaker incubator,

then the mixture was centrifuged at 2850 × g and the supernatant was decanted. The

pellet was mixed again with 1000 ml methanol-water and the entire extraction process

was repeated. The supernatants collected from the two phases were mixed in a round

bottom flask and concentrated under reduced pressure in a rotary evaporator. The

concentrated extract was then lyophilized. The residue was kept at -20°C for future use.

The details of the methodology followed for various analyses of phytochemicals

constituents and antioxidant activity of 70% methanolic extract of Euglena tuba samples

(ESME) alongwith the statistical details has already been discussed in Chapter 3.4.2.8.

Results and Discussion

Phytochemical analysis

The phytochemical screenings of chemical constituents related to biological activity of

the plant extract were summarized in the Table-7.1. The results revealed the presence of

biologically active compounds such as alkaloids, carbohydrates, flavonoids, phenols,

saponins, tannins, terpinoids. The quantitative phytochemical estimation indicate that the

extract contains significant amount of phenolics, flavonoids, carbohydrates, tannins,

ascorbic acids and alkaloids (Table 7.2) which confirms its potent scavenging property.

From Table 7.2 it was found that the extract contains highest amount of flavonoids

(100.78 ± 2.114 mg/100mg extract quercetin equivalent) than the other phytochemicals

tested quantitatively. It also contain considerable amount of alkaloids (2.95 ± 0.3 mg/100

mg extract reserpine equivalent), phenolics (11.15 ± 0.001 mg/100 mg extract gallic acid

equivalent), tannins (5.6 ± 0.0472 mg/100 mg extract catechin equivalent), reduced

carbohydrates (9.99 ± 0.022 mg/100 mg extract glucose equivalent) and insignificant

amount of ascorbic acid content (2.16 ± 0.059 mg/100 mg extract ascorbic acid

equivalent).

Table 7.1: Qualitative analysis of phytochemicals of Euglena

Phytochemicals 70% MeOH

extract

Alkaloid +

Carbohydrate +++

Flavonoid +++

Glycoside -

Phenol ++

Saponin +++

Tannin ++

Terpinoids +++

Anthraquinone -

Table 7.2: Quantitative estimation of some phytochemicals

Phytochemicals Amount Present (mg/100 mg extract)

Total phenolics 11.15 ± 0.001 (gallic acid equivalent)

Total flavonoids 100.78 ± 2.114 (quercetin equivalent)

Carbohydtrates 9.99 ± 0.022 (glucose equivalent)

Ascorbic acid 2.16 ± 0.059 (ascorbic acid equivalent)

Alkaloids 2.95 ± 0.3 (reserpine equivalent)

Tannins 5.6 ± 0.0472 (catechin equivalent)

Data are expressed as mean ± S.D, (n=6).

Total antioxidant capacity

The overall antioxidant activity of the extract was measured by using TEAC assay which

is based on interaction between antioxidant and ABTS.+ radical cation which has a

characteristic blue colored showing maxima at 734 nm. Interaction with the extract or

standard trolox suppressed the absorbance of the ABTS.+ radical cation in a dose

dependant manner and the results, expressed as percentage inhibition of absorbance, are

shown in figure 1(a) and figure 1(b), respectively. The TEAC value of the Euglena

extract was 0.202± 0.001 which demonstrates that the extracts possess convincing anti-

oxidant property (Table-7.3).

Fig. 7.1. Total antioxidant activity of ESME and reference compound trolox on ABTS

radical cation decolorization assay. ( mean ± S.D. , n=6).

Correlation of antioxidant activity with phenolic and flavonoid contents

ABTS with high correlation were established between total phenolics contents, total

flavonoids and total antioxidant activity (TEAC) assay. As showed in Figure 7.2(a), the

total phenolic content of Euglena significantly correlated with antioxidant activity

(correlation coefficient R = 0.9881), which proved that the phenolic content of this plant

is attributed to their antioxidant activity. The correlation coefficient of Euglena for

flavonoid content with its antioxidant capacity was highly significant (correlation

coefficient R = 0.9486), correlated with their antioxidant activity (Figure 2(b)).

(a) (b)

Fig. 7.2: Correlation of total antioxidant activity with phenolic and flavonoid contents.

The correlation analyses were described as linear correlation coefficient (R).

DPPH radical scavenging assay

The extracts of Euglena showed excellent dose-dependent scavenging activity of DPPH

radical (Fig-7.3) quite similar to the standard ascorbic acid. The IC50 values (Table 7.3)

of the extract and standard ascorbic acid were 146.07± 1.80

respectively.

Fig. 7.3 - DPPH radical scavenging activity of Euglena and standard ascorbic acid. Each

value represents mean ± S.D. (n=6). ***p < 0.001 vs 0 µg/ml.

Hydroxyl radicals scavenging activity

The abilities of the extract and standard mannitol to inhibit hydroxyl radical-mediated

deoxyribose degradation in an Fe3+-EDTA-ascorbic acid and H2O2 reaction mixture was

exhibited by this assay. The results of hydroxyl radical-scavenging powers of the extract

and standard mannitol were shown in Figure 4. The IC50 values (Table 7.3) of the extract

and standard mannitol were 41.89 ± 0.41 571.45 ± 20.12

The results, as can be found from Figure 7.4 and Table 7.3, indicate that the extract is

more effective hydroxyl radical scavenger than standard mannitol.

Fig. 7.4. Hydroxyl radical scavenging activity of the Euglena w.r.t. mannitol. (% of

inhibition of deoxyribose degradation, mean ± S.D. (n=6). ***p < 0.001 vs 0 µg/ml.)

Superoxide radical scavenging assay

The decrease in absorbance at 560 nm with the plant extract and the reference compound

quercetin indicate their abilities to quench superoxide radicals in the reaction mixture

(Figure 7.5). The IC50 values of the extract and quercetin on superoxide scavenging

activity were 5.83± 0.07 µg/ml and 42.06 ± 1.35 µg/ml respectively (Table 7.3). The

result indicates that the extract is more potent superoxide radical scavenger than standard

compound quercetin.

Fig. 7.5: Scavenging effect of Euglena extract and standard quercetin on superoxide

Nitric oxide radical scavenging

The extract inhibits the nitrite formation by directly competing with oxygen in the

reaction with nitric oxide. Figure 7.6 shows the percentage inhibition of nitric oxide

radical generation by the extract compared to standard curcumin. The extract of Euglena

sp has been found to be efficient (IC50= 278.46 ± 15.02 µg/ml) in scavenging nitric oxide

in dose dependent manner and is not as potent in scavenging nitric oxide as standard

curcumin (IC50= 90.82 ± 4.75 µg/ml).

Fig. 7.6. The nitric oxide radical scavenging activity of Euglena extract and standard

curcumin. ( % of nitric oxide inhibition). ( mean ± S.D. (n=6). ***p < 0.001 vs 0 µg/ml).

Hydrogen peroxide scavenging

Figure 7.7 shows that the plant extract is mediocre scavenger of H2O2 compared to

standard sodium pyruvate. The extracts were capable of scavenging hydrogen peroxide in

a concentration-dependent manner. The IC50 for scavenging of H2O2 were 47.34± 5.05

mg/ml, 3.24±0.30 mg/ml for the extract and sodium pyruvate respectively (Table 7.3).

Scavenging of H2O2 by extracts may be attributed to their polyphenolics, which can

donate electrons to H2O2, thus neutralizing it to water.

Fig. 7.7: H2O2 scavenging assay of Euglena extract and sodium pyruvate. (mean ± S.D.,

n = 6). *p < 0.05, **p < 0.01 and ***p < 0.001 vs 0 µg/ml.

Peroxynitrite scavenging

When different amounts of extract and standard reference compound gallic acid were

added to the reaction mixture, ONOO2 -mediated oxidising activity was inhibited

markedly in a concentration-dependent manner, as shown in Figure 7.8. The calculated

IC50 values for the extract was 2.821 ± 1.69 mg/ml, in comparison to that of the reference

compound gallic acid (IC50 =0.876 ± 0.057 mg/ml) (Table 3) indicating that the samples

are not as potent scavenger of peroxynitrite as gallic acid.

Fig. 7.8: Peroxynitrite anion scavenging assay of Euglena extract and the standard gallic

acid. .)

Singlet oxygen (1O2) scavenging

Fig.7.9 illustrates the percentage of scavenging of singlet oxygen by the extract and

standard lipoic acid. The IC50 value of the extract were found to be 0.879 ± 0.29 mg/ml

whereas, 50% scavenging of the standard lipoic acid was found to be 0.046 ± 0.01 mg/ml

(Table 3).

Fig. 7.9. Effect of Euglena extract and standard lipoic acid on the scavenging of singlet

oxygen. ( mean ± S.D. of six parallel measurements. ***p < 0.001 vs 0 µg/ml).

Hypochlorous acid (HOCl) scavenging

Dose-dependent hypochlorous acid scavenging activity of Euglena extract and standard

ascorbic acid was found in this study (Figure 7.10). The IC50 values (Table-7.3) of extract

and standard were found to be

Fig. 7.10. Hypochlorous acid scavenging activity of Euglena extract and standard

ascorbic acid. ( mean ± S.D. (n=6). ***p < 0.001 vs 0 µg/ml.)

Reducing power Assay

In the reducing power assay, the presence of antioxidants in the samples would result in

the reducing of Fe3+ to Fe2+ by donating an electron. As illustrated in Figure 7.11, from

Fe3+ to Fe2+ transformation in the presence of the extract and reference ascorbic acid was

performed to measure the reductive capability and found increasing in showing reduction

ability in a dose dependent manner, with increasing concentrations. Although the activity

of ascorbic acid was better than the sample with absorbance values of 0.47 and 0.053 for

the reference and sample, respectively, the sample showed low reducing capability. The

reducing capacity of a compound may serve as a significant indicator of its potential

antioxidant activity.

Fig. 7.11. The reductive ability of the extract and standard ascorbic acid. (mean ± S.D.

(n=6). ***p <0.001 vs 0 mg/ml).

Inhibition of Lipid peroxidation Assay

Lipo-peroxidation inhibitory effect of the extract can be indirectly symbolized as the

antioxidant capacity of the plant, as is also reflected in Figure 7.12 in a dose dependent

manner, similarly like standard trolox. The calculated IC50 values for the extract was

202.49± 33.32 µg/ml, in comparison to that of the reference compound trolox (IC50 =6.76

± 0.17 µg/ml) (Table 7.3) indicating that the samples are not as potent inhibitor of lipid

peroxidation as trolox.

Fig. 7.12. The inhibitory effect of Euglena extract and standard trolox on lipid

peroxidation phenomenon from mice brain homogenate. ( mean ± S.D. of six parallel

measurements. **p < 0.01 and ***p < 0.001 vs 0 µg/ml).

Table 7.3: Comparison of the antioxidant and free radical scavenging capacities of 70%

methanolic extract of Euglena and standard reference compounds.

Name of Assay 70% methanolic

extract of Euglena sp

Standard Values of Standard

compounds

TEAC Values 0.202± 0.001 - -

Ø IC50 values of the extracts for free radical scavenging capacity for

DPPH 146.07± 1.80*** Ascorbic acid 5.29 ± 0.28

Hydroxyl radical (OH•)

scavenging

41.89 ± 0.41*** Mannitol 571.45 ± 20.12

Superoxide anion (O2• )

scavenging

5.83± 0.07*** Quercetin 42.06 ± 1.35

Nitric oxide radical

(NO) scavenging

278.46 ± 15.02*** Curcumin 90.82 ± 4.75

Hydrogen peroxide

(H2O2) scavenging

47.34± 5.05*** Sodium

pyruvate

3.24±0.30

Peroxynitrite (ONOO-)

scavenging

2.821 ± 1.69*** Gallic acid 0.876 ± 0.57

Singlet oxygen (1O2)

scavenging

0.879 ± 0.29*** Lipoic acid 0.046 ± 0.01

Hypochlorous acid

(HOCl) scavenging

223.25± 4.19*** Ascorbic acid 235.96 ± 5.75

Iron chelating activity 261.96 ± 4.73*** EDTA 1.27 ± 0.05

Lipid peroxidation 202.49± 33.32*** Trolox 6.76 ± 0.17

Ø IC50

oxygen scavenging assay where values are expressed in mg/ml). Each value represents

mean ± S.D. (n= 6). *** p < 0.001.

In living systems, free radicals are constantly generated and they can cause extensive

damage to tissues and biomolecules leading to various disease conditions, especially

degenarative diseases and extensive lysis (Halliwell and Gutterridge, 1998). Many

synthetic drugs protect against oxidative damage but they have adverse side effects. An

alternative solution to the problem is to obtain natural antioxidants from biological

resources (Yazdanparast and Ardestani, 2007; Yazdanparast et al. 2008). Several studies

have been made on biological activities of the seaweed (Ehresmann et al., 1977), and

could be potential rich sources of natural antioxidants (Matanjun et al., 2008).

Traditionally, seaweeds have been used in the treatment of various infectious diseases

(Hoppe, 1999), and reports of many active compounds have been isolated and their

structure determined (Vairappan et al., 2001; Mundt et al.,2003). Among the features of

marine algae and their substance, several extracts were screened on an antioxidant

capability (Latham, 2008), and their inhibitory activity on lipoxygenase enzyme (Mori et

al., 2003), as well as by radical scavenging activity, using a stable free radical

(Matsukawa et al., 1997). Among several algal genera, Spirulina and Chlorella are

considered to be important source as nutrients in traditional diet of some population of

Africa and Mexico. These tiny organisms have drawn attention due to their importance as

human food and their in vitro and in vivo antioxidant potential (Dillon and Phan, 1993).

Das et al.(2005) have described the production of antibacterials from the freshwater alga

Euglena viridis (Ehren)Recently, Euglena gracilis Z wild type has already been

considered a potential source of vitamin E (Kusmic et al. 1993). In the present study ,

the antioxidant capacity of Euglena tuba was measured by improved ABTS radical cation

decolorisation assay and gallic acid was used as the standard for phenolic content

measurement; both studies shows promising results. Therefore it is clear that the extracts

of Euglena tuba have good antioxidant activity as well high polyphenolic contents .

ABTS.+ is blue chromophore produced by the reaction between ABTS and Potasium

persulfate. Addition of the Euglena sample to this preformed radical cation reduced it to

ABTS in a concentration-dependent manner. This results were compared with those

obtained using trolox and TEAC value demonstrate that the extract is an extraordinary

potent antioxidant.

Hydroxyl radicals are the major active oxygen species causing lipid peroxidation and

enormous biological damage ( Aurand et al. 1977). The algal extract when added to the

reaction mixture, it removed the hydroxyl radicals from the sugar and prevented the

reaction indicating the algae is a hydroxyl radical scavenger to some extent.

Superoxide anion is alsovery harmful to cellular components( Korycha-Dahl and

Richardson, 1978). Robak and Glyglewski (1988) repored that flavonids are effective

antioxidants mainly because they scavenge superoxide anions. The results suggest that

the Euglena extract shows some scavenging activity of superoxide radical. It is well

known that nitric oxide has an important role in various inflammatory processes.

Sustained levels of production of this radicals are directly tox tissues and contribute to the

vascular collapse associated with septic shock, whereas chronic expression of nitric oxide

radical is associated with various carcinomas and inflammatory conditions including

juvenile diabetes, multiple sclerosis, arthritis and ulcerative colitis (Tylor et al., 1997).

The toxicity of NO increases greatly when it reacts with superoxide radical forming the

highly reactive peroxynitrite anion (ONOO-) (Huie and Padmaja, 1993). The nitric oxide

generated from sodium nitoprusside reacts with oxygen to form nitrite. The Euglena

extract inhibits nitrite formation by directly competing with oxygen in the reaction with

nitric oxide. The present study proved that the alga has extraordinary potent nitric oxide

scavenging activity than the standard curcumin.

Hydrogen peroxide is weak oxidising agent that inactivates a few enzymes directly,

usually by oxidation of essential thiol (-SH) groups. It can cross cell membranes rapidly;

once inside the cell, it can probably react with Fe 2+ and possibly Cu 2+ ions to form

hydroxyl radicals and these may be the origin of many of its toxic effects (Miller et al.,

1993). From the results it appeared H2O2 scavenging activity of the algal extract is

comparatively very high to that of the standard sodium pyruvate.

Peroxynitrite (ONOO-) is relatively stable as compared to other free radicals but once

protonated , it forms the highly reactive peroxynitrous acid (ONOOH) (Balavoine and

Geletti, 1999). Generation of excess ONOO- leads to oxidative damage and tissue injury

(Ischiropoulos et al., 1995). Peroxynitrite bleaches Evans Blue by oxidising it. According

to the present findings the algal extract inhibits Evans Blue bleaching by scavenging

peroxynitrite and its activity which is greater than that of the reference gallic acid.

Singlet oxygen is generated in the skin by ultra violet radiation. It is a high energy form

of oxygen and is known as one of the ROS. Singlet oxygen induces hyperoxidation and

oxygen cytotoxicity and decreases antioxidative acivity (Kochevar and Redmond, 2000).

The present study indicates that Euglena tuba extract has good scavenging activity for

singlet oxygen than that of the standard lipoic acid.

At sites of inflammation, the oxidation of Cl- ions by the neutrophil enzyme

myeloperoxidase results in the production of another harmful ROS, hypchlorous acid

(Auroma et al., 1989). HOCL has the ability to inactivate the antioxidant enzyme catalase

through breakdown of the heme prosthetic group. Catalase inactivation is inhibited in the

presence of the extract, signifying its HOCL scavenging activity. From the results it

appeared that the hypocholorous scavenging activity is comparable with the standard

ascorbic acid as an efficient scavenger.

Iron can stimulate lipid peroxidation by the Fenton reaction (H2O2 + Fe2+ = Fe3+ + OH- +

OH-) and also accelerate lipid peroxidation by decomposing lipid hydroperoxides into

peroxyl and alkoxyl radicals that perpetutate the chain reaction (Halliwell, 1991). Metal

chelating capacity is significant since it reduces the concentration of the transition metal

that catalyzes lipid peroxidation (Duh et al., 1999). According to the present finding, the

Euglena extract shows exceptionally high activity as compared to the EDTA proving to

be a very good iron chelator.

The reducing capacity of a compound may serve as a significant indicator of its potential

antioxidant activity. However, the activities of antioxidants have been attributed various

mechanisms such as prevention of chain initiation, decomposition of peroxides, reducing

capacity and radical scavenging (Yildirim et al., 2000). The result indicates Euglena

extracts contains significant amount of flavonoids and phenolic compounds. Both of

these classes of compounds have antioxidant potential and their effects on human

nutrition and health are significant. The mechanism of action of flavonoids is through

scavenging or chelation (Cook and Samman, 1996). Phenolic compounds are also very

important plant constituents because their hydroxyl groups confer scavenging activity

(Yildirim et al., 2000).

On the basis of the results obtained in the present investigation it is concluded that a 70%

methanolic extract of Euglena tuba, which contains large amounts of flavonoids and

phenolic compounds exhibits high antioxidant and free radical scavenging activities. It

also chelates iron and high reducing power. These invitro assays indicate that this algal

extracts is a significant source of natural antioxidant, which might be helpful in

preventing the progress of various oxidative stresses. However, the components

responsible for the antioxidative activity are currently unclear. Therefore further

investigation is needed to isolate and identify the antioxidant compounds present in the

algal extract. Moreover, the invivo antioxidant activity of this extract need to be assessed

prior to clinical use.