PHYTOCHEMICAL SCREENING AND ANTIOXIDANT POTENTIAL OF ECLIPTA PROSTRATA (L) L-A VALUABLE HERB

SHILPAM SINHA, RICHA RAGHUWANSHI*

Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India
Email: [email protected]
 

Received: 21 Dec 2015 Revised and Accepted: 25 Jan 2016


ABSTRACT

Objective: In this study, phytochemical screening, antioxidant activity, total phenolic, flavonoid contents and reducing potential of Eclipta prostrata (L.) L plant prepared in different solvents (methanolic, ethanolic and double distilled water) was evaluated by various assays.

Methods: Polyphenolic contents, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, Hydrogen peroxide (H2O2) radical scavenging activity and reducing power of the plant was estimated by modified protocol.

Results: In DPPH free radical, methanolic extract of plant was most potent in activity with 50% inhibition at 333.34 µg/ml. and 398.12 µg/ml concentration respectively. Total phenolic content was maximum in the methanolic extract of plant (52.5±0.64 mg/g of gallic acid equivalent) and total flavonoid was maximum in aqueous extract of plant (193.12±1.65 mg/g of rutin equivalent). High reducing capacity of plant was observed in case of methanolic extract. A significant correlation was found between antioxidant activity and polyphenolic content (total phenols and total flavonoids). Moreover, a significant correlation was found between antioxidant activity and reducing potential of plant extract, denoting that reducers are important contributors to antioxidant.

Conclusion: The study shows E. prostrata as an important natural source of antioxidants.

Keywords: Eclipta prostrata, Plant extract, Antioxidant activity, Polyphenolic content, DPPH activity, H2O2 scavenging activity, Reducing potential


INTRODUCTION

Medicinal plants have been used in folk medicine since date back in different part of the world against a variety of diseases. The interest in medicinal plants for their phytochemical constituents, biological activities and antioxidant activity have been studied since long back due to the absence of side effects and its economic viability [1]. The practice of using plants as a source of food and medicine has recently showed revival of interest globally. Oxidative stress produces free radicals. Free radicals like reactive oxygen species (ROS) and reactive nitrogen species (RNS) are constantly formed in an organism during the process of cellular respiration and from sources like pollution, ionizing radiation and drugs. These radicals contribute in the development of many diseases like Alzheimer, cancer, heart disease and rheumatoid arthritis [2]. Thus, when the endogenous antioxidant becomes inadequate for free radicals balancing, a demand for exogenous antioxidants is increased in the body to prevent oxidative damage of cells and tissues [3]. The plant contains numerous free radical trapping molecules like phenols, flavonoids, carotenoids and vitamins which are medicinally important [4]. Phenolic groups quench free radicals by hydrogen donation [5].

Antioxidants are naturally occurring molecule which inhibits the oxidizing chain reactions by delaying or inhibiting the oxidation of molecules [6]. These antioxidants are used to maintain food quality by arresting the oxidative deterioration of lipids. They slow down the chronic disease by enhancing the body’s natural antioxidant defences by adding with dietary antioxidants [7]. Natural antioxidants are preferred over synthetic antioxidants for not only as an eco-friendly approach but also for its low manufacturing than artificial antioxidant having multifarious benefits [8, 9]. Enormous research opportunities exist in the field of plants antioxidant and their effects on human health. Antioxidant activities are present due to biologically active polyphenol components such as flavonoids and phenolic acids [10].

The extracts of various plant species show preventive action against anti-inflammation, cancer, cardiovascular, and neurodegenerative disease [11]. The plant based natural antioxidants like alkaloids, flavonoids, phenols, tocopherols in foods is gaining much attention [12]. These natural biologically active agents exhibit anti-carcinogenic potential and offer diverse health-contributing effects because of their antioxidative and antimicrobial attributes [13].  Eclipta prostrata is a medicinal herb widely distributed in tropical and sub-tropical region of Asia. It is a valuable medicinal herb reported for its various pharmacological and biological activities like hair growth promotion [14], hepatoprotective [15], antidiabetic [16], antimicrobial property [17] and anticancerous activity [18]. Phytochemical study and reducing potential was not earlier reported from the whole plant. The objective of the present work was to study phytochemical screening, polyphenolic content and antioxidant activity of the plant. Reducing potential of E. prostrata was evaluated for the first time in methanolic, ethanolic and aqueous extracts derived from the plant.

MATERIALS AND METHODS

Chemicals

2,2-Diphenyl,1-picryl hydrazyl (DPPH), ascorbic acid, gallic acid, rutin, trichloroacetic acid (TCA), potassium ferricyanide (K3Fe(CN)6), ferric chloride (FeCl3), Folin-Ciocalteu phenol reagent, aluminium chloride (AlCl3), rutin, sodium carbonate (Na2CO3), sodium potassium tartarate (Na-K tartarate) were purchased from Hi-Media Ltd and Merck.

Plant collection

Plant of E. prostrata was collected from the campus of Banaras Hindu University, Varanasi, during the months of June-July. Plant material was collected from a different location to make a composite sample. The plant was authenticated at Botanical Survey of India (BSI), Allahabad. Collection number BHU-171 and voucher number-91926 was given by BSI to the plant flora.

Percentage yield

Plants were washed thoroughly for dust removal under running tap water. The plant material was oven dried at 40-45 °C for 24 h. The plant was crushed in the electrical grinder. Extraction was done from 20 g of whole plant powder consisting of root, stem and leaf in 250 ml of the solvent by using a Soxhlet apparatus for 12 h. Extracts was then filtered and evaporated to dryness at 40 ºC in the rotatory evaporator. Extracts were stored at 4 °C till use. Percentage yield (w/w) of crude extract was calculated with the original amount of coarse powder used for extraction [19].

Preparation of sample extract

One gram of plant extract was dissolved in 10 ml of respective solvent to make a stock solution of 100 mg/ml. Extracts were further diluted as per requirement in different experiments.

Phytochemical screening

The initial phyto-compound testing of the various solvent extract was performed using standard protocol [20-21].

DPPH free radical scavenging activity

The free radical scavenging activity of methanolic, ethanolic and aqueous extracts of the plants was measured by 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) assay [22]. Different concentration of plant extracts (100-600 µg/ml) were added to 3 ml of DPPH solution in methanol (0.25 mM). The mixture was incubated for 10 min at room temperature in the dark, after which the absorbance was measured at 517 nm by using spectrophotometer (Thermo Scientific UV 1). Ascorbic acid was used as a standard and methanol acted as blank.

Hydrogen peroxide radical scavenging activity

The hydrogen peroxide (H2O2), the radical scavenging activity of methanolic, ethanolic and aqueous extracts of the plants, was measured according to Ruch et al., [23]. The plant extracts of different concentration (100-600 µg/ml) in the volume of 1 ml were added to 2 ml of H2O2 (40 mM) prepared in (50 mM, pH-7.4) phosphate buffer. The plant samples were incubated for 10 min at room temperature. The absorbance was determined at 230 nm against a blank solution containing phosphate buffer only (Thermo Scientific UV 1). Ascorbic acid was used as a standard.

Estimation of total phenolic content and flavonoid content

Total phenolic content (TPC) was measured by Folin–Ciocalteu assay [24]. In brief, 0.5 ml of plant extract (200 µg/ml) prepared with different solvent (methanol, ethanol and water) was mixed with 0.5 ml Folin-Ciocalteu reagent and 3 ml of 20% Na2CO3. Absorbance was taken at 650 nm. The standard graph was prepared with gallic acid as a reference compound and expressed as mg/g of gallic acid equivalent (GAE).

Total flavonoid content (TFC) was determined byAlCl3 colorimetric method [25]. The plant extract (1 ml, 200 µg/ml) prepared with different solvent (methanol, ethanol and water) was taken in which 100 µl AlCl3 (10% w/v), 100 µl Na–K tartrate and 2.8 ml distilled water were added and kept for 30 min. Absorbance was taken at 415 nm. The standard graph was prepared with rutin as a reference compound and expressed as mg/g of rutin equivalents (RE).

Reducing potential capacity (RPC)

The reducing capacity assessment of methanolic, ethanolic and aqueous plant extract was determined according to the method of Athukorala et al., [26] with minor modifications. The extracts were used at different concentrations (50-300 µg/ml) prepared in 1 ml of different solvents were mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide [K3Fe(CN)6] (30 mM). The mixture was incubated at 50 °C for 20 min. Then, 2.5 ml TCA (0.6 M) was added to the mixture which was then centrifuged for 10 min at 3000 rpm. The upper organic layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 1 mM) and the absorbance was measured at 700 nm by a spectrophotometer. Ascorbic acid was used as a standard.

Determination of percentage and statistical analysis

Percentage scavenging activity (%) was calculated by the formula.



Where, C is the absorbance of control, T is absorbance of the test samples/standard. The antioxidant activity of the extract was expressed as IC50. IC50 was calculated through linear regression analysis. IC50 value is the concentration (in µg/ml) of extract that inhibits the formation of radical by 50 %. The above experiments were performed four times in independent manner and repeated for thrice. Data was represented as mean±standard error (SE) by applying one-way Analysis of Variance (ANOVA). Tukey’s multiple range tests were used for separation of means when ANOVA was significant (p=0.001) (SPSS 16.0; Chicago, IL, USA). The graphs were prepared in Sigma plot 11.0.

RESULTS AND DISCUSSION

Percentage yield

The maximum percentage yield of extract was 42.8%, 39.2% and 36% for aqueous, methanolic and ethanolic extract respectively. The percentage yield of plant extract varied significantly in different extraction solvents. Variation in % yield in the different solvent may be due to various degree of solubility of crude extract in different polarity solvents. Almost similar 33.66 % yield in methanolic extract of aerial part of the same plant was reported [27]. Percentage yield of various plant extracts depends upon the extraction conditions such as solvent polarity for the production of a fraction with adequate yield [19].

Phytochemical screening in various solvents

In the present study, the phytochemical testing showed the presence of various bioactive compounds like carbohydrate, alkaloids, tannins, steroids and flavonoids (table 1). There are other reports of screening of metabolites from the leaf, stem and roots of this plant in other solvents [28]. The distribution of bioactive compounds varies in plant and extraction solvents play an important role in screening. Medicinal herbs are rich in phenolic compounds, flavonoids, tannins; lignin and other compounds [29] which may be responsible for the antioxidant activities of the extract [30].


Table 1: Phytochemical screening of plant in different solvents

Phytochemicals

Test performed

Methanolic extract

Ethanolic extract

Aqueous extract

Carbohydrate

Fehling test

+

-

+

Phenols

Ferric chloride test

+

+

+

Flavonoids

Ammonia test

+

+

+

Alkaloids

Wagner’s test

+

+

-

Steroids

Salkowski test

+

+

+

Tannins

Lead acetate test

+

+

-

Saponins

Frothing test

+

+

+

Glycosides

Nitroprusside test

+

+

+

Amino acids

Ninhydrin test

+

+

+

Note: += Presence; -= Absence of phytochemicals


Table 2: Antioxidant activity of E. prostrata by DPPH free radical scavenging method in different solvents

Concentration(µg/ml)

Percentage inhibition (mean±SE)

 Methanolic

 Ethanolic

 Aqueous

Ascorbic Acid

 100

24.11±0.39f

18.15±0.56f

15.99±0.42f

25.12±0.29f

200

36.69±0.59e

22.68±0.64e

23.91±0.37e

39.34±0.20e

300

48.86±0.32d

33.61±0.46d

36.09±0.42d

56.25±0.22d

400

58.41±0.24c

55.75±0.82c

47.42±0.24c

65.15±0.14c

500

68.65±0.37b

61.49±0.49b

54.80±0.55b

86.47±0.38b

600

73.40±0.53a

69.34±0.46a

63.92±0.37a

95.22±0.32a

 IC50

 333.34

366.46

 448.16

 271.47

Data represented as mean±SE (n=4). One way ANOVA followed by Tukey’s test. All data is significant at p<0.001; a,b,c = different letter shows significant difference between means.


Antioxidant DPPH activity

Antioxidant capacity is one of the most important parameters for quality check of medicinal bioactive and functional components in the plant. In the present study, different solvents i.e. water; methanol and ethanol were used for extraction. All extracts of E. prostrata plant prepared in different solvent showed significant free radical scavenging activity (table 2). The methanolic extract (IC50=333.34 µg/ml) of the plant showed highest scavenging activity followed by ethanolic (IC50=366.46 µg/ml) and aqueous (IC50=448.16 µg/ml) extract.

DPPH radical scavenging activity is a simple and fast method to screen the scavenging potentials of plant sample [31]. DPPH is neutralized by antioxidants present in extract due to their ability to donate electrons. The degree of DPPH scavenging can be used to signify the hydrogen-donating capacity of antioxidants. The reduction in absorbance is the indicator of the increased antioxidant capacity of the extract [32]. The bioactive compounds present in plant extracts were able to discolour DPPH solution turning its purplish colour to yellowish coloured diphenyl-picryl hydrazine at low concentration. Ascorbic acid has also been used as standard antioxidant to determine the IC50 of extracts in other plants [33]. Similar pattern of antioxidant activity was observed in methanolic leaf extract of E. prostrata [34].

Antioxidant H2O2 scavenging activity

All the extracts of this plant have shown significant scavenging activity (table 3). The scavenging activity of the methanolic extract was found highest (IC50=398.12 µg/ml) followed by ethanolic (IC50=449.12 µg/ml) and aqueous (IC50=473.96 µg/ml) extract.


Table 3: Antioxidant activity of E. prostrata by H2O2 radical scavenging in different solvents

Concentration(µg/ml)

Percentage inhibition (mean±SE)

 Methanolic

 Ethanolic

 Aqueous

 Ascorbic Acid

 100

23.17±0.20f

 20.16±0.45f

19.36±0.24f

 25.86±0.38f

200

31.00±0.23e

28.39±0.32e

24.10±0.42e

36.80±0.30e

300

48.81±0.38d

40.10±0.41d

35.89±0.40d

48.33±0.31d

400

50.64±0.42c

47.87±0.35c

45.69±0.23c

57.07±0.29c

500

57.49±0.44b

56.02±0.47b

52.90±0.23b

61.87±0.39b

600

60.95±0.37a

58.89±0.43a

59.30±0.20a

74.16±0.38a

 IC50

 398.12

 449.12

 473.96

 342.56

Data represented as mean±SE (n=4). One way ANOVA followed by Tukey’s test. All data is significant at p<0.001. a,b,c = different letters shows significant difference between means.


The plant extracts scavenge H2O2 by donating an electron to hydrogen peroxidase, thereby converting it into the water. In this assay, methanolic extract of E. prostrata showed greater inhibition in comparison to aqueous extract. It may be due to the presence of higher phenolics in methanolic extract of the plant. This suggests that aqueous extract required significantly high concentration to show its potency. H2O2 radical scavenging activity was also reported from different extracts of Crataegus monogyna [35].

Total phenolic and flavonoid content

Total phenolic content was reported as mg/g of GAE in reference to a standard curve (y=0.001x+0.05, R2=0.997). TPC was found higher in methanolic (52.5±0.64) followed by ethanolic (44.25±1.75) and aqueous (13.25±0.85) extract of E. prostrata. All TPC value was represented as mg/g of GAE.

Total flavonoid content was calculated by a standard curve (y=0.0008x+0.198, R2=0.994) and reported as mg/g of RE. Total flavonoids were found higher in aqueous (193.12±1.65 mg/g) extract followed by ethanolic (162.5±1.73 mg/g) and then methanolic extract (144.62±1.62 mg/g) (table 4).

The phenolic and flavonoids present in the plants are secondary metabolites which act as antioxidants and help in free radical scavenging, metal ions chelation [36] and protects against human disease like cardiac-disorder, thrombosis, hepatotoxicity, anti-carcinogenic, anti-mutagenic, etc [37]. The phenolic content was found to be maximum in the methanol extract of the plant. Similarly, higher phenolic content in an organic solvent has also been reported [38]. The presence of secondary metabolites like phenol and flavonoid contents in plant extract depend on the solvent used [39, 40].

In this study, flavonoids content showed the highest activity in aqueous extract, it might be due absence of tannin and alkaloid in an aqueous solvent as observed in screening experiment. Flavonoids reported to be responsible for antioxidant activities of the plant through their scavenging activity [41]. Flavonoids might show higher antioxidant activity in aqueous extract due to structure and substitution pattern of hydroxyl group.

Reducing potential capacity

The reducing potential capacity of the plant extracts increased as per their concentration in all the three solvents. The methanolic extract showed maximum reducing power followed by ethanolic extract and the minimum was found in aqueous extract. Increased absorbance of the reaction mixture indicated an increase in reducing power capacity (fig. 1).

In this assay, the colour of plant extracts solution changes from yellow to a range of green and blue colour. The reducing capacity of plant extract acts as an indicator of its antioxidant activity [42]. Similar to antioxidant activity, the reducing potential also increases in a concentration-dependent manner [43].

Correlation between antioxidant activity and polyphenolic compounds

The positive, significant and linear correlation was found between total antioxidant activity and polyphenolic contents (TPC & TFC) of various extracts (fig. 2, A-C). Correlation coefficient (R2) values of different extract showed very high correlation between antioxidant activities and TPC and TFC content (R2 ranges from 0.965 to 0.999). Thus, it can be said that there found good correlation with antioxidant activity and total phenolic and flavonoid content of E. prostrata in this study.

A significant correlation between the polyphenolics and total antioxidant capacity has been reported in another plant system [44].


Table 4: Total phenolic and flavonoid content in E. prostrata in different solvent

Total polyphenolic content

Plant extract

 TPC (mg/g GAE)

 TFC (mg/g RE)

Methanol

52.5±0.64a

144.62±1.62c

Ethanol

44.25±1.75b

162.5±1.73b

Aqueous

13.25±0.853c

193.12±1.65a

Data represented as mean±SE (n=4); One-way ANOVA followed by Tukey’s test. All data is significant at p<0.001; a,b,c = different letters shows significant difference between means.



Fig. 1: Reducing power capacity of plant extract of E. prostrate




 


Fig. 2: Correlation between antioxidant activity and polyphenols (TPC and TFC) of E. prostrata (A) Methanolic extract of plant (B) Ethanolic extract of plant and (C) Aqueous extract of plant

Correlation between antioxidant activity and reducing power

A significant correlation was obtained between total antioxidant activity and reducing the potential of plant extract.

The correlation coefficient (R2) between antioxidant activity and reducing potential was found (R2=0.849), (R2=0.962) and (R2=0.963) for methanolic, ethanolic and aqueous extract respectively (fig. 3A-C).



(A) Methanolic extract



(B) Ethanolic extract



(C)Aqueous extract


Fig. 3: Correlation between antioxidant activity and reducing power of E. prostrata (A) Methanolic extract of plant (B) Ethanolic extract of plant and (C) Aqueous extract of plant

In this study reducing potential and antioxidant activity of extract increased with increase in extract concentration. So, very high and significant correlation was observed between reducing power and antioxidant activity. A similar relationship was also found between antioxidant potential and reducing the power of different plant extract [45].

CONCLUSION

Our study report that whole plant extract of E. prostrata plant is a rich source of antioxidant compounds. The antioxidant property, reducing power and polyphenolic content varied significantly in the different extraction solvent. For most of the activity, organic solvents gave more promising results than aqueous one. Thus, extracts of this plant can be used for relieving the oxidative damage caused by stress and may help in preventing aging. Therefore, this plant can be recommended to be used for the production of nutraceuticals and pharmaceuticals.

ACKNOWLEDGEMENT

University Grants Commission is acknowledged for financial support.

CONFLICT OF INTERESTS

The authors declare no conflict of interest

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