IAETSD JOURNAL FOR ADVANCED RESEARCH IN APPLIED SCIENCES ISSN NO: 2394-8442

COMPARATIVE SCREENING AND QUANTIFICATION OF TOTAL

FLAVONOIDS AND TOTAL PHENOLICS OF SOME COMMON
HYDROPHYTIC AND WETLAND PLANTS FROM EAST
SINGBHUM, JHARKHAND (INDIA).

*Kumari Roma1, Kiran Shukla2, Debasish Sahoo3

1(Research
Scholar, University Department of Botany, Kolhan University, Chaibasa)
2(Head
of Department, University Department of Botany, Kolhan University, Chaibasa)
3(Sr.R.A. R&D, Vitilux Organics (P) Ltd., Hyderabad.)

1romak1007@gmail.com, 2kiranshukla310@gmail.com, 3sahoodebasish3125@gmail.com

Abstract

The aqueous methanolic phase for solvent extraction and soxhlet extraction found out to be a good extraction
phase for Flavonoids and Phenolics. Flavonoids and Phenolics are present in good quantity in different hydrophytes that
have been studied. From the Quantitative analysis for aqueous methanolic extract of different plants both for solvent and
Soxhlet extract, solvent extract of Peperomia pellucida have the highest concentration of Flavonoids followed by its soxhlet
extract with 219 mM equivalent of Quercetin and 192.33 mM equivalent of Quercetin respectively. The soxlet extract of
Peperomia pellucida and soxlet extract of Leaves of Nymphaea nouchali contains highest concentration of Phenolics with 147.66
mM equivalent of gallic acid and 131 mM equivalent of gallic acid respectively.

Keywords: Hydrophytes, Quantitative Estimation, Flavonoids, Phenolics, Quercetin, Gallic acid.

I. INTRODUCTION
Hydrophytes and wetland plants play a very important role in our ecosystem. Though the potential of aquatic plants as
food and feed has been emphasized by several authors they often seem to be neglected by the masses [1,2]. Huge
anthropogenic pressure has led to the extinction of a number of natural water bodies, thereby also leading to the extinction
of a number of hydrophytes and wetland plants with immense ethno-medicinal values [3-5]. Colocasia esculenta (L.) Schott,
Hydrilla verticillata (L.f.) Royle, Ipomoea aquatica Forssk, Peperomia pellucida Kunth, Nymphaea nouchali Burm.f. are some
common hydrophytes [6-11] that are present in East Singbhum, Jharkhand that have good nutritional and medicinal
properties.

II. MATERIALS AND METHODOLOGY

A. Sample collection

Different hydrophytic samples were collected from waterbodies and marsh lands of East Singbhum, Jharkhand, India.
These plant resources were authenticated by The Botany of Bihar and Orissa by H.H Haines. The different plants were
Colocasia esculenta (L.) Schott, Hydrilla verticillata (L.f.) Royle, Ipomoea aquatica Forssk, Peperomia pellucida Kunth, Nymphaea
nouchali Burm.f.

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IAETSD JOURNAL FOR ADVANCED RESEARCH IN APPLIED SCIENCES ISSN NO: 2394-8442

B. Sample Processing

The leaves of different hydrophytes were collected and washed with distill water to remove dirt and other contaminants.
Then they were washed with 10% saline solution and again with distill water. Then the leaves were shade dried until the
moisture is reduced to 10%. The dried leaves were blended into powder form.

C. Crude Extract Preparation

The crude extract was prepared from the leaves of different hydrophytes by Solvent extraction [12] and Soxhlet extraction
[13]. In solvent extraction, the dried plant sample were taken and required solvent in the ratio (1gm : 20ml) respectively in
a amber colored glass container with air tight lids for 4 days with frequent agitation. Then they were filtered and the filtrate
is the crude extract. In soxhlet extraction, the sample was loaded into the thimble and solvent in the bottom flask/boiling
flask. The cycles were continued until all the extract has been exhausted from the plant sample (generally 10 cycles).

D. Qualitative Screening for Flavonoids and Phenolics

The presence of flavonoids and phenolics can be tested qualitatively using the standard procedures to identify the
constituents. The qualitative test for flavonoids and phenolics were done by alkaline reagent Test and Ferric Chloride test
respectively. In Alkaline reagent test, formation of intense yellow coloration or fluorescence in Alkaline reagent Test
indicates presence of flavonoids whereas formation of deep blue color in Ferric Chloride test indicates presence of
phenolic compound. Every test is done in triplicates [14-15].

E. Quantitative Estimation of Flavonoids and Phenolics

Total flavonoids [16] and Total phenolics [17] were estimated from Quercetin standard calibration curve and Gallic acid
standard calibration curve respectively. The quantitative estimation were expressed as mM equivalent of Quercetin and
mM equivalent of gallic acid.

III. RESULTS
The plant samples were coded as A: Colocasia esculenta (L.) Schott, B: Hydrilla verticillata (L.f.) Royle, C: Ipomoea aquatica
Forssk, D: Peperomia pellucida Kunth, E1: Flowers of Nymphaea nouchali Burm.f, E2: Leaves of Nymphaea nouchali Burm.f.
The Plant samples were dried and the solvent system used was Methanol:Water (70:30), both for solvent extract and
soxhlet extract. The Plant crude extract for solvent extract (Table I) and Soxhlet extract (Table II) were then analyzed
qualitatively for presence/absence of Flavonoids and Phenolics.

Table I: Qualitative estimation of Flavonoid and Phenolic compound for Solvent extract.

Sl.NO Metabolite Observations A B C D E1 E2

Yellow
Flavonoids colouration Absent Absent Present Present Present Absent
1. (Alkaline reagent on addition of
Test) Ammonium - - ++ ++ ++ -
hydroxide)
Deep blue or Absent Present Absent Absent Present Absent
Phenols (Ferric Black colour
2.
Chloride Test) on addition of - ++ - - ++ -
aq.FeCl3

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Table II: Qualitative estimation of Flavonoid and Phenolic compound for Soxhlet extract.

Sl.NO Metabolite Observations A B C D E1 E2

Yellow
Flavonoids coloration on Absent Present Present Present Absent Absent
1. (Alkaline reagent addition of
Test) Ammonium - ++ ++ ++ - -
hydroxide)
Deep blue or Absent Absent Present Present Present Present
Phenols (Ferric Black color
2.
Chloride Test) on addition of - - ++ ++ ++ ++
aq.FeCl3

The standard curve for Quercetin (Fig. 1) and Gallic acid (Fig. 2) was first derived for Quantitative estimation of unknown
concentration of Flavonoids (in mM equivalent of Quercetin) and Phenolics (in mM equivalent of Gallic acid).

Standard Curve _Quercetin

1.8 1.63
1.55
1.6 1.45 y = 0.0033x + 0.0833
R = 0.9918
Absorbance at 415nm

1.4 1.25
1.1
1.2
1 0.87
0.72
0.8 Absorbance at 415nm
0.56
0.6 0.45 Linear (Absorbance at 415nm)
0.4 0.2
0.2
0
0 100 200 300 400 500 600
Concentration of Quercetin (mM)

Fig. 1- Standard Calibration Curve for Quercetin.

Standard Curve _Gallic acid

2.5
y = 0.0033x + 0.3173
2 1.75 1.83
Absorbance at 765nm

R = 0.9786
1.66
1.57
1.4
1.5
1.11
0.96
0.79 Absorbance at 765nm
1
0.67
Linear (Absorbance at 765nm)
0.4
0.5

0
0 100 200 300 400 500 600
Concentration of Gallic Acid (mM)

Fig. 2- Standard Calibration Curve for Gallic acid.

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The Plant extracts that had shown positive for the presence of Flavonoid and Phenolics in Qualitative Test (Table I and
Table II) were then quantified by using quantitative methods by standard curve. The unknown concentration of
Flavonoids and Phenolics from Aqueous methanolic extracts (solvent extract and soxhlet extract) of different plants
(Table III) coded as A: Colocasia esculenta (L.) Schott, B: Hydrilla verticillata (L.f.) Royle, C: Ipomoea aquatica Forssk, D:
Peperomia pellucida Kunth, E1: Flowers of Nympheae nouchali Burm.f, E2: Leaves of Nymphaea nouchali Burm.f. was
determined by plotting the absorbance on the Standard curve of Quercetin (Fig. 1) (for flavonoids) and Gallic acid (Fig. 2)
(for Phenolics).

Table III- Quantitative Estimation of Flavonoids and Phenolics from different plant extracts.
FLAVONOID CONTENT PHENOLIC CONTENT
Absorbance at Conc.(mM Absorbance at Conc.(mM
Sl. No. Sample code. Sample Extract type 415nm equivalent 765nm equivalent
Quercetin) Gallic acid)

1. A. Colocasia esculenta Solvent -- -- -- --

Extract
2. A. Colocasia esculenta Soxhlet -- -- -- --
Extract
3. B: Hydrilla verticillate Solvent -- -- 0.54 74.33
Extract
4. B: Hydrilla verticillate Soxhlet 0.32 79.00 -- --
Extract
5. C: Ipomoea aquatic Solvent 0.45 122.33 -- --
Extract
6. C: Ipomoea aquatic Soxhlet 0.23 49.00 0.69 124.33
Extract
7. D: Peperomia pellucida Solvent 0.74 219.00 -- --
Extract
8. D: Peperomia pellucida Soxhlet 0.66 192.33 0.76 147.66
Extract
9. E1: Flowers of Solvent 0.56 159.00 0.61 97.66
Nymphaea nouchali Extract
10. E1: Flowers of Soxhlet -- -- 0.42 34.33
Nymphaea nouchali Extract
11. E2: Leaves of Nymphaea Solvent -- -- -- --
nouchali Extract
12. E2: Leaves of Nymphaea Soxhlet -- -- 0.71 131.00
nouchali Extract

From the Quantitative analysis for aqueous methanolic extract of different plants both for solvent and Soxhlet extract,
solvent extract of Peperomia pellucida have the highest concentration of Flavonoids followed by its soxhlet extract with 219
and 192.33 mM equivalent of Quercetin respectively. The soxhlet extract of Peperomia pellucida and soxhlet extract of
Leaves of Nympheae nouchali contains highest concentration of Phenolics with 147.66 and 131 mM equivalent of gallic acid
respectively. (Fig.3).

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Concentration of Flavonoids and Phenolics 250

219
192.33
200
159
147.66
150 131
122.33 124.33
Flavonoids (mM
97.66 equivalent of
100
74.33 79 Quercetin)
49 Phenolics (mM
50 34.33 equivalent of Gallic
acid)
0

Aqueous Methanolic Extract of different Hydrophytes.

Fig. 3- Comparative analysis of Flavonoid and Phenolic content for aqueous methanolic extract of different hydrophytes.

IV. DISCUSSION
Phenolic compounds possess in common an aromatic ring bearing one or more hydroxyl groups. There are about 8000
naturally occurring plant phenolics and about half of this number are flavonoids [18]. Phenolics possess a wide spectrum
of biochemical activities such as antioxidant, antimutagenic, anticarcinogenic as well as ability to modify the gene
expression [19]. Phenolics are the largest group of phytochemicals that account for most of the antioxidant activity in
plants or plant products [20].

Flavonoids are the largest group of naturally occurring phenolic compounds, which occurs in different plant parts both in
Free State and as glycosides. They are found to have many biological activities including antimicrobial, mitochondrial
adhesion inhibition, antiulcer, antiarthritic, antiangiogenic, anticancer, protein kinase inhibition, etc [21]. The flavonoids
have two benzene rings separated by a propane unit. The flavones and flavones are the most widely distributed of all the
phenolics [22]. Flavonoids are particularly beneficial, acting as antioxidants and giving protection against cardiovascular
disease, certain forms of cancer and age-related degeneration of cell components. Their polyphenolic nature enables them
to scavenge injurious free radicals such as super oxide and hydroxyl radicals [23]. A variety of dietary plant flavonoids
inhibits tumour development in experimental animal models [24]. The bioflavonoids possess pharmacological effects like
ability to inhibit the release of histamines, the adhesion of blood platelets and the action of lens aldose reductase, to block
the inflammatory effects of hepatotoxins, and to act as a heart-stimulant [25].

V. CONCLUSION
The local hydrophytes need to be explored more for better natural source and potential alternative to conventional
medicine system that can be a safe and promising for the healthcare system. They can be used as local cuisine and sources
of compounds.

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