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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Neeru et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041
Volume 5, Issue 6, 1884-1891 Research Article ISSN 2278 – 4357
DETERMINATION OF TOTAL PHENOLIC CONTENT, TOTAL

FLAVONOID CONTENT AND TOTAL ANTIOXIDANT CAPACITY OF
DIFFERENT EXTRACTS OF ROYLEA ELEGANS WALL (AERIAL
PARTS)
Neeru1*, Shilpi Kashyap1, Esha Vatsa2, Jitendra Singh3 and

Ankush Sundriyal4
1
Himachal Institute of Pharmacy, Paonta Sahib District Sirmour.
2
Manav Bharti University, Solan, Himachal Pradesh.
3,4
SBSPGI, Balawala Dehradun.
Article Received on ABSTRACT

13 April 2016,
Petroleum ether, chloroform, ethyl acetate, methanol and water
Revised on 01 May 2016,
Accepted on 22 May 2016 extracts of Roylea elegans (aerial parts) was screened for assessing
DOI: 10.20959/wjpps20166-7002 bioactive phytochemical constituents. The preliminary phytochemical
screening using the standard phytochemical tests detected the presence
*Corresponding Author of alkaloids, steroids, flavonoids, terpenoids and phenols in different
Neeru
solvent extracts. Total phenolic and flavonoid contents along with total
Himachal Institute of
antioxidant capacity of the ethyl acetate extract of Roylea elegans
Pharmacy, Paonta Sahib
District Sirmour. (aerial parts) was evaluated to explore the reliable and potential
sources of novel natural antioxidants. The ethyl acetate extract showed
high flavonoid content. Due to high flavonoid content it showed better antioxidant activity.
KEYWORDS: Roylea elegans, Total phenolic content, total flavonoid content, antioxidant
activity.
INTRODUCTION
In nature plants gives more products which are useful for human beings. These natural
products are used for several diseases and also used as antioxidant. Plants give secondary
metabolites which are used for several diseases. These secondary metabolites are alkaloids,
glycosides, terpenoids, flavonoids, phenolic compound and tannins. These secondary
metabolites are produced from various parts of plants like stem, leaves, flowers, fruits and
www.wjpps.com Vol 5, Issue 6, 2016. 1884

roots. These secondary metabolites are biologically active and show better activity. Flavonoid
and phenolic content shows better antioxidant activity.
Roylea elegans a plant belonging to family lamiaceae shows better antioxidant activity due to
presence of good flavonoid content. Roylea elegans is a shrub of monotypic genus. The
family lamiaceae contain 22 species. Plant Roylea elegans belonging to family lamiaceae.
Leaves part of this plant (decoction) is traditionally used as a bitter tonic and also as a
febrifuge. It is also used as a tonic in contusions. Leaves are used in skin disease and fever.
The leaves of Roylea elegans contains various phytoconstituents like betulin, beta-sitosterol,
beta-amyrin, glucose, sti gmasterol, cetyl alcohol, and palmitic, stearic, oleic, gallic, oxalic,
fructose, arabinose and tartaric acids.
MATERIALS AND METHODS

chemicals and reagents
All the chemicals which were used in study are of analytical grade and purchased from the
Himedia Lab. Pvt. Ltd, Rankem.
Plant selection and identification

The plant material (Roylea elegans) investigated in the present study was collected from
Nauni, near Solan, Himachal Pradesh. The plant material was identified and authenticated at
the Herbarium of Council of Scientific and Industrial Research - National Institute of Science
Communication and Information Resources (CSIR-NISCAIR), Delhi vide reference no.
NISCAIR/RHMD/Consult/2014/2781/160.
Preparation of plant extract

The dried aerials parts of plant were collected. Then aerial parts of the plant were pulverized
through a mechanical grinder. Powder material was dried in hot air oven at moderate
temperature. The Powdered material was subjected to successive Soxhlet extraction by
solvents in increasing order of polarity viz. Petroleum ether, chloroform, ethyl acetate then
macerate with water for 24hour. The extract was concentrated and stored at 40c until further
use in the experiment (Chatwal 2011).
Phytochemical evaluation of the extract

The preliminary phytochemical screening of the aerial parts extract mainly done for the
evaluation of the various phytoconstituents such as steroids, tannin, Alkaloids, Flavonoids
and glycosides were present in the aerial parts of different extracts of R. elegans.
Determination of total phenolics content

The Total Phenolic content of extract of R. elegans was estimated according to the method
described in Ayurvedic Pharmacopoeia of India. The total phenol content was expressed in
milligrams of gallic acid equivalents per gram of extract. Prepare a stock solution (1mg/ml)
of the extract in methanol. From the stock solution, take suitable quantity of the extract into
25ml volumetric flask and add 10 ml of water and 1.5ml of folin ciocalteau reagent. Keep the
mixture for 5min and then add 4ml of 20% sodium carbonate solution and make up to 25ml
with double distilled water Keep the mixture for 30min and record absorbance at 765nm.
Calculate percentage of total phenolics from calibration curve of gallic acid prepared by
using the above procedure and express total phenolics as percentage of gallic acid.[5]
Total phenolic content was calculated with the help of standard curve equation and the
formula was below
Total phenolic content (%w/w)= GAE×V×D×10-6×100

W
Where, GAE – Gallic acid equivalent (µg/ml)
V – Total volume of sample (ml)
D – Dilution factor
W – Sample weight (gm)
Determination of total flavonoid content

The total flavonoid content of extract of R. elegans was estimated according to the aluminium
chloride method as follows: The aluminium chloride method was used for the determination
of the total flavonoid content of the sample extracts. Rutin was used to make the calibration
curve. Ten milligrams of rutin was dissolved in 80% ethanol and then diluted to 25, 50 and
100 μg/mL. The standard solutions (0.5 mL) were prepared by mixing 1.5 mL of 95%
ethanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1M potassium acetate and 2.8 mL of
distilled water. The absorbance of the reaction mixture was measured after 30 min at 415 nm.
The amount of 10% aluminum chloride was substituted by the same amount of distilled water
in blank.[11]

Total flavonoid content was calculated with standard curve equation and the formula
Total flavonoid content (% w/w)= RE×V×D×10-6×100

W
Where, RE – Rutin equivalent (µg/ml)
V – Total volume of sample (ml)
D – Dilution factor
W – Weight of sample (gm)
Antioxidant activity
DPPH
DPPH will be assayed as described by Elizabeth and Rao. The reaction mixture contained 1.0
ml of 0.3mM DPPH in 50 ml of methanol will be added with concentrations ranging from 20-
100 μg/ml .The mixture of DPPH in methanol used as positive control and methanol alone
served as blank. When DPPH reacts with antioxidants in the sample, it was reduced and the
colour changed from deep violet to light yellow and measured at 517 nm. Quercetin will be
used as a reference standard. Scavenging activity (%) = [A517 (control) – A517 (sample)/
A517 (control)] X 100.[4]
RESULT AND DISCUSSION

Table 1: Phytochemical screening of different extracts of Roylea elegans Wall. (aerial
parts)
S. Phytochemical Pet. Ether Chloroform Ethyl acetate Methanol Water
no. tests extract extract extract extract extract
1. Carbohydrate - - - - -
2. Protein - - - - -
3. Amino acid - - - + +
4. Fats + - - - -
5. Steroid - - + - -
6. Triterpenoides + - - - -
7. Glycosides - - - - -
8. Flavonoids - - + + +
9. Alkaloids - + - - +
10. Tannins - - - - +
Total Phenolic Content
Table 2: Standard curve of gallic acid

S. no. Concentration(µg/ml) Absorbance

1. 0.1 0.292
2. 0.2 0.329
3. 0.4 0.402
4. 0.8 0.614
5. 1.6 0.869
Figure 1: Standard curve of gallic acid
Table 3: Total phenolic content in different extracts

S. no. Extracts Total phenolic content (%w/w)
1. Petroleum ether 0.0887
2. Chloroform 0.267
3. Ethyl acetate 0.416
4. Methanol 0.565
5. Water 0.737
Figure 2: Total phenolic content
Total Flavonoid Content

Table 4: Standard curve of rutin

S. no. Concentration (µg/ml) Absorbance

1. 25 0.103
2. 50 0.180
3. 100 0.344
Figure 3: Standard curve of Rutin
Table 5: Total flavonoid content in different extracts.

S. no. Extracts Total flavonoid content (%w/w)
1. Petroleum ether 7.5
2. Chloroform 33.43
3. Ethyl acetate 64.37
4. Methanol 18.75
5. Water 11.56
Figure 4: Total flavonoid content
Total Antioxidant Capacity

Table 6: DPPH scavenging activity

% Inhibition
S. Concentration
Petroleum Ethyl
no. (µg/ml) Rutin Chloroform Methanol Water
ether acetate
1. 40 28.73 0.74 2.98 1.49 12.31 0.37
2. 60 49.25 6.34 4.85 10.44 19.02 3.35
3. 80 55.59 11.56 7.46 17.16 21.64 20.89
4. 100 63.43 15.67 10.82 29.10 24.25 29.85
Figure 5: DPPH scavenging activity
CONCLUSION
In our present study total phenolic content of in different extracts were showing that water
extract has high phenolic content and total flavonoid content of different extract showing
ethyl acetate has high. Total antioxidant activity of different e extracts was showing that ethyl
acetate has high antioxidant power due to high flavonoid content.
REFERENCES
1. Benzie, I. F. F. & Strain, J. J.,The ferric reducing ability of plasma (FRAP) as a measure
of ‘‘antioxidant power”: The FRAP assay, Analytical Biochemistry, 1996; 70–76.
2. Bhandari, S. L. Bisht, S. S. Bisht, N. S. (Department of Botany, H.N.Bal University,
Campus Pauri,(Garhwal), Micro propagation of Roylea elegans Wall. an important
medicinal herb, Journal of Medicinal and Aromatic Plant Sciences v, 2008; 30(4): 370-
374.
3. Dobhal, M. P. & Joshi B. C., 1979. Chemical investigations of Roylea elegans Wall. Part
I, Herba Pol., 1979; 95-97.
4. Elizabeth, K. & Rao, M. N. A., 1990. Oxygen radical scavenging activity of curcumin. Int
J Pharmaceut, 1990; 237-240.

5. Indian Pharmacopoeia. I.P, Vol I. Ministry of Health and Family Welfare, Government of
India, Published by the Indian Pharmacopoeia Commission, Ghaziabad, India, 2010; 82-
83: 139 & 201.
6. Khandelwal, K.R. Practical pharmacognosy, 19th edition, 2008; 9: 149- 156, 158.
7. Ranganayaki S. Gusain P. S. Singh A. K. The chemical constituents of leaves of Roylea
elegans. J Sci Res Plants & Medicines, 1985; 5-9.
8. Sai Khan, M. S. Howard, L. R. Miller, J. C. antioxidant activity and total phenolics in
Solanum, J Food Sci, 1995; 60(2): 341- 343.
9. Upadhyay, G. Kamboj, P. Malik, J. International Journal of Research in Pharmaceutical
and Biomedical Sciences, 2011; 2(4).
10. Upadhyay, G. Maurya, H. Chauhan, N. Traditional claim of Roylea elegans Wall. On
liver disorder., 2012.
11. Woisky, R. and Salatino, A. Analysis of propolis: some parameters and procedures for
chemical quality control. J. Apic. Res., 1998; 37: 99-105.

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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Neeru et al. World Journal of Pharmacy and Pharmaceutical Sciences

Volume 5, Issue 6, 1884-1891 Research Article ISSN 2278 – 4357

DETERMINATION OF TOTAL PHENOLIC CONTENT, TOTAL

Neeru1*, Shilpi Kashyap1, Esha Vatsa2, Jitendra Singh3 and

Article Received on ABSTRACT

www.wjpps.com Vol 5, Issue 6, 2016. 1884

MATERIALS AND METHODS

Plant selection and identification

Preparation of plant extract

Phytochemical evaluation of the extract

www.wjpps.com Vol 5, Issue 6, 2016. 1885

Determination of total phenolics content

Total phenolic content (%w/w)= GAE×V×D×10-6×100

Determination of total flavonoid content

www.wjpps.com Vol 5, Issue 6, 2016. 1886

Total flavonoid content (% w/w)= RE×V×D×10-6×100

RESULT AND DISCUSSION

Table 2: Standard curve of gallic acid

www.wjpps.com Vol 5, Issue 6, 2016. 1887

Figure 1: Standard curve of gallic acid

Table 3: Total phenolic content in different extracts

Figure 2: Total phenolic content

Total Flavonoid Content

www.wjpps.com Vol 5, Issue 6, 2016. 1888

S. no. Concentration (µg/ml) Absorbance

Figure 3: Standard curve of Rutin

Table 5: Total flavonoid content in different extracts.

Figure 4: Total flavonoid content

Total Antioxidant Capacity

www.wjpps.com Vol 5, Issue 6, 2016. 1889

Figure 5: DPPH scavenging activity

www.wjpps.com Vol 5, Issue 6, 2016. 1890

www.wjpps.com Vol 5, Issue 6, 2016. 1891

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