Published by : International Journal of Engineering Research & Technology (IJERT)

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Vol. 5 Issue 08, August-2016

Extraction, Isolation and Quantification of

Bioactive Compound (Fisetin) and its Product
Formulation
Ms. Sharvari A. Surnis Mr. Pratik S. Patil
Department of Biotechnology Engineering Mr. Rohan H. Jadhav
T.K.I.E.T.,Warananagar, Maharastra , India Department of Biotechnology Engineering
T.K.I.E.T.,Warananagar, Maharastra , India

Abstract— Flavonoids are the class of plant secondary to show anti-cancerous, anti-diabetic [8], antioxidant, anti-
metabolites. Flavonoids are widely distributed into plants, inflammatory and memory stimulator effects.
fruits and vegetables. Fisetin is a flavonol, belongs to Many studies and researches have been done on the
flavonoid group of polyphenols. Berries contain more amount physical, chemical and medicinal properties of strawberries
of fisetin as compared to other fruits and vegetables. Among
and even many literatures have been published on its
the berries strawberries contains the highest amount of the
fisetin (i.e. 160 μg/ gm. of strawberry). Fisetin has many extraction, but none has been actually incorporated as
medicinal properties including anti-cancerous, anti- application bases in food form or any other medicinal or
inflammatory, anti-oxidant, memory stimulator, improve potent form. Fisetin is one amongst them. Strawberry
detoxification capacity of the liver etc. so it is necessary to contains more amount of fisetin (160μg/g of strawberry).
integrate this property into daily consumable form or 37 strawberries are needed to be consume daily to fulfill
product. The strawberries were oven dried, then were dietary needs. So it is necessary to integrate this property
subjected to extraction with methanol as solvent system. The into a daily consumable product .
liquid- liquid extraction technique was used for extraction
followed by HPLC for quantification. Then it was formulated
MATERIALS AND METHODS
in the edible form that is biscuits.
Sample preparation
Keywords— Flavonoids; fisetin; medicinal properties; 411 gms of strawberries were cut in fine slices and were
extraction; HPLC.
kept for oven drying for 55 hrs at 500°c, the strawberries
I. INTRODUCTION were grinded in electrical grinder and 30gm fine powder
Flavonoids and their conjugates form a very large group of was prepared. This powder was used as sample.
natural products. They are found in many plant tissues,
where they are present inside the cells or on the surfaces of Methanol Extraction
different plant organs. The chemical structures of this class 30gm strawberry powder was taken in a petri plate
of compounds are based on a C6-C3-C6 skeleton [7]. The followed by adding hydro-alcoholic mixture (methanol /
flavonoids may be modified by hydroxylation, water 80/20: v/v) for 72 hours, in the dark at room
methoxylation, or O-glycosylation of hydroxyl groups as temperature, with renewal of solvent every 24 hours (110
well as C-glycosylation directly to carbon atom of the ml x 3).
flavonoid skeleton. Different classes of flavonoids and their Solution was filtered by using cheese cloth and evaporated
conjugates have numerous functions during the interactions at 50°C. Refrigeration for 48hr after adding 200ml distilled
of plant with the environment, both in biotic and abiotic water followed filtration [5].
stress conditions. Additionally, flavonoid conjugates,
because of their common presence in plants, are important Liquid-liquid extraction
components of human and animal diet. Due to the different Chloroform used as a solvent system [5]. The filtrate was
biological activities of plant secondary metabolites, their poured in separating funnel. 30ml of chloroform was added
regular consumption may have serious consequences for in the apparatus and left for 2 min.The colourless part of
health, both positive and negative. For the mentioned the mixture was taken which was supposed to be
reasons, methods for the efficient and reproducible analysis containing flavonoids.
of flavonoids play a crucial role in research conducted in
different fields of the biological and medical sciences. Quantification of extract
Fisetin manufactured by Doctor’s best, Inc. was used as
Fisetin is a flavonol, a structurally distinct chemical
standard. 10 ppm fisetin standard was prepared by
substance that belongs to flavonoid group of polyphenols.
dissolving 0.2 mg of fisetin powder in 20 ml of extra pure
It can be found in many plants, fruits and vegetables.
methanol. The quantification of samples were done by
Berries contain more amount of fisetin as compared to
using HPLC of column C18 (250mm*4.6mm, 5μm), flow
other fruits. Eg.Blueberry, strawberry, raspber. It is known
rate was 1ml/min, 210 nm and methanol (HPLC grade) was
used as mobile phase.

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Published by : International Journal of Engineering Research & Technology (IJERT)
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Vol. 5 Issue 08, August-2016

Biscuit preparation
The extract was evaporated at 65°c to remove excess of
chloroform. Preheat oven to 230°c. In a large mixing bowl
we sift together 150 gm flour, 10 gm baking powder and
salt. cut in shortening with fork until mixture resembles
coarse crumbs. Then we pour milk into flour mixture while
stirring with the fork, mix in milk followed by adding
extract until dough is soft, moist and pulls away from the
side of bowl. Roll the dough out and cut with a knife. Then
we placed biscuit on ungreased baking sheet and baked in
preheated oven until golden brown, about 20 min [10].

Biscuit analysis
Prepared biscuit was sent for analysis of polyphenol
content, nutritional value and shelf life.

Sodium Hydroxide test

5mg of strawberry powder was added in a 10 ml D/W. The
solution was held on a burner till the solution become
warm and it was filtered through whatmann filter paper no
41. The filtrate was collected in another test tube. 2 ml of
10% aq. Sodium hydroxide was added to this solution
which gives yellow colour. A change in colour from yellow
to colourless on addition of dilute HCl occurred which is an
indication for the presence of flavonoid.

RESULT AND DISCUSSION

A. For Sodium Hydroxide test
Test tube Shows formation of yellow color due to
addition of filtrate and NaOH followed by adding Hcl until Fig.2 HPLC graph for Fisetin extract.
the color become colorless which is the indication of
presence of flavonoids. C. For Biscuit analysis
Sr No. Parameter Unit Value
B. For Quantification of extract
Nutritional value
1 Moisture % 03.04
2 Total minerals % 04.13
3 Crude Protein % 09.91
4 Crude Fat % 19.08
5 Crude Fiber % 00.12
6 Carbohydrate % 63.72
7 Energy Kcal/100g 466.24
m
Other Analysis
1 Shelf Life Month 1
2 Total polyphenols Mg/100gm 412.44
Nutritional value

Fig. 1 HPLC graph for Flavonoid standard (10ppm)

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Published by : International Journal of Engineering Research & Technology (IJERT)
http://www.ijert.org ISSN: 2278-0181
Vol. 5 Issue 08, August-2016

CONCLUSION
Fisetin has many medicinal properties so it is necessary
to incorporate these properties in human consumable form.
Fisetin is present abundantly in strawberry. The
confirmation test of strawberry powder for presence of
flavonoid was done and the result was positive. The
extraction of fisetin was successfully carried out by using
methanol as solvent system. And isolation was done in
chloroform by liquid-liquid extraction. The HPLC results of
sample was compared with fisetin standard, shows that
fisetin was present in sample. The Biscuits that was
formulated using fisetin is a functional food since it has the
beneficial effect of flavonoid (Fisetin). Biscuit was sent for
nutritional analysis and the results came positive. The idea
of combining all these ingredients and making a product
with the goodness of each of the ingredients gives the
synergistic effect in the functional food.

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