WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Hijazi et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 7, 1889-1898. Research Article ISSN 2278 – 4357

EXTRACTION OF PHENOLIC COMPOUNDS AND FLAVONOIDS

FROM ERYNGIUMCRETICUM L. BY CONVENTIONAL AND NON-
CONVENTIONAL EXTRACTION TECHNIQUES

Saeed Zeidan1, Akram Hijazi1*, Hassan Rammal1, Ahmad Kobaissi1,Bassam Badran1

1
Doctoral School of Science and Technology, Research Platform for Environmental Science
(PRASE), Lebanese University, Lebanon.

ABSTRACT
Article Received on
24 May 2014, Phenolic compounds are well-known phytochemicals found in all
Revised on 17 June
2014, plants. They can be broadly divided into non-soluble compounds such
Accepted on 03 July 2014
as condensed tannins, lignin, and cell-wall bounded hydroxycinammic
acid, and soluble phenolics such as phenolic acids, phenylpropanoids,
*Correspondence for Author flavonoids and quinones. In this work the extraction of total phenolic
Dr. Akram Hijazi compounds (TPC) from LebaneseEryngiumcreticum was performed by
Doctoral School of Science and
two techniques: Conventional such as maceration, reflux and soxhlet
Technology, Research Platform
extraction, and non-conventional extraction like microwave assisted
for Environmental Science
(PRASE), Lebanese extraction using ethanol as a solvent. The results shows that among the
University, Lebanon conventional extraction techniques reflux and Soxhlet techniques gave
approximately the same yield of 687.8 mg and 685.23 mg respectively
higher than that obtained by maceration technique (410.93 mg), however, Microwave assisted
extraction gave the highest yield among both the conventional and non-conventional
extraction techniques (950 mg).

Keywords: Eryngiumcreticum, phenolic compounds, extraction, conventional and

nonconventional techniques.

INTRODUCTION
EryngiumcreticumL. is a perennial plant belonging to the family Umbellifereae, mainly found
in Lebanon, Palestine, Jordan, also spread in Spain, France, Germany, Balkan and other
scattered localities in Europe, and in Africa as well. E.creticum is cultivated for use as
vegetables mainly in salads; it is also used traditionally as laxative and to treat skin infections

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Hijazi et al. World Journal of Pharmacy and Pharmaceutical Sciences

and tumors. It is an antidote, used in the treatment of snake bites. E. creticum also showed
anti-inflammatory and anti-microbial activities, it was also used in the treatment of poisoning,
anemia and infertility. This plant has also an antioxidant property by inhibiting the lipid
peroxidase in the liver of rats [1, 2]. This plant includes a wide range of chemicals such as
phenolic acids and flavonoids.

Phenolic compounds are considered as secondary metabolites that are synthesized by plants
during normal development and in response to stress conditions such as infection, wounding,
and UV radiation [3]. These compounds can be classified into non-soluble or soluble
compounds which are involved in many processes of plants and animals [4]. Phenolics are
utilized as coloring agents, flavoring, aromatizens and antioxidants. They are found
ubiquitously in fruits, nuts, seed, flowers, vegetables, barks and herbs [5]. On the other hand,
flavonoids form a large group of natural products found in many plant tissues, where they are
present inside the cells or on the surface of different plant organs. Flavonoids carry different
physiological and biochemical roles in the various cell types and organs; they consist of more
than 6.000 different compounds that have numerous functions during the interactions of plant
with the environment, both in biotic and abiotic stress conditions [6]. Flavonoids are the most
common phenolics responsible also alongside the carotenoids and chlorophylls for their blue,
purple, yellow, orange and red colors [7].

Plant extracts are widely used in the food, pharmaceutical and cosmetics industry. Extraction
techniques have been widely investigated to obtain such valuable natural compounds from
plants for commercialization [8]. Conventional methods for extraction of phenolic
compounds include maceration, reflux, and soxhlet extraction.These techniques have been
used for many decades; however, they are often time-consuming and require relatively large
quantities of polluting solvents [9]. So, the substitution of these methods allow the use of
lesser amount of solvents for the extraction of organic and in-organic compounds from plant,
so, the alternative method is Microwave Assisted Extraction (MAE) which is economically
promising (simple and efficient) technique which is referred to as non-conventional
extraction technique [10].

The purposes of this study were to determine at first the phytochemical screening of
Eryngiumcreticum and to give a comparison between the conventional and non-conventional
extraction techniques for the extraction of phenolic compounds.

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MATERIALS AND METHODS

1.Collection and preparation of plant material
Fresh plants were gathered from Rowiest Al Ballout (Mount Lebanon) on February 2014.
Then, plants were well-cleaned and washed with water and left to shade dried under room
temperature away from sun light. After this period, the collected plants have been grinded by
a grinder to obtain a powder form; the powder is then preserved in a container away from
light, heat and moisture for later use.

2.Preparation of crude extracts

In the preparation of crude extraction each extraction technique is repeated for three times.

2.1.Maceration method
5 g of E.creticum powder was placed in a beaker with 250 ml ethanol. The solution is
macerated under room temperature for 48 hours with agitation of 360 rpm. After maceration
the solution is filtrated under vacuum and concentrated by a rotary evaporator under 40°C
and a low pressure. Then, the obtained extracts were stored in the refrigerator for later
analysis [11].

2.2. Reflux method

5 g of E.creticum powder was placed in a round bottom flask with 250 ml ethanol. The
solution is refluxed for 4 hours under heat. After reflux the solution is filtered under vacuum
and concentrated by a rotary evaporator under 40°c and a low pressure. Then, the obtained
extracts were stored in the refrigerator for later analysis [11].

2.3. Soxhlet method

5 g of E.creticum powder was placed in a round bottom flask with 250 ml ethanol. The
solution is subjected for soxhlet extraction for 4 hours under heat. After soxhlet, the solution
is filtered under vacuum and concentrated by a rotary evaporator under 40°c and low
pressure. Then, the obtained extracts were stored in the refrigerator for later analysis. (Note
that: the experiment is repeated two times) [12].

2.4.Microwave Assisted Extraction (MAE)

A domestic microwave oven (KOG-3767, DAEWOO), was used in this study, the microwave
has a total capacity of 850 W. 1 g of Eryngiumcreticum powder was placed in threaded round
bottom top PFA vials with 50 ml ethanol. The vials were inserted into the microwave oven in

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to PFA beaker. The resulting mixtures were irradiated with microwaves (750 powers). The
irradiation is achieved for 2 min with 45s power on, 30s power off, then 15s power on. After
the irradiation, the samples were allowed to cool at room temperature, then, filtrated under
vacuum and concentrated by a rotary evaporator under 40°C and low pressure. Then, the
obtained extracts were stored in the refrigerator for later analysis [13].

3.Preliminary phytochemical screening

a.Detection of alkaloids
5 drops of the Dragendorff reagent was added to 1 ml of the extracts obtained from each
method. The appearance of red to orange precipitates indicates the presence of alkaloids [14].
b. Detection of tannins
10 ml of the extracts obtained by different extraction methods were filtered by a filter paper
(0.45 ). Many drops of ferric chloride (FeCl3 1%) have been added to each of the different
filtered extracts. The presence of tannins was indicated by the appearance of blue color [14].
c. Detection of Terpenoids
1 ml of acetic anhydride was added to 2 ml of concentrated sulfuric acid and then mixed with
1 ml of each of the previously filtered extracts. The presence of reddish brown color on the
interface indicates the presence of terpenoids [14].
d. Detection of resins
10 ml of each of the previous filtered extracts were added to 20 ml of HCl (4%). The
appearance of turbidity indicates the presence of resin in the extracts [15].
e. Detection of phenolic acids
In beakers, 5 ml of each of the pervious filters extracts were taken and 1 ml of FeCl3 (1%)
and 1 ml of K3(Fe(CN)6) (1%) were added. The appearance of fresh radish blue color
indicates the presence of polyphenols [16].
f. Detection of flavonoids
Two solutions (A) and (B) were prepared:
Solution A: 5 ml of each of the extracted obtained previously.
Solution B: 5 ml of ethanol (50%) added to 5 ml of KOH (50%).
The two solutions were then mixed together and the appearance of yellow color indicates the
presence of flavonoids [17].
g.Detection of Quinones
To 1 ml of the previously obtained extracts, 1 ml of sulfuric acid was added. Formation of red
color indicates the presence of Quinones [18].

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h. Detection of Saponins
The test tubes containing each of the previous filtered extracts were shacked for 5 min using a
vortex. The appearance of big foamy indicates the presence of saponins [19].

RESULTS AND DISCUSSION

Figure 1: conventional extraction techniques, A:

maceration, B: reflux, C: soxhlet

Figure 1: amount of extracts obtained by A: maceration method, B: reflux method, C:

soxhlet method

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Amount of extracts obtained by the conventional extraction techniques

The amount of extracts obtained varies from one extraction method to another in which
maceration method gave 420, 400.2, and 412.6 mg (M1, M2 and M3) of extracts in the three
replicates respectively (figure 1A), while reflux method gave 683.8, 691.4, and 688.2 mg
(R1, R2, and R3) of extracts in the three replicates respectively (figure 1B).On the other
hand,soxhlet method gave 690.6, 678.9, and 686.2 mg (S1, S2, and S3) of extracts in the
three replicates respectively (figure 1C).

Comparison between the conventional extraction techniques for the amount of extracts
obtained
Among the three conventional extraction methods, reflux and soxhlet gave approximately the
same results after comparing the means of the amount of extracts obtained by these methods,
in which reflux and soxhlet gave a mean of 687.8 mg and 685.23 mg respectively.These
amounts of extracts are higher than that obtained by maceration extraction technique (410.93
mg) (figure 2). So, we can say that among the conventional extraction techniques, both reflux
and soxhlet can be used efficiently for the extraction of phenolic compounds and flavonoids
from plant material.

As a comparison among reflux and soxhlet, soxhlet is the preferred method for extraction
because the plant materials are not subjected to direct heat as that in the reflux method, so
there is no lose or degradation of the thermo liable of volatile compounds.

Figure 2: mean of the amount of extracts obtained by the three conventional extraction
techniques(maceration: M, reflux: R, and soxhlet: S)

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Microwave assisted extraction and comparison between conventional and non-

conventional extraction techniques
The amounts of extracts obtained by (MAE) were 188.84, 199.3, and 181.86 mg respectively
for 1 g of powder used.So, we can say that the amounts of extracts obtained from 5 g of
powder are 944.2, 996.5, and 909.3 mg (Mic1, Mic2, and Mic3) respectively (figure 3). The
mean of the amounts of extracts obtained by MAE (Mic= 950) is higher than those obtained
by other extraction techniques. So, MAE as a non-conventional extraction gave the highest
amount of extracts (950 mg) than those obtained maceration, reflux, and soxhlet (410.93,
687.8, and 685,23 respectively) is more effective for extraction of phenolic compounds and
flavonoids from plant material (figure 4).

Figure 3: amount of extracts obtained by MAE

Figure4: mean of extracts obtained by conventional and non-conven

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Preliminary phytochemical screening

Table 1 shows the phytochemical screening of Eryngiumcreticum.Mainly resin, phenolic acid
and flavonoids are found in higher concentrations in microwave assisted extraction.
However, tannins, saponins, and terpenoids are absent in all extraction techniques. Alkaloids
are found in all the extraction techniques except in maceration and with the higher amounts in
microwave assisted extraction.Quinones are only present in microwave assisted extraction
and absent in all the other extraction techniques. So, microwave assisted extraction contains
not only the higher amount of extracts but also the higher amounts of different chemical
compounds as shown in table 1.

Table 1: Phytochemical screening of E.creticum using different extraction techniques

M1 M2 M3 R1 R2 R3 S1 S2 S3 Mic1 Mic2 Mic3
Alkaloids − − − + + + + + + ++ ++ ++
Tannins − − − − − − − − − − − −
Resin + + + + + + + + + + + +
Saponins − − − − − − − − − − − −
Phenolic
++ ++ ++ ++ ++ ++ ++ ++ ++ +++ +++ +++
acid
Terpenoids − − − − − − − − − − − −
Flavonoids + + + + + + + + + ++ ++ ++
Quinones − − − − − − − − − + + +
+++ = high amount after the addition of reagent immediately; ++ = moderate amount after 5
minutes of reagent added; + = low amount after 10 minutes of reagent added and - = absence
of active compound after 20 minutes

Figure5: effect of time on extraction by different extraction techniques

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Effect of extraction time on the amount of extracts of extracts obtained

The extraction time varies form one technique to another in which even though maceration
had the highest extraction time (2880 min), it gave the lowest amount of extracts (410.93
mg). Reflux and soxhlet are performed for 240 min and they gave approximately the same
amount of extracts, while MAE which is only performed for 2 min gave the highest amount
of extracts (950 mg)(figure 5). So, MAE is the most effective extraction technique.

CONCLUSION
According to the results obtained in conventional extraction techniques, reflux and soxhlet
had the highest extraction yield and maceration had the lowest one. The results also proved
that microwave assisted extraction is the most effective technique as compared to other
methods. The extraction time was reduced, less solvent was used and the amount of extracted
compounds was increased. So, microwave assisted extraction is the best extraction techniques
for bioactive compounds from Eryngiumcreticum L.

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