Quantitation of phytochemical constituents of Fumaria vaillantii L. with different extract methods

ARTICLE
TMR | September 2019 | vol. 4 | no. 5 |237
Submit a manuscript: https://www.tmrjournals.com/tmr
doi: 10.12032/TMR20190905134
Persian Medicine
Quantitation of phytochemical constituents of Fumaria vaillantii L.
with different extract methods
Fahimeh Mohajerani1, 2
, Zeinab Pourjabbar2
, Fatemeh Zamani Mazdeh3
, Roja Rahimi1
, Gholam-Reza
Amin2
, Tayebeh Toliyat4
, Sareh Kargar2
, Mannan Hajimahmoodi1, 2, 3
*
1
Persian Medicine and Pharmacy Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran 1416753955, Iran. 2
Drug and Food Control Department, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran 1416753955, Iran. 3
Food and Drug Administration, Tehran University of Medical Sciences,
Tehran 1416753955, Iran. 4
Pharmaceutical Department, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran 1416753955, Iran.
*Corresponding to: Mannan Hajimahmoodi, Drug and Food Control Department, Faculty of Pharmacy, Tehran
University of Medical Sciences, 21 Dameshgh St., Vali-e Asr Ave., Tehran 1416753955, Iran. Email:
hajimah@sina.tums.ac.ir.
Highlights
Total phenolic, total flavonoid, total alkaloid, ascorbic and organic acids yields in Fumaria vaillantii L.
aerial parts with different extract methods are evaluated and compared by spectrophotometric and HPLC
methods.
Traditionality
The genus fumaria (Fumariaceae or Papaveraceae) includes more than 40 species in the world. Seven
species are found in Iran. Fumaria vaillantii L. is one of the species which grow in a wide variety of areas
of Iran with the common name of fumitory or earth smoke. The aerial parts of the plant which harvested
during flowering time are used for medicinal purposes.

ARTICLE
TMR | September 2019 | vol. 4 | no. 5 |238
Submit a manuscript: https://www.tmrjournals.com/tmr
doi: 10.12032/TMR20190905134
Abstract
Objective: The genus fumaria includes more than 40 species in the world. The aim of this study was to quantify the
phytochemical constituents of Fumaria vaillantii L. aerial parts and compare the different methods of extraction.
Total phenol, total flavonoid, total alkaloid, ascorbic and organic acids (oxalic, maleic, citric, succinic and fumaric
acids) yields were evaluated in terms of the temperature effect, type of solvent and maceration time. Methods:
Dried plant samples were extracted by different procedures. Total phenolic, total flavonoid, total alkaloid and
ascorbic acid yields were determined by spectrophotometric methods. Also, the organic acid yields were analyzed
using high performance liquid chromatography method. Results: With the same extraction method, the natural
flora extract was showed more yields of oxalic, maleic and citric acids than the commercial one, while the
commercial extract was showed more yields of total phenol, ascorbic, succinic and fumaric acids than the natural
flora one. The water-boiled extract was showed more yields of total phenol and total flavonoid. The macerated in
ethanol 80% extract was also demonstrated more amounts of total alkaloid and ascorbic acid. Among different
aqueous macerated extracts of the commercial sample, as the maceration time increased, total phenol, total
flavonoid, oxalic, maleic, succinic, fumaric and ascorbic acids yields decreased. Macerated commercial dried
fumitory in double-distilled water for 24 hrs resulted in an extract with the highest possible fumaric acid yield.
Conclusion: It can be concluded that both water-boiled and macerated in ethanol 80% extracts can be used as rich
sources of total phenolic and total flavonoid, which are considered as the important antioxidants.
Keywords: Total phenolic, Total flavonoid, Total alkaloid, Organic acid, Fumaria vaillantii L.
Acknowledgments:
This work was supported by a grant from Persian Medicine and Pharmacy Research Center, Faculty of
Pharmacy, Tehran University of Medical Sciences, Tehran, Iran numbers 94-03-96-30194 & 95-03-96-32757.
Abbreviations:
ROS, Reactive oxygen species; HPLC, High performance liquid chromatography; ANOVA, Analysis of
variance; AtE, Atropine equivalent; GaE, Gallic acid equivalent; QE, Quercetin equivalent; DW, Dried weight.
Competing interests:
The authors declare that they have no conflict of interest.
Citation:
Fahimeh Mohajerani, Zeinab Pourjabbar, Fatemeh Zamani Mazdeh, et al. Quantitation of phytochemical
constituents of Fumaria vaillantii L. with different extract methods. Traditional Medicine Research 2019, 4 (5):
237-245.
Executive Editor: Cui-Hong Zhu, Nuo-Xi Pi.
Submitted: 27 February 2019, Accepted: 12 May 2019, Online: 18 August 2019.

ARTICLE
Submit a manuscript: https://www.tmrjournals.com/tmr TMR | September 2019 | vol. 4 | no. 5 |239
doi: 10.12032/TMR20190905134
Background
The genus fumaria (Fumariaceae or Papaveraceae)
includes more than 40 species in the world [1]. Seven
species are found in Iran [2]. Fumaria vaillantii L. is
one of the species which grow in a wide variety of
areas of Iran with the common name of fumitory or
earth smoke [3]. The aerial parts of the plant which
harvested during flowering time are used for medicinal
purposes. The whole plant are claimed to be
anti-allergic drug, antipyretics, choleretic, diuretic,
laxative and blood-puriﬁer in traditional and folkloric
medicine and used for controlling of hepatobiliary,
dermatological, and gastrointestinal disorders [4, 5].
Today, fumaria extracts are components of several
phytotherapeutic preparations, which are used mostly
in cases of minor hepatobiliary dysfunction,
gastrointestinal diseases and skin disorders [6].
Typically, reactive oxygen species (ROS) are often
by-products of biological and metabolic reactions in
body cell or due to external factors [7]. ROS can cause
tissue damage which results in inflammatory,
cardiovascular, neurodegenerative diseases and even
cancers. Antioxidants are capable of inhibiting or
delaying oxidative activity of free radicals [8]. In
recent decades, there has been an increasing interest
for natural antioxidants especially herbal ones due to
carcinogenic potential of synthetic antioxidants [9].
The protective action of medicinal plants against
oxidative stress is due to the presence of antioxidants,
especially phenolic, flavonoids compounds and
antioxidant vitamins [10]. Different antioxidant
compounds have been reported from fumaria species
[11]. These compounds include alkaloids (often
referred to as fumitory alkaloids or protopine-like
alkaloids), flavonoids, glycosides, tannins, saponins,
anthraquinones, steroids and triterpenoids [12].
Moreover, the presences of organic acids in different
parts of the fumitory plant have been established by
several recent studies [13-15]. Organic acids have
basic functions in plant organisms such as respiration,
reproduction, storage, cell division and growth [16].
Likewise, organic acids have been identified as the
metabolites of Krebs cycle in the plants [17]. They are
naturally found in vegetables and fruits [18]. The
profile and concentration level of organic acids depend
on plant variety, region, extracting techniques and
aging process [19, 20].
In the present study, total phenolic, total flavonoid,
total alkaloid and ascorbic acid yields in different
extracts of Fumaria vaillantii L. aerial parts were
determined by spectrophotometric methods.
Furthermore, simultaneous determination of organic
acids including oxalic, maleic, citric, succinic and
fumaric acids in these extracts was developed using a
cation-exchange column high performance liquid
chromatography (HPLC) method.
Methods
Chemicals and reagents
All solvents, reagents and standards were purchased
from Sigma-Aldrich Chemical (St. Louis, MO, USA)
and Merck (Darmstadt, Germany).
Plant material
Two types of Fumaria vaillantii L. were used in this
study. The herbal market sample was purchased from
the herbal drug market of Tehran, Iran (originated from
Shiraz city, Fars province, Iran), which is commonly
used in traditional medicine of Iran. The natural flora
sample was collected from the natural flora of Alamut
Mountain, Qazvin Province, in the north of Iran in
April 2015 when the aerial parts of the plant were at
the flowering and fruit setting stage. The natural flora
sample was compared with the herbal market one
extracted by same method in terms of targeted
phytochemical properties. They were authenticated at
the herbarium of the Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran and were
the same species of fumaria. Their voucher specimens
were deposited at the herbarium of the Faculty of
Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran (Reference number: PMP-335 for the
commercial sample and 6530-THE for the natural flora
one). The plant was dried in shade at room temperature
and ground to get a coarse powder.
Sample preparation
All the samples were dried and ground. The
commercial sample (100 g) was extracted through
different methods including boiling in double distilled
water for 15 mins (extract no.1), maceration in ethanol
80% for 24 hrs (extract no.2) and maceration in double
distilled water (600 mL) for 72, 48 and 24 hrs (extracts
no.3, no. 4 and no.5). The natural flora sample was
only extracted by maceration in double distilled water
for 24 hrs (extract no.6) in order to be compared with
the commercial one with the same extraction method.
In all the extraction methods, the solvent to material
ratio was 6 : 1. Aqueous extraction was investigated in
24, 48 and 72 hrs in order to determine the effect of
maceration time on the yields of targeted
phytochemical components. The ethanolic extracts
were prepared in order to be compared with the
distilled water extracts in the same extraction condition
(maceration for 24 hrs). One method of extraction was
only used for the fresh sample in order to be compared
with the herbal market extract with the same extraction
condition (maceration for 24 hrs in aqueous solvent).
All the extracts were filtered through sieve no.20 (20
mesh size) and concentrated in a rotary evaporator
under reduced pressure and low temperature (45°C)
conditions. Drying aqueous extracts was done by using

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doi: 10.12032/TMR20190905134
freeze dryer. Then, the yield value of each extract was
calculated.
Spectrophotometric analysis
Determination of total phenolic yield. Total phenolic
yield was determined according to the
Folin-Ciocalteau method with slight modification [21].
0.1 g of dried extract was transferred to a 10 mL
volumetric flask and diluted with 50% methanol
solution (dilution factor = 100). After being sonicated
for 15 mins, the solution was filtered through
Whatman paper. A set of gallic acid standard solutions
covering the concentration range between 25 and 125
μg mL-1
were prepared (diluted with 50% methanol
solution). 200 µL of the dilute solution (50% methanol
solution) and each of the standard solutions were
separately transferred into test tubes. After adding 1.5
mL of Folin-Ciocalteu reagent (previously diluted 1 :
10 with double distilled water) into all the tubes, they
were incubated at room temperature for 5 mins. Then,
1.5 mL of sodium bicarbonate solution (60 g L-1
) was
added to all the tubes and incubated again in darkness
at room temperature for 90 mins. All the samples were
sonicated for 15 mins to evacuate the gases. To prepare
the blank, 400 µL of double distilled water was
transferred to a test tube. Next that, 3 mL of
Folin-Ciocalteu reagent were added to the tube, and
were incubated at room temperature for 5 mins. Then,
3 mL of sodium bicarbonate solution (60 g L-1
) was
added to the tube and incubated again in darkness at
room temperature for 90 mins. The absorbance was
measured against the blank at 725 nm with an UV/VIS
spectrophotometer. All determinations were performed
in triplicate. Total phenol yield was expressed as mg of
gallic acid equivalent (GaE)/g of dried plant.
Determination of total flavonoid yield. Total
flavonoid yield was measured by using aluminum
chloride colorimetric method with some modification.
This method is based on the formation of
flavonoid-aluminum complex [22]. 0.05 g of dried
extract was transferred to a 10 mL volumetric flask and
diluted with 50% methanol solution (dilution factor =
200). The solution was sonicated for 15 mins and then
was filtered through Whatman papers. A set of
quercetin standard solutions covering the concentration
range between 5 and 100 μg mL-1
were prepared
(diluted with 50% methanol solution). 10 mL of 50%
methanol solution and each of the standard solutions
were separately transferred to 25 mL volumetric flasks.
To prepare the blank, 10 mL of 50% methanol solution
was transferred to a 25 mL volumetric flask. After
adding 1 mL of aluminum chloride reagent 2% (w/v)
to all volumetric flasks, they were diluted with
methanolic acetic acid 5% (v/v). All samples were
incubated at room temperature for 30 mins. Next they
were sonicated for 15 mins to evacuate the gases. The
absorbance was measured against the blank at 415 nm
with an UV/VIS spectrophotometer. All determinations
were performed in triplicate. Total flavonoid yield was
expressed as mg of quercetin equivalent (QE)/g of
dried plant.
Determination of total alkaloid yield. Total alkaloid
yield was determined by using a UV
spectrophotometer method with some modification.
This method is based on the reaction between alkaloid
and bromocresol green [23, 24]. The concentration
range of standard atropine solutions was between 10
and 50 μg mL-1
. 0.1 g of dried extract was transferred
to a 10 mL volumetric flask and diluted with
chloroform (dilution factor = 100). All experiments
were performed in triplicate. Total alkaloid yield was
expressed as mg of atropine equivalent (AtE)/g of
dried plant.
Determination of ascorbic acid yield. Ascorbic acid
yield was measured by using the 2,4-dinitrophenyl
hydrazine method [25]. For preparing
2,4-dinitrophenyl hydrazine, thiourea, copper sulphate
(DTC solution), 3 g of powdered 2,4-dinitro-phenyl
hydrazine, 0.4 g of tiourea and 0.05 g of copper sulfate
were transferred to a 100 mL volumetric flask and then
diluted with sulfuric acid 9 N solution. This solution
was stable for at least one week.
0.1 g of ascorbic acid was transferred to a 100 mL
volumetric flask and diluted with trichloroacetic acid
5% solution. A set of reference standard solutions of
ascorbic acid covering the concentration range
between 5 and 50 µg mL-1
were prepared (diluted with
methanol). 0.1 g of dried extract was transferred to a
10 mL volumetric flask and diluted with double
distilled water (dilution factor = 100). After being
sonicated for 15 mins, the solution was filtered through
Whatman paper. 400 µL of the extract solution and
each concentration of standard solutions were
separately transferred into the test tubes. To prepare the
blank, 200 µL of double distilled water was transferred
to a test tube. Next, 80 µL of DTC solution (2,
4-dinitrophenyl hydrazine, thiourea, copper sulphate)
added into all the tubes and they were incubated in
water bath (37°C) for 3 hrs. The samples were then
placed in ice for 10 mins and then 600 µL of sulfuric
acid 65% solution was added to all the tubes and were
shaken vigorously. All the tubes were incubated at
room temperature for 30 mins. The absorbance was
measured against the blank at 520 nm with an UV/VIS
spectrophotometer. All determinations were performed
in triplicate. Ascorbic acid yield was expressed as mg
of ascorbic acid/g of dried plant.
HPLC analysis
Apparatus and chromatographic conditions.
Organic acids including oxalic, maleic, citric, succinic
and fumaric acids were analyzed by using a Knauer
HPLC system (Germany) coupled to a UV Detector
K-2500 monitored at the 210 nm, equipped with a
degassor, an injection valve (10-µL sample loop), a
quatry pump, an oven Knauer, a cation-exchange

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doi: 10.12032/TMR20190905134
Eurokat H, vertex plus column (300 × 8 mm, 10 µm)
as the stationary phase and the Chem32 software. 10
μL aliquots of the diluted samples were injected into
the system. Separation was performed at the pressure
of 3 MPa with an isocratic flow rate of 0.7 mL min-1
of
0.003 N sulfuric acid in deionized water (pH 3.2) as
the mobile phase. In order to achieve complete
separation of peaks in the chromatogram, this analysis
would need to be run at 60°C.
Standard preparation. The standard organic acids
were precisely weighed (5 mg) and dissolved in 5 mL
of deionized water. A set of serial dilutions of each
organic acid was prepared. The concentration ranges of
standard solutions were 1-30 μg mL-1
for fumaric acid
and oxalic acid, 5-60 μg mL-1
for citric acid and
succinic acid and 0.5-20 μg mL-1
for maleic acid. Then,
a 10-µL aliquot of each serial dilution was injected into
the HPLC system.
Determination of yield of organic acid. The organic
acids were analyzed in different dried extracts of
Fumaria vaillantii L., 10 mg of each dried extract was
precisely weighed and dissolved in 5 mL of deionized
water in a 5 mL volumetric flask by using a sonicator
for 2 mins (dilution factor = 500). The solution was
filtered through a 0.45 µm PVDF membrane. Then, it
was injected into the HPLC system.
Statistical analysis
The obtained data were considered with the SPSS
statistical package, version 21 (SPSS Inc. Chicago, IL,
USA). Analysis of variance (ANOVA) was applied for
evaluation the differences of distribution between
different methods of extraction.
Results
Determination of yield for different methods of
extraction
According to the results (Table 1), the highest
extractive value (43.9%) was obtained in the extraction
method of boiling at 100°C for 15 mins. Among the
different maceration methods, the ethanolic (80%)
extract had slightly higher extractive value (20.3%) in
comparison with the aqueous macerated extracts.
Furthermore, in the method of maceration with double
distilled water, as the maceration time increased, the
amount of extractive value decreased from 24 to 48 hrs,
and then remained approximately consistent from 48 to
72 hrs.
Yields of total phenolic, total flavonoid, total
alkaloid, ascorbic acid and organic acids
In the present study, we investigated the yields of total
phenolic, total flavonoid, total alkaloid, ascorbic acid
and organic acids in different extracts of Fumaria
vaillantii L. growing in Iran. Calibration equations,
correlation coefficients and validation parameters for
different analysis methods were shown in Table 2 for
the yields of total phenolic, total flavonoid, total
alkaloid and ascorbic acid and in Table 3 for the
organic acids.
The results for yields of total phenolic, total
flavonoid, total alkaloid and ascorbic acid extracted
with different methods were presented in Table 4.
These results show mg of the active ingredient per 1 g
of dried plant [mg/g dried weight (DW)]. According to
One way ANOVA analysis, there are significant
differences between the yields of total phenol,
flavonoid, alkaloid and ascorbic acid in various
methods of extraction (all P < 0.001). The water-boiled
extract is showed more yields of total phenol and total
flavonoid (P < 0.001). The macerated in ethanol 80%
extract is also demonstrated more amounts of total
alkaloid and ascorbic acid (P < 0.001). Flavonoid has
not significant difference in commercial samples
macerated in double distilled water for 72 hrs and 48
hrs.
The results for organic acids were demonstrated in
Tables 5. These results show mg of the active
ingredients per 1 g of dried plant (mg/g DW). One way
ANOVA analysis approves that the yields of fumaric,
citric, succinic, oxalic and maleic acids had significant
differences in various methods of extraction (all P <
0.001). Macerated commercial dried fumitory in
double-distilled water for 24 hrs results in an extract
with the highest possible yield of fumaric acid (P <
0.001).
Discussion
Regarding different methods of extraction, the yields
of total phenolic, total flavonoid, total alkaloid and
ascorbic acid in dried commercial sample of Fumaria
vaillantii L. ranged in the limits of 0.999-5.481 mg
GaE/g DW, 0.558-5.585 mg QE/g DW, 0.266-1.009
mg AtE/g DW and 0.106-0.737 mg ascorbic acid/g DW,
respectively. Water-boiled extract had the highest
yields of total phenol and total flavonoid among all the
investigated extracts when the extractive values were
considered in different extraction methods.
Furthermore, the highest ascorbic acid and alkaloid
yield was found in ethanolic extract. With the same
extraction method, the commercial extract showed
higher yields of total phenol and ascorbic acid than
natural flora one.
According to Soušek et al, methanolic extract of
Fumaria vaillantii L showed a high yield of total
phenol as 20.41 mg GaE/g DW [6]. Orhan et al. found
that total phenolic and flavonoid yields in the ethanolic
extracts of four fumaria species including F. cilicica
Hausskn., F. densiflora DC., F. kralikii Jordan and F.
parviflora Lam., growing in Turkey, were in the range
of 0.015-0.030 mg GaE/g DW and 0.006-0.017 mg
RutinE/g DW, respectively [26]. Abbasi et al. was
carried out a research on total phenolic yield in the
aqueous extract of F. indica and was reported 0.047 mg

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doi: 10.12032/TMR20190905134
LOD: Limit of detection; LOQ: Limit of quantitation.
Table 2 Analytical characteristics of the method validation for different spectrophotometric methods
Analyzed
content
Reference
standard
Concentration
range of
standard
solutions
(μg mL-1
)
Calibration equation
Correlation
coefficient
(R2
)
LOD
(μg mL-1
)
LOQ
(μg mL-1
)
Total phenolic Gallic acid 25-125 y = 0.0061x － 0.0184 0.998 6.087 18.446
Total flavonoid Quercetin 10-100 y = 0.0216x + 0.0748 0.9997 2.139 6.481
Total alkaloid Atropine 10-50 y = 0.0165x － 0.0263 0.998 2.623 7.948
Ascorbic acid Ascorbic acid 10-50 y = 0.0242x + 0.1581 0.999 2.697 8.174
GaE/g DW for total phenolic yield [9]. In a recent
study, Jaberian et al. indicated that the methanolic
extract of F. vaillantii had more total phenol in
comparison to methanol-water extract (10.5 compared
to 4.27 mg GaE/g DW), whereas total flavonoid yield
of the methanol-water extract was higher than the
methanolic extract (3.11 in comparison with 2.07 mg
QE/g DW) [1]. Ivanov et al. reported high total
phenolic and flavonoid yields for ethanolic extracts of
F. officinalis, F. thuretii, F. kralikii, F. rostellata and F.
schrammii, growing in Bulgaria, which ranged in the
limits of 20.20-30.30 mg GAE/g DW and 8.70-16.62
mg QE/g DW, respectively [21].
In the current study, total alkaloid yield of
commercial Fumaria vaillantii L dried aerial parts
ranged in the limit of 0.266-1.009 mg AtE/g DW, while
Maiza-Benabdesselam et al. reported total isoquinoline
alkaloid yield of methanolic extract of two different
fumaria species including F. capreolata L. and F.
bastardi L., were 4.26 and 5.21 mg AtE/g DW,
respectively [22]. According to a research carried out
by Rathi et al, the concentration of protopine in the
hydroalcoholic (ethanol 80%) extract of Fumaria
vaillantii L. was reported 0.2 mg/g DW, whereas in
this study, using the same solvent for the extraction,
total alkaloid yield was found 1.009 mg AtE/g DW
[23]. According to Wagner and Bladt, total alkaloid
yield in F. officinalis which expressed as
protoberberine type alkaloid was reported 5-10 mg/g
DW [24]. In the present study, ascorbic acid yield of
commercial F. vaillantii Liosel. dried aerial parts
ranged in the limit of 0.106-0.737 mg ascorbic acid/g
DW. As comparison, in a study done by Kanaujia et al.
ascorbic acid yield in F. indica was reported in the
range of 0.3-0.65 mg ascorbic acid/g DW [25]. It can
be concluded that Fumaria vaillantii L. is rich in total
phenol, total flavonoid, total alkaloid and ascorbic acid
yields and can be used as an antioxidant plant. In the
present study, regarding different extracting methods,
oxalic, maleic, citric, succinic and fumaric acid yields
in dried commercial sample of Fumaria vaillantii L.
ranged in the limits of 0.0703-0.2551, 0.0352-0.0905,
0.5818-2.3596, 0.685-12.8921 and 0.6529-1.3372 mg
organic acid/g DW, respectively. Citric, succinic and
fumaric acids were found in all the extracts. The
obtained results showed different levels of organic acid
yields in two different samples of Fumaria vaillantii L.
which were extracted by the same method (aqueous
macerated for 24 hrs). The natural flora sample extract
had more oxalic, maleic and citric acid yields than
commercial one, whereas the commercial sample
extract showed more succinic and fumaric acid yields
than natural flora one. Among five different extracts of
the commercial sample, the water-boiled extract were
showed the highest yields of oxalic, citric and succinic
acids. Also, the highest maleic and fumaric acid yields
were found for the aqueous macerated (for 24 hrs)
extract. The results also indicated that among different
aqueous macerated extracts of the commercial sample,
by increasing the maceration time the yields of oxalic,
Table 1 Yields in the extraction of 100 g of dried aerial parts
Extract no. Extract method Yields (%)
1 Commercial sample boiled in double distilled water for 15 mins 43.9
2 Commercial sample macerated in ethanol 80% for 24 hrs 20.3
3 Commercial sample macerated in double distilled water for 72 hrs 14.2
6 Natural flora sample macerated in double distilled water for 24 hrs 12.8

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Table 3 Analytical characteristics of the method validation for HPLC method
Analyzed
organic
acid
Concentra-
tion range
of standard
solutions
(μg mL-1
)
Calibration
equation
Correlation
coefficient
(R2
)
LOD
(μg mL-1
)
LOQ
(μg mL-1
)
Concentration
of spiked
solution
(μg mL-1
)
Recovery
(%)
Oxalic acid 1-30 y = 0.4142x - 0.2759 0.9999 0.254 0.769 10 93.60
Maleic acid 0.5-20 y = 2.9136x + 0.423 0.9999 0.155 0.471 5 90.34
Citric acid 5-60 y = 0.0394x - 0.0031 0.9998 1.101 3.065 20 92.49
Succinic
acid
5-60 y = 0.016x + 0.0188 0.9997 0.993 3.010 20 94.20
Fumaric
acid
1-30 y = 2.9187x + 0.8682 1 0.117 0.355 10 97.80
Table 5 Yields of organic acid (mg/g) DW of Fumaria vaillantii L. aerial parts
Extract no. Oxalic acid Maleic acid Citric acid Succinic acid Fumaric acid
1 0.2551 ± 0.0008 0.0325 ± 0.0006 2.3596 ± 0.021 12.8921 ± 0.08 0.9224 ± 0.011
2 ND ND 0.7166 ± 0.012 3.9926 ± 0.039 0.9306 ± 0.015
3 0.0703 ± 0.0009 ND 0.5818 ± 0.010 0.6855 ± 0.013 0.1879 ± 0.003
4 0.0895 ± 0.0017 0.0627 ± 0.0012 1.9393 ± 0.025 1.4637 ± 0.028 0.6529 ± 0.005
5 0.0896 ± 0.0016 0.0905 ± 0.0017 1.3977 ± 0.026 8.5063 ± 0.044 1.3372 ± 0.023
6 0.2107 ± 0.0040 0.1785 ± 0.0026 2.9703 ± 0.024 4.0427 ± 0.037 0.0244 ± 0.0002
Oxalic acid: all P < 0.001, 1 vs 2, 3, 4, 5 or 6, P < 0.001, 5 vs 6, P < 0.001; maleic acid: all P < 0.001, 6 vs 1, 2, 3,
4 or 5, P < 0.001; citric acid: all P < 0.001, 6 vs 1, 2, 3, 4 or 5, P < 0.001; succinic acid: all P < 0.001, 1 vs 2, 3, 4,
5 or 6, P < 0.001, 5 vs 6, P < 0.001; fumaric acid: all P < 0.001, 5 vs 1, 2, 3, 4 or 6, P < 0.001.
1: Commercial sample boiled in double distilled water for 15 mins; 2: commercial sample macerated in ethanol
80% for 24 hrs; 3: commercial sample macerated in double distilled water for 72 hrs; 4: commercial sample
macerated in double distilled water for 48 hrs; 5: commercial sample macerated in double distilled water for 24
hrs; 6: natural flora sample macerated in double distilled water for 24 hrs. DW: Dried weight (of the plant); ND:
Not detected.
Table 4 Yields of total phenol, flavonoid, alkaloid and ascorbic acid (mg/g DW) of Fumaria vaillantii L.
Extract
no.
Total phenolic
(mg GaE/g DW)
Total flavonoid
(mg QE/g DW)
Total alkaloid (mg
AtE/g DW)
Ascorbic acid (mg
ascorbic acid/g DW)
1 5.481 ± 0.005 5.585 ± 0.085 0.537 ± 0.001 0.106 ± 0.004
2 2.526 ± 0.007 3.646 ± 0.061 1.009 ± 0.001 0.737 ± 0.039
3 0.999 ± 0.002 0.558 ± 0.040 0.352 ± 0.003 0.342 ± 0.012
4 1.078 ± 0.008 0.592 ± 0.038 0.266 ± 0.002 0.352 ± 0.017
5 1.108 ± 0.005 0.855 ± 0.028 0.615 ± 0.001 0.546 ± 0.005
6 0.722 ± 0.004 0.892 ± 0.026 0.650 ± 0.001 0.486 ± 0.014
Total phenol: all P < 0.001, 1 vs 2, 3, 4, 5 or 6, P < 0.001, 5 vs 6, P < 0.001; total flavonoid: all P < 0.001, 1 vs
2, 3, 4, 5 or 6, P < 0.001; total alkaloid: all P < 0.001, 2 vs 1, 3, 4, 5 or 6, P < 0.001, 5 vs 6, P < 0.001; ascorbic
acid: all P < 0.001, 2 vs 1, 3, 4, 5 or 6, P < 0.001, 5 or 6, P = 0.023.
1: Commercial sample boiled in double distilled water for 15 mins; 2: commercial sample macerated in ethanol
80% for 24 hrs; 3: commercial sample macerated in double distilled water for 72 hrs; 4: commercial sample
macerated in double distilled water for 48 hrs; 5: commercial sample macerated in double distilled water for 24
hrs; 6: natural flora sample macerated in double distilled water for 24 hrs. DW: Dried weight (of the plant);
GaE: Gallic acid equivalent; QE: Quercetin equivalent; AtE: Atropine equivalent.

ARTICLE
doi: 10.12032/TMR20190905134
maleic, fumaric, succinic, and ascorbic acids and total
phenol and total flavonoid would decrease.
Fumitory is a major source of fumaric acid, which
due to its anti-inflammatory properties is widely used
for the treatment of skin disorders such as psoriasis and
itching. This study aimed to achieve a fumitory extract
containing the highest possible yield of fumaric acid.
Considering the extractive values, among different
extraction methods, macerating in double-distilled
water for 24 hrs resulted in an extract with the highest
fumaric acid yield.
According to Guerrant et al, short-chain acids such
as pyruvic, succinic, lactic, fumaric, formic, acetic and
propionic acids were identified and determined in
anaerobic bacteria in the United States using a HPLC
system equipped with a cation-exchange column and
UV detector at the wavelength of 210 nm. Furthermore,
GC-MS method was used to confirm the presence of
fumaric acid in the cultivated extract [27]. Stein et al.
determined the short-chain fatty acids such as formic,
acetic, propionic and n-butyric acids in biological
materials in Germany using HPLC system equipped
with UV detector at 214 nm [28]. The concentrations
of low molecular weight organic acids such as lactic,
formic, acetic, propionic, n-butyric and iso-butyric
acids in seawater samples were determined by Albert
and Martens in the United States of America, using
HPLC method [29]. Organic acids including citric,
coumaric, ferulic, fumaric, malic, 3-hydroxybenzoic,
protocatechuic and caffeic acids (and its methylester)
were investigated by Soušek et al. in Czech Republic
in seven fumaria species: F. agraria, F. capreolata, F.
densiflora, F. muralis, F. officinalis, F. parviflora, and
F. vaillantii. using GC-MS technique. The presence of
fumaric, malic, citric and caffeic acids were exhibited
in the aerial parts of Fumaria vaillantii L. [6]. Lian et
al. carried out an investigation on simultaneous
determination of organic acids including oxalic,
fumaric, maleic and succinic acids in pharmaceutical
compounds in China, using ion-suppression RP-HPLC
system equipped with UV detector at 210 nm [30].
According to a study done by Khalighi-Sigaroodi et al,
fumaric acid yield in F. parviflora L. was reported as
9.3 mg fumaric acid/g DW in Iran, using HPLC
method [31]. In the present study, fumaric acid yield of
commercial Fumaria vaillantii L. was in range of
0.1879-1.3372 mg fumaric acid/g DW, which is lower
than the value reported in the previous study (with
different species of fumaria). According to a research
conducted by Jaberian et al. in Iran, fumaric acid
yields of aqueous, methanolic-aqueous and methanolic
extracts of Fumaria vaillantii L. were determined by
using GC-MS method. The methanolic-aqueous extract
showed higher fumaric acid yield (1.966 mg fumaric
acid/g DW) in comparison with aqueous (1.49 mg
fumaric acid/g DW) and methanolic (0.875 mg fumaric
acid/g DW) extracts [1], whereas in our study, the
highest fumaric acid yield were found for the aqueous
macerated (for 24 hrs) extract with commercial
Fumaria vaillantii L., and then it was decreased in
80% of ethanol, water-boiled, aqueous macerated (for
48 hrs) and aqueous macerated (for 72 hrs) extracts,
respectively. Vrancheva et al. identified six organic
acids including citric, iso-citric, malic, succinic,
glyceric and threonic acids in Bulgaria in five species
of fumaria: F. officinalis L., F. thuretii Boiss., F.
kralikii Jord., F. rostellata Knaf. and F. schrammii
Velen. using GC-MS method [32]. In the present study,
it was revealed that among the identified organic acids,
succinic acid was the dominant organic acid in all the
extracts except the aqueous macerated (for 48 hrs)
extract.
Conclusion
Based on the results of the current study, it can be
concluded that high total phenol and flavonoid yield in
boiled extract and high levels of total alkaloid and
ascorbic acid in 80% of ethanol macerated extract.
Moreover, among different aqueous macerated extracts
of the commercial sample, as the maceration time
increased, the yields of total phenolic, total flavonoid,
ascorbic, oxalic, maleic, succinic and fumaric acids
would decrease.
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