Future Journal of Pharmaceutical Sciences 4 (2018) 57e62

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Future Journal of Pharmaceutical Sciences

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pharmaceutical-sciences/

Supercritical fluid extraction of g-Pyrones from Ammi visnaga L. fruits

Mokhtar Bishr a, Mohamed El-Degwy a, Mohammed Abdel Hady a, Mohamed Amin a,
Osama Salama b, *
a
Arab Company for Pharmaceuticals and Medicinal Plants (Mepaco-Medifood), Cairo 11361, Egypt
b
Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Cairo 11835, Future University in Egypt, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Extraction with supercritical fluid technique has proved to be effective in many applications including
Received 6 August 2016 extraction and separation of various active principals from medicinal plants. It was used due to its ad-
Accepted 10 September 2017 vantages especially safety, specificity, selectivity and ease of component recovery.
Available online 15 March 2018
Ammi visnaga, L. belongs to the family Apiaceae. The fruits are used specifically for the treatment of
kidney stones depending on its g-Pyrones (mainly khellin and visnagin) [2]. The supercritical fluid
Keywords:
extraction technique of khellin and visnagin was investigated and the operating conditions for their
Ammi visnaga fruits
extraction were optimized. The effect of different pressure (150, 200, 300, 400 and 500 bars), temper-
g-Pyrones (khellin and visnagin)
Supercritical fluid extraction and HPLC
ature (35, 40, 45, 50 and 55
C), and particle sizes of the raw material (0.5, 1, 1.4 mm and entire fruits) on
the extract yield was studied under dynamic conditions for extraction for a run time of 90 min. Optimum
supercritical extraction condition was found to be 200 bars at 45
C and optimum particle size was found
to be 1.4 mm. The yield is yellowish white bitter powder and measures 1.74% w/w relative to the dried
weight of the fruits containing 38.414% w/w average g-Pyrones content of which 29.4%w/w khellin, and
9.014%w/w visnagin.
The obtained extracts were analyzed by reversed phase HPLC.
© 2017 Production and hosting by Elsevier B.V. on behalf of Future University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Ammi visnaga L. belongs to the family of Apiaceae. Ammi visnaga
is known under a diversity of names including Bishop's weed,
Khella and Toothpick weed or herb [2].
Ammi visnaga fruit preparations, such as teas prepared from
crushed or powdered seeds, have traditionally been used in the
Middle East to ease urinary tract pain associated with kidney stones
and to promote stone passage [3]. Visnagin and khellin are among
the main compounds of Ammi visnaga fruits, belonging to the
category of furanochromones (2e4%). Many properties have been
attributed to Ammi visnaga and its constituents such as antibacte-
rial, antifungal, antiviral, antidiabetic, anti-inflammatory and neu-
roprotective [4e8]. The most prominent effects of Ammi visnaga
extract and its compounds are being peripheral and coronary
vasodilator and antispasmodic, where it relaxes the smooth mus-
cles; also it acts as bronchodilator and for urolithiasis [9e14].
* Corresponding author.
E-mail address: osalama@fue.edu.eg (O. Salama).
Peer review under responsibility of Future University.
Visnadin, khellin and visnagin are thought to be responsible for the
calcium channel blocking activity [10e14]. Vasodilatory and anti-
spasmodic effects on the urinary tract could possibly explain the
usage for urolithiais, acting as expulsive therapy, thereby facili-
tating and reducing time for passage of smaller stones. Khan et al.
reported a reduction of oxalate and calcium content in rat kidneys
after administration of Ammi visnaga tea preparation [15].
Solvent extraction methods were applied to extract the active
ingredients (g-Pyrones mainly khellin and visnagin) from Ammi
visnaga fruits. The solvent used are methanol, ethanol and hydro-
ethanol. Actually, the solvent methods have many disadvantages
such as toxicity, health hazards, unfriendly to environment, high
cost and unavailability. Therefore, we shifted to an advanced
method to extract the (g-Pyrones from Ammi visnaga fruits; that is
supercritical fluid carbon dioxide extraction technology (SCFE).
SCFE technology has been replacing the traditional solvent
extraction methods as it overcomes the drawbacks of using the
solvents; in addition it has many other advantages such as it gives
an extract free from residual solvents and produces uniform stable
extract. Supercritical fluid extraction technique is more selective,
efficient, cheap, and afford mean for simple recovery of the com-
pounds without degradation [1,16e22].

https://doi.org/10.1016/j.fjps.2017.09.001
2314-7245/© 2017 Production and hosting by Elsevier B.V. on behalf of Future University. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
58 M. Bishr et al. / Future Journal of Pharmaceutical Sciences 4 (2018) 57e62
The objective of the present work was to investigate the opti-
mum condition for supercritical fluid extraction of g-Pyrones from
the fruits of Ammi visnaga L. The obtained g-Pyrones extract was
analyzed by reversed phase HPLC.
2. Materials and methods
2.1. Materials
Ammi visnaga fruits samples were supplied by Mepaco-
Medifood Pharmaceutical Company, Egypt. Khellin and Visnagin
reference standards were purchased from Sigma Aldrich, Germany.
HPLC grade Methanol and Water were acquired from Merck,
Germany.
2.2. Equipments
Lab-scale supercritical fluid extraction unit, ISCO SFX™ 220,
ISCO Inc., USA. Ultra-Fast Liquid Chromatography (UFLC), Shimadzu
Model Prominence LC-20 ADXR, equipped with auto sampler, SIL-
20ACXR and PDA detector (Shimadzu Model: SPD-M20A), Kyoto,
Japan.
2.3. Experimental
2.3.1. Supercritical fluid extraction method
Optimization trials were conducted on lab-scale SCFE equip-
ment through changing or fixing parameters to get the most opti-
mum extraction procedure; these parameters were:
 Pressure: the critical pressure (Pc) was changed from 150, 200,
300, 400 and finally 500 bars; together with fixing temperature
and particle size all over the trials.
 Temperature: the critical pressure (Pc) and particle size were
fixed; with changing critical temperature from 35, 40, 45, 50 and
55
C.
 Particle size of the sample: optimum critical pressure (Pc) and
temperature (Tc) were fixed; then different particle sizes were
used i.e. entire fruit and crushed fruit (1.4, 1 and 0.5 mm).
2.3.1.1. Selection of optimum pressure. Fixed weight samples of
crushed Ammi visnaga fruits on sieve 1.4 mm (fixed particle size)
were used for each trial; to be filled in the 7 ml extraction column.
The effect of five different extraction pressures (150, 200, 300, 400,
and 500 bars) was investigated using fixed temperature (Tc, 45
C)
together with constant flow rate of the fluidized carbon dioxide
over a fixed time ranges of 1.5 h under dynamic mode of extraction.
Each experiment was repeated three times and the average of the
outcome was recorded.
2.3.1.2. Selection of optimum temperature. Fixed weight samples of
crushed Ammi visnaga fruits on sieve 1.4 mm (fixed particle size)
were used for each trial; to be filled in the 7 ml extraction column.
The effect of five different extraction temperatures (Tc, 35, 40, 45,
50 and 55
C) was investigated using fixed pressure (Pc, 200 bars)
together with constant flow rate of the fluidized carbon dioxide
over a fixed time ranges of 1.5 h under dynamic mode of extraction.
Each condition was repeated three times and the average of the
outcome was recorded.
2.3.1.3. Selection of optimum sample particle size. In a similar way,
in each experiment, a fixed weight of crushed Ammi visnaga fruits is
to be filled in the 7 ml extraction column. The effect of different
particle sizes of the material used (entire fruits, 1.4 mm, 1 mm and
0.5 mm) was investigated using fixed optimum extraction tem-
perature (Tc, 45
C), fixed optimum pressure (Pc, 200 bars) and were
fixed and constant flow rate of the fluidized carbon dioxide over a
fixed time ranges of 1.5 h under dynamic mode of extraction. Each
experiment was repeated three times and the average of the
outcome was recorded.
2.3.2. Optimization of the supercritical fluid extraction condition
In order to find out the effect of different extraction pressures
and effect of different extraction temperatures on the yield of g-
Pyrones with various particle sizes mentioned above; extraction
pressures around the optimum (Pc) obtained with particle size
1.4 mm, viz: (150, 200 and 300 bars) and the effect of extraction
temperatures around the optimum (Tc) obtained with particle size
1.4 mm, viz: (40, 45 and 45
C) were investigated alongside with
the different sizes (entire fruits, 1.0 mm and 0.5 mm).
2.3.2.1. Effect of different extraction pressures on the yield of g-
Pyrones from Ammi visnaga fruits at three different particle sizes.
In all experiments, a fixed weight sample of Ammi visnaga fruits of
three different particle sizes (entire fruits, 1.0 mm and 0.5 mm)
were separately used and to be filled in the 7 ml extraction column.
Three different extraction pressures (150, 200 and 300 bars), fixed
temperature (Tc,45
C), constant flow rate of the fluidized carbon
dioxide over a fixed time ranges of 1.5 h under dynamic mode of
extraction were chosen. Each condition was repeated three times
and the average of the outcome was recorded.
2.3.2.2. Effect of different extraction temperatures on the yield of g-
Pyrones from Ammi visnaga fruits at three different particle sizes.
In all experiments, a fixed weight sample of Ammi visnaga fruits of
three different particle sizes (entire fruits, 1.0 mm and 0.5 mm)
were separately used and to be filled in the 7 ml extraction column.
Three different extraction temperatures (40, 45 and 50
C), fixed
pressure (Pc, 200 bars), constant flow rate of the fluidized carbon
dioxide over a fixed time ranges of 1.5 h under dynamic mode of
extraction were chosen. Each condition was repeated three times
and the average of the outcome was recorded.
2.3.3. HPLC analysis of the obtained extracts
2.3.3.1. Chromatographic conditions. The g-Pyrones content of
Ammi visnaga extracts obtained by supercritical carbon dioxide
fluid extraction were analyzed by liquid chromatography (UFLC)
(Shimadzu Model Prominence LC-20 ADXR), equipped with auto
sampler, SIL-20ACXR, PDA detector (Shimadzu Model: SPD-M20A);
equipped with a Hypersil C18 column, (Thermo BDS, 5 mm,
(250  4.6 mm); SN: 10185576, USA). Column temperature was
40
C. The mobile phase consists of methanol: water (50: 50)% at
flow rate 1.5 ml/min. Detection wavelength was 245 nm and the
injection volume was 20 ml.
2.3.3.2. Standard solution preparation. Twenty milligrams of stan-
dard khellin were weighed accurately and transferred into 100 ml
volumetric flask. The standard khellin was dissolved in methanol
till volume; then sonicate for 20 min to obtain a concentration of
20 mg/dl. The standard solution was filtered over 0.45 mm syringe
filter prior to injection.
2.3.3.3. Sample preparation. Fifteen milligrams of the obtained
Ammi visnaga extracts were weighed accurately and transferred
into 20 ml volumetric flask. The extract was dissolved in methanol
till volume; then sonicate for 20 min to obtain a concentration of
75 mg/dl. The obtained solution was filtered over 0.45 mm syringe
filter prior to injection.
 M. Bishr et al. / Future Journal of Pharmaceutical Sciences 4 (2018) 57e62 59

Fig. 1. HPLC-chromatogram of Ammi visnaga supercritical fluid extract.

Table 1
Effect of the Critical Pressure (Pc) on the Yield of g-Pyrones from Crushed Ammi visnaga Fruits (Particle Size 1.4 mm) at Tc ¼ 45
C.

Sample no. Pressure Average outcome Average Outcome appearance HPLC assay of g-Pyrones
(bars) Wt. (mg) outcome %
Total % (Khellin þVisnagin % of Khellin

1 150 84.4 1.298% Slightly yellowish white, bitter, faint odour, fatty 29.09 22.72
2 200 113.3 1.74% Yellowish white powder, bitter, characteristic odour 35.46 27.73
3 300 95.7 1.47% Yellowish white powder, slightly fatty, 34.16 27.72
bitter, characteristic odour
4 400 89.8 1.38% Yellowish white powder, fatty, bitter, characteristic odour 34.58 28.16
5 500 87.9 1.352% Yellowish powder, fatty bitter, characteristic odour 34.41 27.66

3. Results and discussion
3.1. HPLC method
A simple, precise, accurate, specific HPLC method was devel-
oped into to analyze the Ammi visnaga supercritical fluid extracts
content. This method enables simultaneous determination of
khellin and visnagin, the major g-Pyrones found in Ammi visnaga
because of good separation and resolution of the chromatographic
peaks. The retention time of khellin was 6.25 min while that of
visnagin was 7.5 min at wavelength ¼ 245 nm (Fig. 1).
3.2. Supercritical fluid extraction method
3.2.1. Selection of optimum pressure
Upon increasing the pressure from 150 bars to 200 bars at fixed
temperature of 45
C, the obtained extract percent increased with
an increase of their total g-Pyrones content. However, upon further
increase of the pressure (300 bars), a decrease in the outcome
percent with relative decrease in total g-Pyrones content and the
outcome becomes slight fatty. Also on increasing the pressure to
400 and 500 bars, more fatty materials were extracted concurrent
with theg-Pyrones.
From Table 1, it is clear that the best pressure used was
200 bars at 45
C from the g-Pyrones content and extract percent.
3.2.2. Selection of optimum temperature
Upon increasing the temperature from 35 bars to 45 bars at fixed
pressure of 200 bars, the obtained extract percent increased with
an increase of their total g-Pyrones content with decrease in
chlorophyll which appeared at 35
C as greenish color and dis-
appeared at 45
C. However, upon further increase of the temper-
ature, a decrease in the outcome percent with relative decrease in
total g-Pyrones content. However, fatty materials were extracted
concurrent with theg-Pyrones when the temperature increased to
50 or 55
C. From Table 2; it is clear that the best temperature used
was 45
C.
3.2.3. Selection of optimum sample particle size
Effect of sample particle size on the yield of totalg-Pyrones at
(Pc, 200 bars) and (Tc, 45
C) is shown in Table 3 where; it is clear
that the optimum particle size was 1 mm. Upon reducing the

Table 2
Effect of the Extraction Temperature (Tc) on the Yield of g-Pyrones from Crushed Ammi visnaga Fruits (Particle Size 1.4 mm) at PC ¼ 200 bars.

Sample no. Temp. Outcome Outcome % Outcome appearance HPLC assay of g-pyrones
(
C) Wt. (mg)
Total % (Khellin þVisnagin % of Khellin

1 35 59.3 0.91% Greenish white, faint characteristic odour 32.68 26.00

2 40 78.2 1.2% Greyish white powder, characteristic odour 33.09 25.90
3 45 113.3 1.74% Yellowish white powder, bitter, characteristic odour 35.46 27.73
4 50 108 1.66% Yellowish white powder, slightly fatty, bitter, characteristic odour 34.21 27.08
5 55 72.1 1.109% Yellowish white powder, slightly fatty, bitter, characteristic odour 31.63 24.87
60 M. Bishr et al. / Future Journal of Pharmaceutical Sciences 4 (2018) 57e62

Table 3
Effect of Sample Particle Size of Ammi visnaga Fruits on the Yield of g-Pyrones at Tc ¼ 45
C and PC ¼ 200 bars.

Sample no. Particle Outcome Outcome % Outcome appearance HPLC assay of g-Pyrones
size (mm) Wt. (mg)
Total % % of Khellin
(Khellin þVisnagin

1 Entire 41.25 0.75 Greyish white powder, bitter taste, faint characteristic odour 29.01 23.57
2 1.4 113.3 1.74 Yellowish white powder, bitter, characteristic odour 35.46 27.73
3 1 156.2 2.605 Yellowish white, fatty, bitter, Strong characteristic odour 31.17 21.31
4 0.5 329.45 5.99 Yellowish white, fatty, bitter, Strong characteristic odour 24.60 16.97

Table 4
Effect of Different Pressures on the Yield of g-Pyrones from Entire Ammi visnaga Fruits at Tc ¼ 45
C.

Sample no. Particle Pressure Average Average Outcome appearance HPLC assay of g-pyrones
size (mm) (bars) outcome outcome %
Total % (Khellin þVisnagin % of Khellin
Wt. (mg)

1 Entire seeds 150 0 0 NA NA NA

2 200 41.25 0.75 Yellowish white powder, bitter, characteristic odour 29.01 23.57
3 300 36 0.613 Yellowish white, powder, bitter, characteristic odour 26.64 23.06

Table 5
Effect of Different Pressures on the Yield of g-Pyrones from Crushed Ammi visnaga Fruits (1.0 mm) at Tc ¼ 45
C.

Sample no. Particle Pressure Average Average Outcome appearance HPLC assay of g-pyrones
size (mm) (bars) outcome outcome %
Total % (Khellin þVisnagin % of Khellin
Wt. (mg)

1 1 150 127.6 2.126 Yellowish white, slightly fatty, bitter, characteristic odour 28.38 18.05
2 200 156.2 2.605 Yellowish white, slightly fatty, bitter, characteristic odour 31.17 21.31
3 300 156 2.6 Yellowish white, slightly fatty, bitter, characteristic odour 27.17 19.31

particle size of the fruits, the total outcome percent of the obtained
extract increased due to the increase of the surface area subjected
to the extraction media of supercritical carbon dioxide. However,
with decreasing the particle size, more fatty materials were
extracted together with the major bioactive materials (g-Pyrones)
which negatively impacted on the nature of the obtained extract.
This was very clear with particle size 0.5 mm gives the highest yield
but lower overall g-Pyrones relative to the outcome weight.
3.3. Optimization of the supercritical fluid extraction condition
3.3.1. Effect of different extraction pressures on the yield of g-
Pyrones from Ammi visnaga fruits at three different particle sizes
The impact of different pressure was obvious especially upon
using the entire fruits. Although the major bioactive ingredients
(g-Pyrones) are present in the pericarp of the fruits; however at
150 bars, the extraction pressure wasn't sufficient to penetrate
and extract them. Upon increasing the pressure to 200 bars in-
crease in the outcome percent and the g-Pyrones content was
observed. On further increase of the extraction pressure, relative
decrease in the outcome percent and g-Pyrones content as
shown in Table 4.
With the decrease of particle size to 1 mm, the outcome percent
increased with increase of the pressure used to 200 bars but was
nearly the same at 300 bars; however the total g-Pyrones content
was maximum at 200 bars and decreased at 300 bars revealing that
200 bars was selectively optimum for extraction the required
bioactive materials as shown in Table 5.

Table 6
Effect of Different Pressures on the Yield of g-Pyrones from Crushed Ammi visnaga Fruits (0.5 mm) at Tc ¼ 45
C.

Sample no. Particle Pressure Average Average Outcome appearance HPLC assay of g-Pyrones
size (mm) (bars) outcome outcome %
Total % (Khellin þVisnagin % of Khellin
Wt. (mg)

1 0.5 150 180.3 3.27 Yellowish white, slightly fatty, bitter, slightly 20.98 14.87
Characteristic odour
2 200 329.45 5.99 Yellowish white, fatty, bitter, Characteristic odour 24.60 16.97
3 300 373.9 6.8 Yellowish white, fatty, bitter, characteristic odour 21.41 17.27

Table 7
Effect of Different Temperatures on the Yield of g-Pyrones from Entire Ammi visnaga Fruits at PC ¼ 200 bars.

Sample no. Particle Temp. (
C) Average Average Outcome appearance HPLC assay of g-pyrones
size (mm) outcome Wt. (mg) outcome %
Total % (Khellin % of Khellin
þVisnagin)

1 Entire seeds 40 39.1 0.714 Yellowish white powder, bitter, characteristic odour 18.20 15.56
2 45 41.25 0.75 Yellowish white powder, bitter, characteristic odour 29.01 23.57
3 50 37.7 0.689 Yellowish white powder, bitter, characteristic odour 17.15 14.42
 M. Bishr et al. / Future Journal of Pharmaceutical Sciences 4 (2018) 57e62 61

Table 8
Effect of Different Temperatures on the Yield of g-Pyrones from Ammi visnaga Crushed Fruits (1.0 mm) at PC ¼ 200 bars.

Sample no. Particle Temp. (
C) Average Average outcome % Outcome appearance HPLC assay of g-pyrones
size (mm) outcome
Total % (Khellin %
Wt. (mg)
þVisnagin) of Khellin

1 1 40 121 2.106 Yellowish white, slightly fatty, bitter, Characteristic odour 20.83 15.77
2 45 156.2 2.605 Yellowish white, slightly fatty, bitter, Characteristic odour 31.17 21.31
3 50 134.4 2.24 Yellowish white, slightly fatty, bitter, Characteristic odour 19.64 14.19

Table 9
Effect of Different Temperatures on the Yield of g-Pyrones from Ammi visnaga Crushed Fruits (0.5 mm) at PC ¼ 200 bars.

Sample no. Particle Temp. (
C) Average Average outcome % Outcome appearance HPLC assay of g-pyrones
size (mm) outcome Wt. (mg)
Total % (Khellin % of Khellin
þVisnagin)

1 0.5 40 278.5 5.06 Yellowish white, fatty, bitter, Characteristic odour 21.74 14.88
2 45 329.45 5.99 Yellowish white, fatty, bitter, Characteristic odour 24.60 16.97
3 50 285.6 5.19 Yellowish white, fatty, bitter, Characteristic odour 23.7 16.71

Upon further decrease of particle size to 0.5 mm, the outcome
percent increased with increase of the pressure used which may be
due to high surface area available for extraction upon size reduction
however the total g-Pyrones content was lower relative to the
outcome percent revealing that the size reduction enables extrac-
tion of other non-active materials resulting in diluting the total
outcome as shown in Table 6.
3.3.2. Effect of different extraction temperatures on the yield of g-
Pyrones from Ammi visnaga fruits at three different particle sizes
Upon using different temperature at different particle sizes, the
outcome percent increased to optimum from at 45
C; then
decreased again at 50
C with collateral decrease in theg-Pyrones
content as shown in Tables 7e9.
4. Conclusion
The results showed that the yields obtained under the different
conditions studied were ranging from 0% to 6.8%. The results
indicate that the pressure, temperature and particle size of the
fruits had a significant effect on the yield and nature of the extracts
obtained with supercritical CO2 extraction technique. On the other
hand, the temperature and pressure had also significant effect on
the g-Pyrones contents of the obtained extracts. The particle size of
the fruits had also significant effect on the g-Pyrones contents of
the obtained extracts. The more decrease the particle size of the
crushed fruits, the more outcome percent was obtained. However
with decrease of the particle size, the fatty nature of the extract
increases significantly which in turn decreases the total bioactive
content of g-Pyrones relative to the total outcome percent.
Optimum condition for extraction of g-Pyrones from Ammi
visnaga fruits was found to be at pressure of 200 bars at tempera-
ture of 45
C under dynamic conditions for 90 min at particle size of
crushed Ammi visnaga fruits of 1.4 mm.
From the above mentioned method and its results, it is very
clear that it is the first time to apply a pilot scale SCFE method for
the g-Pyrones from Ammi visnaga Fruits. So, we can say that this
method is a novel one.
Acknowledgement
We would like to show our deep gratitude for Science Tech-
nology Development, Egypt (STDF) for supporting and funding our
research through project ID: 5715 entitled “Application of Super-
critical Fluid Extraction in Pharmaceutical Industry”.
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