Food Anal.

Methods
DOI 10.1007/s12161-015-0375-4

Optimization of Anthocyanin Extraction from Saffron Petals

with Response Surface Methodology
K. Mahdavee Khazaei 1 & S. M. Jafari 1,2,4 & M. Ghorbani 1 & A. Hemmati Kakhki 3 &
M. Sarfarazi 1

Received: 15 July 2015 / Accepted: 30 November 2015

# Springer Science+Business Media New York 2015

Abstract Optimum extraction conditions of anthocyanins Introduction

from petals of saffron (Crocus sativus) using acidified ethanol
as the solvent were revealed. The investigated factors were These days, agricultural by-products removed from food pro-
solvent to sample ratio (20:1–80:1), ethanol concentration duction lines that were considered as useless materials and
(%), extraction temperature (25–45 °C), and time (8–24 h). produced big and complicated problems for the environments
Response surface methodology with Box–Behnken design in the past are now mostly known as functional foods due to
was applied to determine optimum processing conditions the necessity of use of natural resources in food industries
leading to maximum extraction efficiency (mg cyanindin-3- (Galanakis 2012, 2013; Galanakis et al. 2015). Most of the
glucoside/l). Obtained coefficients of variance showed that the synthetic colors that are used in the food industry have chem-
linear effect of temperature was more pronounced for extrac- ical sources with harmful health effects. Since the anticancer
tion yield than three other variables at 5 % level. Optimum and antivirus properties of natural colorants are proven, today
extraction conditions that maximize the extracted anthocya- there are more tendencies to use natural colorants instead of
nins were found to be a ratio of solvents to sample 20 ml/g, synthetic ones (Andersen and Jordheim 2010).
ethanol concentration of 25.02 %, temperature 25.8 °C, and Anthocyanins as natural pigments are found in roots, leaves,
extraction time 24 h which gave 1609.11 mg/l anthocyanins. fruits, and flowers of plants (Chen 2008). Attractive color and
A quadratic regression equation describing the effects of in- functional properties (like prevention of neuronal and cardio-
dependent process variables on anthocyanin extraction from vascular, cancer, and diabetes illnesses) of anthocyanins make
saffron petals can be used for finding optimum conditions to them a suitable substitute for synthetic pigments in the food
achieve desired extraction yield in similar conditions. industry (Castaoveda-Ovando et al. 2009). There are different
sources of anthocyanins like grapes, berries, red cabbage, ap-
ples (Castaoveda-Ovando et al. 2009), red potatoes, and black
Keywords Saffron petal . Conventional extraction . carrots (Kirca et al. 2006). Saffron (Crocus Sativus) which
Anthocyanin . Response surface methodology produces largely in Iran (Esfanjani et al. 2015). with a share
of annually more than 90 % of total saffron in the world
(Rajabi et al. 2015). has cyanic color flowers with major col-
* S. M. Jafari orant of anthocyanins (Norbark et al. 2002). Saffron petals
[email protected]
consist a large portion of saffron flower total weight
(Hemmati Kakhki et al. 2001). then, these petals could be a
1
Department of Food Materials and Process Design Engineering, big by-product of agriculture and a good source of natural
University of Agricultural Sciences and Natural Resources, anthocyanins. Many studies have investigated the biomedical
Gorgan, Iran
and cancer therapy properties of saffron’s petal (Kubo and
2
Cereals Health Research Center, Golestan University of Medical Kinst-Hori 1999; Hadizadeh et al. 2003; Lee et al. 2008;
Sciences, Gorgan, Iran
Fatehi et al. 2003; Moshiri et al. 2006; Akhondzadeh Basti et
3
Research Institute of Food Science and Technology, Mashhad, Iran al. 2007; Agha-Hosseini et al. 2008; Khalili et al. 2010). In
4
Pishro Food Technology Research Group, Gorgan, Iran these studies, it has been reported that the major antioxidant
 Food Anal. Methods

compounds of petals such as flavonoids, anthocyanins, and purpose of this technique is to optimize the response sur-
flanonons are responsible for these functional properties. face that is influenced by factors of the process (Raissi and
Since nearly 86.4 % (wb) or 96.36 % (db) of total weight of Eslami Farsiani 2009).
saffron flowers belong to their petals (Hemmati Kakhki et al. In this study, four conventional extraction parameters in
2001) and large scale of saffron flowers is released in nature in saffron petal’s extraction including ethanol percent, ratio of
Iran after picking stigmas annually, potentially, anthocyanins solvent to sample, extraction time, and temperature were op-
in saffron’s petal extract can be used as a natural resource of timized by RSM, in order to achieve maximum extraction
colorants in food products, adding to its other medicinal/ yield of anthocyanins.
industrial applications (Kafi 2006).
Generally, extraction of anthocyanins takes place at low
temperatures (below 30 °C), preferably under vacuum (to Materials and Methods
minimize degradation) and in an acidic environment.
Solvents like ethanol, methanol, N-butanol, cold acetone, pro- Samples and Chemicals
pylene glycol, mixture of methanol—acetone and water, or
boiled water can be used in extraction of anthocyanins. Saffron flowers were collected before sunlight from a farm
Among the various solvents, ethanol is usually preferred for near Torbat-E-Heydariyeh (Iran) on November 2013. After
its low toxicity. Use of mineral acids such as hydrochloric acid removing stigmas and anther, the petals were dried in a dark
preserves this pigment through extraction in its stable form and warm room (37 °C) in front of a fan. This method of
(ion flavylium) by reducing pH of solvents) Gould et al. drying was compared with three other methods of drying in
2008 .(In a study by Kirca et al (2007) which was carried oven, 70 °C for 6 h, vacuum oven drying, 40 °C for 24 h, and
out on the anthocyanin extraction from black carrot, it was conventional drying, 25 °C for 3 days, and mentioned that
observed that rising extract pH from 4.3 to 6 destroyed antho- method was chosen due to the most stability of anthocyanins
cyanins, and generally, in solutions with pH > 5, monomeric through drying. Dried petals were crushed and sieved (16
meshes) and were kept in airtight bags in around 5 °C.
anthocyanins significantly (P < 0.05) decreased. Hydrochloric
Analytical grade hydrochloric acid (HCL) and ethanol were
acid may cause deformation of anthocyanins including hydro-
purchased from Merck (Darmstadt, Germany). Distilled water
lysis in acidic conditions during evaporation in a rotary evap-
was used for preparation of all solutions. Potassium chloride
orator (40 °C) )Van Sumere et al. 1985)or deacylation of ac-
buffer pH 1.0 and sodium acetate buffer pH 4.5 used in this
ylated anthocyanins with aliphatic acids which can occur at
study were of analytical grade.
room temperature )Harbourne et al. 2013.( Acetylated antho-
cyanins are more stable than their non-acetylated analogues
Saffron Petal Analysis
(Yoshida et al. 1991.(
Temperature is another factor in conventional solvent ex-
Some physicochemical properties of the saffron petal were
traction of anthocyanins. According to Einstein’s equation,
  analyzed including moisture content, proteins, total ash, total
D∝ Tη rising temperature increases diffusion coefficient, fiber and total sugar, lipids (AOAC 2006), and total mono-
and thus, ingredients can be extracted faster. Furthermore, meric anthocyanin content (Lee et al. 2008).
denaturation of plant cell walls which occurs at high temper-
atures makes removal of anthocyanins from plant tissues eas- Extraction of Anthocyanins from Saffron Petals
ier (Cacace and Mazza 2003a). However, the vulnerability of
anthocyanins to heat and deterioration of these pigments into In order to extract anthocyanins, the dried petals were added to
brown or colorless polymeric pigments and disappearance of 30 ml of acidic ethanol (pH = 2). Amount of petals, extraction
desired color of extract exposed to heat treatment cannot be time, temperature, and ethanol percentage which were select-
easily ignored (Pala and Toklucu 2012; Patras et al. 2010; ed based on RSM experimental design are presented in Table
Stintzing and Carle 2004). One solution could be encapsula- 1. Experiments were done in brown color bottles with screwed
tion of anthocyanins by natural biopolymers (Akhavan et al. caps. After extraction, samples were filtered through filter
2014; Khazaei et al. 2014). paper (Whatman No. 1). Total anthocyanins of extracts were
Response surface methodology (RSM) is as an approach measured with pH differential method which was adopted
to build approximated models based on data collected dur- from Giusti and Wrolstad (2001). In this method, the extracts
ing physical examination, simulated by computer and were added to buffers 1.0 and 4.5 and allowed to equilibrate
experimented observations (Sarfarazi et al. 2015). In for 20 min. The absorbance of each equilibrated solution was
RSM, a set of mathematical-statistical models are used then measured at 520 nm (λ max) and 700 nm for haze cor-
for engineering and modeling procedures. The main rection, using an UV–Vis spectrophotometer (Shimatzu-160A
Food Anal. Methods

Table 1 Total anthocyanin

content of saffron petal’s extract No. Independent variables Total anthocyanin
through conventional extraction content
in Box-Behnken design X1 X2 X3 X4
Time (h) Temperature (°C) Ethanol percent (%) Solvent ratio (ml/g) mg/l extract

1 8 (−1)a 45 (1) 50 (0) 50 (0) 1248.24

2 8 (−1) 35 (0) 50 (0) 20 (−1) 1050.01
3 8 (−1) 25 (−1) 50 (0) 50 (0) 1511.25
4 8 (−1) 35 (0) 25 (−1) 50 (0) 1214.46
5 8 (−1) 35 (0) 75 (1) 50 (0) 1419.4
6 8 (−1) 35 (0) 50 (0) 80 (1) 1495.46
7 8 (−1) 35 (0) 50 (0) 50 (0) 1190.54
8 16 (0) 45 (1) 50 (0) 20 (−1) 1011.95
9 16 (0) 35 (0) 50 (0) 50 (0) 1202.03
10 16 (0) 45 (1) 75 (1) 50 (0) 993.67
11 16 (0) 25 (−1) 50 (0) 20 (−1) 1110.24
12 16 (0) 35 (0) 50 (0) 50 (0) 1200.95
13 16 (0) 25 (−1) 75 (1) 50 (0) 1287.29
14 16 (0) 45 (1) 25 (−1) 50 (0) 1178.47
15 16 (0) 35 (0) 50 (0) 50 (0) 1196.04
16 16 (0) 45 (1) 50 (0) 80 (1) 1035.02
17 16 (0) 35 (0) 75 (1) 80 (1) 1163.94
18 16 (0) 25 (−1) 50 (0) 80 (1) 1436.1
19 16 (0) 35 (0) 50 (0) 50 (0) 1201.36
20 16 (0) 35 (0) 75 (1) 20 (−1) 852.54
21 16 (0) 35 (0) 25 (−1) 20 (−1) 1210.54
22 16 (0) 25 (−1) 25 (−1) 50 (0) 1340.92
23 16 (0) 35 (0) 25 (−1) 80 (1) 1157.2
24 24 (1) 25 (−1) 50 (0) 50 (0) 1275.09
25 24 (1) 35 (0) 75 (1) 50 (0) 962.65
26 24 (1) 45 (1) 50 (0) 50 (0) 1042.23
27 24 (1) 35 (0) 25 (−1) 50 (0) 1446.23
28 24 (1) 35 (0) 50 (0) 80 (1) 1175.27
29 24 (1) 35 (0) 50 (0) 20 (−1) 1300.14
a
Numbers in parenthesis show independent variables in coded units

,Japan). Pigment content in acidic ethanol was calculated delphinidin MW (Giusti and Wrolstad 2001). DF is the dilu-
based on cyanidin-3-glucoside (Lee et al 2008). The absor- tion factor, and α is the molar absorptivity.
bance of the diluted sample (A) was calculated as follows:
Total Pigment Content of Saffron’s Petal
A ¼ ðAvis−max –A700 ÞpH 1:0 –ðAvis−max –A700 ÞpH 4:5 ð1Þ
Total pigment content of petals was measured base on the meth-
The total anthocyanin content in the original sample was od by Hemmati Kakhki et al. (2001) with some modifications. A
calculated using the following formula: total of 5 ± 0.01 g powdered sample was mixed with 200 ml
HCL-50 % ethanol (pH = 2). Extraction temperature was set at
25 ± 1 °C. Experiment was carried out for 8 h while it was mixing
Total anthocyanin contentðmg=lÞ ¼ ðA  MW  D F  1000Þ=ðα  LÞ
slowly (100 rpm). Extract was filtered through Whatman No. 1
ð2Þ paper under vacuum and collected in a volumetric flask. The
residue was taken back and extracted again in the same condi-
where MW is the molecular weight of delphinidin. Since ma- tions. The anthocyanin contents of double extractions were
jor anthocyanin content of saffron petal was not clear, we used mixed and used for determination of the total anthocyanins.
 Food Anal. Methods

Statistical Analysis variable (Y). X1, X2, X3, and X4 were independent variables,
namely, solvent ratio, ethanol percent, temperature, and time,
In order to estimate effect of independent variables on total respectively. Based on ANOVA test (Table 4), four studied
anthocyanin content, response surface methodology was ap- variables had a significant (P < 0.01) linear effect on the mod-
plied using a commercial package, Design-Expert version el. Quadratic effect of solvent ratio and temperature (x21, x24;
8.0.1 (Statease Inc., Minneapolis, USA). Box-Behnken design P < 0.01) and the interaction effects of all independent vari-
was used, and the design consisted of 29 experiments shown ables except interaction of ethanol percent and temperature
in Table 1 with five replicates in center point and two repli- (X2*X3) and solvent and ethanol percent (X1*X2), with 99 %
cates in every experiment. Statistical significance of the terms confidence, were significant.
in the regression equation was examined. Response surface
Anthocyanin ¼ 1198:18−61:44x1 −120:19x2 −73:11x3
plots were generated with the same software. Optimum levels
þ77:31x4 −172: 13 x1 x3 −142:61x1 x4
of independent variables for the procedure (solvent ratio, ex- ð3Þ
−75:7x2 x4 þ 91:18x3 x4 þ 88:92x21
traction time, ethanol percent, and temperature) were deter-
−53:84 x24
mined through optimization by RSM.
Represented model (Eq. (3)) is the second-order polynomi-
al equation (regression model) after omitting insignificant co-
Results and Discussion efficients in 99 % confidence level. Constant number
(1198.18) is constant coefficient of polynomial model. Y is
Physicochemical Characteristics of Saffron’s Petal total anthocyanin content (mg/l) based on delphinidin. As it
can be seen, among four independent variables, only the sol-
In Table 2, some physicochemical properties of the saffron’s vent had a positive effect and other three independent vari-
petal are represented. Total anthocyanin content of extract was ables had negative effects on total anthocyanin content in ex-
equal to 1712.19 ± 60 mg delphinidin/l. Lee et al (2008) in perimental conditions. Also among mentioned factors, time
their investigation reported that total anthocyanin contents in and temperature had highest and lowest effects on response,
some natural resources based on mg cyanidin-3-glocoside/l respectively. Coefficient of determination, R2, was 0.948, and
were as cranberry juice, 13.6; red wine, 201.6; natural color- adjusted R2 was 0.92. When R2 is near to unity, it means that
ant, 640.8; raspberry juice, 336.7; and elderberry 3006.8 mg/l. fitted empirical model is suitable for actual data and the model
Total anthocyanin content in black carrot was determined as can explain the relation among variables (Koocheki et al.
1750 mg/kg (wb) by Kirca et al. (2007). As it can be seen, the 2009). In Fig. 1, the model predicted and actual values of
saffron petal in comparison with others is a good natural anthocyanins obtained during the extraction have been
source of anthocyanin with high capacity. compared.

Extraction of Total Anthocyanin Content from Saffron

Petal Response Surface Plots of Extraction Efficiency

Obtained total anthocyanin content through conventional sol- The interaction of time and temperature on total anthocyanin
vent extraction based on mg delphinidin/l (per liter of extract) content is shown in Fig. 2a. In all response surface plots, two
is represented in Table 1. After analysis of variance and based other variables which are not mentioned are in their midpoints.
on F and P values, a second-order polynomial response sur- During all extraction times, increasing the temperature re-
face model (Eq. (3) and Table 3) was fitted to the response duced the total anthocyanin content linearly. Monomeric

Table 2 Physicochemical
properties of saffron petals Properties Results Unit Method

Protein 8.73 ± 0.09 g/100 g dry matter AOAC (2006)

Lipid 10.25 ± 0.09 g/100 g dry matter AOAC (2006)
Fiber 11.39 ± 0.11 g/100 g dry matter AOAC (2006)
Total sugar 13.05 ± 0.12 g/100 g dry matter AOAC (2006)
Total ash 5.76 ± 0.003 g/100 g dry matter AOAC (2006)
Moisture 87.11 ± 0.04 g/100 g dry matter AOAC (2006)
Total anthocyanins 1712.19 ± 60 mg/l extract (Lee et al. 2008)
Food Anal. Methods

Table 3 Regression coefficients

for total anthocyanin content of Term Coefficient Standard error for the coefficient F P value
saffron petal’s extract in quadratic
model with conventional solvent Intercept 1198.18 20.07 25.05 <0.0001
extraction X1 time −61.44 12.96 22.49 <0.0003
X2 temprature −120.19 12.96 86.07 <0.0001
X3 ethanol (%) −73.11 12.96 31.85 <0.0001
X4 solvent ratio 77.31 12.96 35.61 <0.0001
x21 88.92 17.62 25.46 <0.0002
x22 4.03 17.62 0.052 0.8223
x23 −26.36 17.62 2.24 0.1568
x24 −53.84 17.62 0.33 0.0086
X1X2 7.54 22.44 0.11 0.7419
X1X3 −172.13 22.44 54.84 <0.0001
X1X4 −142.61 22.44 40.39 <0.0001
X2X3 −30.54 22.44 1.85 0.1959
X2X4 −75.7 22.44 11.38 0.0045
X3X4 91.18 22.44 16.51 0.0012

anthocyanins in high temperatures could have been deteriorat- declined. Temperature around 45 °C deteriorated extracted
ed and polymerized to form brown or colorless pigments. Due anthocyanins; therefore, increasing the temperature irrespec-
to stability of polymeric anthocyanins in different pHs, they tive of the solvent ratios reduced total anthocyanin content of
cannot be measured by pH differential method, and as a result, the extract. Moreover, as previously mentioned, higher densi-
total measured anthocyanins decreased (Giusti and Wrolstad ty of ingredients in extract at lower solvent ratios results in
2001). At each temperature, loss of total anthocyanin content further reactions between monomeric anthocyanins and other
occurred with increasing extraction time to about 18 to 20 h, compounds, thus declines the stability and shortens half-life of
which could be due to the dominance of the damaging effects the anthocyanins.
of temperature on the extraction of monomeric anthocyanins Interaction between the temperature and ethanol concentra-
in the interval, and then, in 24-h extraction time, total antho- tion on the extraction rate (although it was not significant with
cyanin content was stable or even slightly increased. 99 % confidence) is presented in Fig. 2c. As the surface plot
The interactive effect of solvent ratio and temperature on shows, in general, increasing the percentage of ethanol and
total anthocyanin content is shown in Fig. 2b. The maximum temperature reduced total anthocyanin content. Its reason
pigment content was extracted in 25 °C and in 80-ml/g solvent could be the detrimental effects of temperature and ethanol
ratio. The higher solvent ratio made higher-density gradient (in the interval of 16 h) on total anthocyanin content of the
and higher distribution coefficient, thereby resulting faster re- samples. In another similar study conducted by Cacace and
lease from petal tissue and hence higher extracted anthocya- Mazza (2003b) on the anthocyanins extracted from
nins. This trend continued till the solvent ratio reached to blackcurrant, the interaction between ethanol percentage and
approximately more than 50 ml/g. With rising solvent propor- temperature (150-min extraction time) was investigated on the
tion, due to lower solute weight ratio and decreasing the den- anthocyanin content. They observed that by increasing the
sity of extracted anthocyanins, total anthocyanin content ethanol concentration (from 20 to 85 %), the critical temper-
ature of anthocyanins (which is the temperature at which an-
thocyanins start to degrade) increased from 25 to 35 °C, and
Table 4 Predicted optimum conditions for the extraction of saffron they reported that at all temperatures, with the rise in ethanol
petal anthocyanins and the response values percentage up to 60 %, the extraction efficiency initially in-
creased and then decreased. In another work of these two
Factors Low High Optimum
scientists (Cacace and mazza 2003a) carried out on the extrac-
Solvent ratio (ml/g) 20 50 20 tion optimization of phenolic compounds from milled berries,
Ethanol concentration (%) 25 75 25.02 the highest amount of phenolic regardless of temperature was
Extraction temperature (°C) 25 45 25.8 extracted in 60 % ethanol (150 min), and at higher or lower
Time (h) 8 24 24
ethanol concentrations, the extraction rate decreased.
Total monomeric anthocyanin (mg/l) 1200 1680 1609.11
Differences between the results obtained in our study and
others could be due to long time of extraction (16 h) as well as
 Food Anal. Methods

Fig. 1 Predicted and actual

values of total anthocyanin
content of saffron petal’s extract

differences in the physical characteristics or chemical structure Fig. 2f. According to the plot, total anthocyanin content was a
of the saffron petals. function of both investigated variables. In all time intervals up
In scrutinizing the effects of extraction time and solvent to around 12 h, with increasing the ethanol percentage, the
ratio (Fig. 2d), it was observed that the highest extraction extraction rate increased, and in an opposite trend, in intervals
efficiency was obtained in minimum time with maximum sol- more than 12 h of extraction, with increasing the ethanol per-
vent ratio. In general, the most total anthocyanin content centage, total anthocyanin content was reduced. Therefore, the
changes through different solvent ratios were observed be- extract which was extracted in 24 h showed the highest total
tween 8 and 12 h of extraction. Cacace and Mazza (2003b) anthocyanin content by 25 % ethanol. In this diagram, totally
reported that increasing solvent to solute ratio increased the at ethanol percentages more than 50, the declining trend of
amount of extracted phenolic compounds from blackcurrant total anthocyanin content over time intervals had a higher
and the time of extraction decreased. These researchers also speed in comparison with the lower concentrations of ethanol.
found similar results in extraction of phenols from milled
berries (Cacace and Mazza 2003a). Optimization of the Anthocyanin Extraction
In Fig. 2e, the interaction between solvent ratio and
ethanol concentration on total anthocyanin content (al- In order to achieve the highest level of anthocyanins (total
though it was not significant with 99 % confidence) is anthocyanin content), optimal conditions of the extraction
illustrated. As the chart indicates, lower ratios of solvent process were evaluated (Table 4). To define the variables,
and higher percentages of ethanol led to a decrease in usage of the least amount of solvent and the least percentage
total anthocyanin content. Higher percentages of ethanol of ethanol were considered. The lower limit of anthocyanin
made a solution with lower polarity and thereby reduced was equal to 1200 mg/l, which was less than approximately
the solubility of anthocyanins. As it can be seen in the 60 % of the obtained data from different experiments. The
surface plot, maximum total anthocyanin content was ob- upper limit of total anthocyanin content was considered as
tained approximately in 50 % ethanol and 50 ml/g of 1680 mg/l, which was equal to the total anthocyanin content
solvent to solute ratio. Cacace and Mazza (2003a) report- obtained from two-time extraction (in 16 h, 35 °C, 50 % eth-
ed that increasing the ethanol concentration up to about anol, and 50 ml/g solvent to petals) from the petals. Solution
50 regardless of the solvent ratio, in extract of milled was provided by the software considering highest desirability,
berries whereas transcending ethanol percent, reduced and the optimal extraction conditions were as follows: extrac-
the extraction efficiency which have been followed in tion time of 24 h, temperature of 25.8 °C, solvent ratio of
our study too. 20 ml/g, and ethanol concentration of 25.02 % (Table 4).
Effects of the extraction time and ethanol concentration on Under these conditions, the amount of extracted anthocyanin
total anthocyanin content of the extract have been displayed in was 1609.11 mg/l.
Food Anal. Methods

(A) (B)

(C) (D)

(E) (F)
Fig. 2 Response surface plots for the effect of extraction parameters on percent (solvent ratio 50 ml/g and temperature 35 °C), d time and solvent
total anthocyanin content of saffron petal’s extract. a Temperature and ratio (ethanol 50 % and temperature 35 °C), e ethanol percent and solvent
time (ethanol 50 % and solvent ratio 50 ml/g), b temperature and solvent ratio (ethanol 50 % and solvent ratio 50 ml/g), and f ethanol percent and
ratio (ethanol 50 % and temperature 35 °C), c temperature and ethanol extraction time (temperature 35 °C and solvent ratio 50 ml/g)

In order to assess the compliance of the optimal extraction Conclusion

conditions with laboratory conditions, solvent extraction took
place in the optimal extraction conditions provided by soft- The results showed that the process variables, including tem-
ware. The average obtained anthocyanins from three repli- perature, extraction time, solvent ratio, and the percentage of
cates were equal to 1542.14 ± 67 mg/l (90.06 % of total an- ethanol, had statistically significant effects on anthocyanin
thocyanins in saffron petal). Considering standard deviation, extraction from the saffron petals. Quadratic polynomial mod-
obtained total anthocyanin content in lab conditions was close el was fitted to the experimental data to predict the amount of
to the value predicted by the model and showed good fitness extracted anthocyanins. Increasing temperature, regardless of
to the predicted data. the other variables, led to a reduction in the total anthocyanin
 Food Anal. Methods

content. Rising the solvent ratio to the solute increased con- AOAC International (2006) Official methods of analysis of AOAC
International
centration gradient of anthocyanins and further increased the
Cacace JE, Mazza G (2003a) Mass transfer process during extraction
extraction efficiency. On the other hand, due to the low pH of of phenolic compounds from milled berries. J Food Eng 59(4):
the extract in high ratios of solvent to solute, the stability of 379–389
extracted anthocyanins was increased. Total anthocyanin con- Cacace JE, Mazza G (2003b) Optimization of extraction of anthocyanins
tent was a function of both variables of ethanol percent and from black currants with aqueous ethanol. J Food Sci 68(1):240–248
Castaoveda-Ovando A, Ma. de Pacheco-Hernondez L, Ma. Priez-
extraction time. In the first 12 h of extraction, as the percent- Hernandez E, Rodriguez JA, Galoan-Vidal CA (2009) Chemical
age of ethanol solvent was increased, total anthocyanin con- studies of anthocyanins: a review. Food Chem 113(4):859–871
tent increased too. In contrast, in the second 12 h of extraction, Chen Y (2008) Analysis on main nutrient content of black soybean [J].
total anthocyanin content decreased with increasing the etha- Journal of Anhui Agricultural Sciences 34:48
Esfanjani AF, Jafari SM, Assadpoor E, Mohammadi A (2015) Nano-
nol concentration. Maximum monomeric anthocyanin content
encapsulation of saffron extract through double-layered multiple
was obtained in 24 h extraction time, 25.8 °C, 20 ml/g solvent, emulsions of pectin and whey protein concentrate. J Food Eng
and 25.02 % ethanol, and extracted anthocyanins was 165:149–155
1609.11 mg/l. Compared with other sources of natural antho- Fatehi M, Rashidabady T, Fatehi-Hassanabad Z (2003) Effects of Crocus
cyanins, the saffron petal is very rich in anthocyanins and it sativus petals’ extract on rat blood pressure and on responses in-
duced by electrical field stimulation in the rat isolated vas deferens
can be used as a potential replacement for the natural color and guinea-pig ileum. J Ethnopharmacol 84(2–3):199–203
resources of anthocyanins for food and pharmaceutical Galanakis C (2012) Recovery of high added-value components from food
industries. wastes: conventional, emerging technologies and commercialized
applications. Trends Food Sci Technol 26:68–87
Galanakis C (2013) Emerging technology for the production of nutraceu-
Acknowledgments Iran National Science Foundation and the Research tical and challenges. Food Bioprod Process 91(4):575–579
Institute of Food Science and Technology in Mashhad (Iran) should be Galanakis CM et al (2015) Phenolic content and antioxidant capacity of
acknowledged for their financial support. cypriot wines. Czech J Food Sci 33(2):126–136
Giusti MM, Wrolstad RE (2001) Characterization and measurement of
Compliance with Ethical Standards
anthocyanins by UV-visible spectroscopy, current protocols in food
analytical chemistry. John Wiley & Sons, Inc
Conflict of Interest Katayun Mahdavi Khazaei declares that he has no
Gould K, Davies K, Winefield C (2008) Anthocyanins: biosynthesis,
conflict of interest.
functions, and applications. Springer
Seid Mahdi Jafari declares that he has no conflict of interest.
Hadizadeh F, Khalili N, Hosseinzadeh H, Khair-Aldine R (2003)
Mohammad Ghorbani declares that he has no conflict of interest.
Kaempferol from saffron petals. Iran J Pharm Res 2:251–252
Abbas Hemmati Kakhki declares that he has no conflict of interest.
Messiah Sarfarazi declares that he has no conflict of interest. Harbourne N, Marete E, Jacquier JC, O’Riordan D (2013) Stability of
phytochemicals as sources of anti-inflammatory nutraceuticals in
beverages, A review. Food Res Int
Human and Animal Rights We must include the following sentence
Hemmati Kakhki A et al (2001) Optimization of effective parameters of
to make sure that readers are aware that there are no ethical issues with
production of food color from saffron petal. Agric Sci 13(20):16–23
human or animal subjects:
Kafi M (2006) Saffron (Crocus Sativus) production and processing.
This article does not contain any studies with human or animal
Science Publishers
subjects.
Khalili M, Roghani M, Ekhlasi M (2010) The effect of aqueous crocus
sativus L. extract on intracerebroventricular streptozotocin-induced
cognitive deficits in rat: a behavioral analysis. Iranian Journal of
Pharmaceutical Research 185–191
Khazaei K, Jafari SM, Ghorbani M, Hemmati Kakhki A (2014)
Application of maltodextrin and gum Arabic in microencapsulation
References
of saffron petal’s anthocyanins and evaluating their storage stability
and color. Carbohydr Polym 105:57–62
Agha-Hosseini M, Kashani L, Aleyaseen A, Ghoreishi A, Rahmanpour Kirca A, Özkan M, Cemeroglu B (2006) Stability of black carrot antho-
H, Zarrinara AR, Akhondzadeh S (2008) Crocus sativus L. (saffron) cyanins in various fruit juices and nectars. Food Chem 97(4):598-
in the treatment of premenstrual syndrome: a double-blind, 605
randomised and placebo-controlled trial. BJOG Int J Obstet Kirca A, Auzkan M, Cemeroulu B (2007) Effects of temperature, solid
Gynaecol 115(4):515–519 content and pH on the stability of black carrot anthocyanins. Food
Akhavan S, Jafari SM, Ghorbani M, Assadpoor E (2014) Spray drying Chem 101(1):212–218
microencapsulation of anthocyanins by natural biopolymers: a re- Koocheki A, Taherian AR, Razavi SMA, Bostan A (2009) Response
view. Dry Technol 32:509–518 surface methodology for optimization of extraction yield, viscosity,
Akhondzadeh Basti A, Moshiri E, Noorbala A-A, Jamshidi A-H, Abbasi hue and emulsion stability of mucilage extracted from Lepidium
SH, Akhondzadeh S (2007) Comparison of petal of Crocus sativus perfoliatum seeds. Food Hydrocoll 23(8):2369–2379
L. and fluoxetine in the treatment of depressed outpatients: a pilot Kubo I, Kinst-Hori I (1999) Flavonols from saffron flower: tyrosinase
double-blind randomized trial. Prog Neuro-Psychopharmacol Biol inhibitory activity and inhibition mechanisms. J Agric Food Chem
Psychiatry 31(2):439–442 47(10):4121–4125
Andersen ØM, Jordheim M (2010) Chemistry of flavonoid-based colors Lee J, Rennaker C, Wrolstad RE (2008) Correlation of two anthocyanin
in plants, comprehensive natural products II. Chemistry and biology quantification methods: HPLC and spectrophotometric methods.
(3):547–614 Food Chem 110(3):782–786
Food Anal. Methods

Moshiri E, Basti AA, Noorbala A-A, Jamshidi A-H, Hesameddin Rajabi H, Ghorbani M, Jafari SM, Sadeghi AR, Rajabzadeh G (2015)
Abbasi S, Akhondzadeh S (2006) Crocus sativus L. (petal) in Retention of saffron bioactive components by spray drying encap-
the treatment of mild-to-moderate depression: a double-blind, sulation using maltodextrin, gum Arabic and gelatin as wall mate-
randomized and placebo-controlled trial. Phytomedicine rials. Food Hydrocoll 51:327–337
13(10):607–611 Sarfarazi M, Jafari SM, Rajabzadeh G (2015) Extraction optimization of
Norbark R, Brandt K, Nielsen JK, Girgaard M, Jacobsen N (2002) Flower saffron nutraceuticals through response surface methodology. Food
pigment composition of Crocus species and cultivars used for a Anal Methods 8:2273–2285
chemotaxonomic investigation. Biochem Syst Ecol 30(8):763–791 Stintzing FC, Carle R (2004) Functional properties of anthocyanins and
Pala KU, Toklucu AK (2012) Effect of UV-C light on anthocyanin con- betalains in plants, food, and in human nutrition. Trends Food Sci
tent and other quality parameters of pomegranate juice. J Food Technol 15(1):19–38
Compos Anal in press, corrected proof Van Sumere C, Vande Casteele K, De Loose R, Heursel J (1985)
Patras A, Brunton NP, Donnell C, Tiwari BK (2010) Effect of thermal Reversed phase HPLC analysis of flavonoids and the biochemical
processing on anthocyanin stability in foods; mechanisms and kinet- identification of cultivars of evergreen Azalea. Biochem Plant
ics of degradation. Trends Food Sci Technol 21(1):3–11 Phenolics 25:17–43
Raissi S, Farsani RE (2009) Statistical process optimization through Yoshida K, Kondo T, Goto T (1991) Unusually stable monoacylated
multi-response surface methodology. World Acad Sci Eng Technol anthocyanin from purple yam Dioscorea alata. Tetrahedron Lett
51(46):267–271 32(40):5579–5580