Determination of D-pinitol in carob syrup

Abstract

Carob syrup is a traditional product native to the Mediterranean region, containing a high concentration of sugar, phenolic compounds and minerals. d-pinitol is a bioactive component extracted from legumes and has some beneficial effects on human metabolism. In this research, the d-pinitol content and sugar profile of 10 different carob syrup samples purchased from Turkish markets were determined. Mean d-pinitol, sucrose, glucose and fructose contents of samples were found to be 84.63 ± 10.73, 385.90 ± 45.07, 152.44 ± 21.72 and 162.03 ± 21.45 g/kg dry weight, respectively. Carob syrup has a considerable amount of d-pinitol compared with the other d-pinitol-including legumes. Consequently, this study showed that carob syrup may be a suitable source of d-pinitol for medical use and d-pinitol may be an indicator for the detection of any adulteration in carob syrup.

Full text

Determination of D-pinitol in carob syrup
NEDIM TETIK, IRFAN TURHAN, HATICE R. OZIYCI, & MUSTAFA KARHAN
Akdeniz University, Faculty of Engineering, Department of Food Engineering, Antalya, Turkey
Abstract
Carob syrup is a traditional product native to the Mediterranean region, containing a high concentration of sugar, phenolic
compounds and minerals. D-Pinitol is a bioactive component extracted from legumes and has some beneficial effects on human
metabolism. In this research, the D-pinitol content and sugar profile of 10 different carob syrup samples purchased from Turkish
markets were determined. Mean D-pinitol, sucrose, glucose and fructose contents of samples were found to be 84.63 ^10.73,
385.90 ^45.07, 152.44 ^21.72 and 162.03 ^21.45 g/kg dry weight, respectively. Carob syrup has a considerable amount of
D-pinitol compared with the other D-pinitol-including legumes. Consequently, this study showed that carob syrup may be a
suitable source of D-pinitol for medical use and D-pinitol may be an indicator for the detection of any adulteration in carob syrup.
Keywords: Carob syrup, legume, D-pinitol, sugar profile, bioactive components, nutrition
Introduction
The carob bean (Ceratonia siliqua L.), belonging to the
Caesalpinaceae subfamily of the Leguminosae, is from a
tree grown in the Mediterranean region. Carob trees
produce fruits of which the pods are 90% and the seeds
10% of the total fruit weight (Karkacier and Artik 1995,
Fletcher 1997). Whole carob fruit is not convenient to
consume because of the hard structure that comprises
the fleshy part. Carob seeds have been used for
production of a powder called locust bean gum (LBG),
which is a commercial product of high value that has
been used as a stabilizer in emulsions and dispersions.
Additionally, enzymatic extracts of carob seeds can be
used a biofertilizer due to the high proportion of water-
insoluble protein (Parrado et al. 2008). Because of the
high commercial value of LBG, carob seeds have
primarily been used for LBG production. Crushed and
deseeded carob pods have been evaluated as a cacao
substitute after a powdering process (Baumgartner et al.
1986), feed for livestock (Khair et al. 2001), feedstock
for the production of bioethanol (Roukas 1995, Turhan
et al. 2010) and as a substrate for citric acid production
(Roukas 1995). Most carob pods have been used for
pekmez production, a traditional concentrated
syrup native to Turkey, after a liquid–solid extraction
process with water (Petit and Pinilla 1995, Batu 2005,
Turhan et al. 2006).
The sugar profile (sucrose, glucose and fructose)
(Karkacier and Artik 1995, Biner et al. 2007), total
phenolic content (Turhan et al. 2006, O
¨zcan et al.
2007), minerals (Karhan et al. 2005, O
¨zcan et al.
2007), amino acid profile (Artik and Erbas 1988), and
protein and fat content (O
¨zcan et al. 2007) of carob
products have been determined by many researchers.
Recently, the demand for functional foods and food
components with health benefits has increased
(Verbeke 2005). Studies on the bioactive components
of carob products have been conducted. One of the
important bioactive components of carob is (3-O-
methyl-D-chiro-inositol) (Figure 1), a cyclitol that is
highly soluble in water (Dowd and Stevens 2002,
Dozois et al. 1938). D-initol is the most dominant
component of the low molecular weight carbohydrate
fraction of legumes (Baumgartner et al. 1986, Kim
et al. 2005), and approximately 99% of total chiro-
inositol (40.0 g/kg) in carob pod exists as D-pinitol
(Kim et al. 2005). D-Pinitol has an insulin-like effect
(Bates et al. 2000) and dietary intake of D-pinitol
represents the major metabolic source for being a
ISSN 0963-7486 print/ISSN 1465-3478 online q2011 Informa UK, Ltd.
DOI: 10.3109/09637486.2011.560564
Correspondence: Nedim Tetik, Akdeniz University, Faculty of Engineering, Department of Food Engineering, 07058 Antalya, Turkey.
Tel: 90 242 310 6535. Fax: 90 242 227 4564. E-mail: nedimtetik@akdeniz.edu.tr
International Jour nal of Food Sciences and Nutrition,
September 2011; 62(6): 572–576
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precursor of D-chiro-inositol in vivo (Davis et al. 2000,
Kim et al. 2005). Also, only 10 mg D-pinitol/kg body
weight was found by Narayanan et al. (1987) to lower
blood glucose significantly during a period of 0.5 –2 h
following administration. In another study, the anti-
inflammatory effect of D-pinitol was demonstrated as
compared with phenylbutazone in the carrageenin-
induced paw edema in rats (Singh et al. 2001). D-
Pinitol may play a critical role in the amelioration of the
pathogenetic process of asthma in rats (Lee et al.
2007). Park et al. (2005) reported that D-pinitol could
be effective in preventing cataract and cornea edema
caused by oxidative stress in a hyperglycemic environ-
ment. D-Pinitol may act as a lipid-lowering, antihyper-
lipidemic, antioxidant and hepatoprotective action in
rats fed a high-fat and high-cholesterol diet (Geethan
and Prince 2008, Choi et al. 2009). It is reported that
D-pinitol has no toxicity (Davis et al. 2000).
However, there is a lack of data on the D-pinitol
content of carob syrup. It is speculated that D-pinitol is
extracted along with the sugars, phenols, and minerals
in syrup production. Therefore, the goal of the present
study was to determine D-pinitol in the carob
syrup samples purchased from Turkish markets.
Materials and methods
Materials
Ten different syrup samples in glass jars were obtained
from local markets in August 2009. The samples
represented all the brands of syrup available on the
market. The sample jars were stored at þ48C until
analysis. All samples were analyzed in duplicate.
Methods
Total soluble solids (8Brix) were determined using an
Abbe refractometer (Atago, Tokyo, Japan). Titratable
acidity (anhydrous citric acid), the formol number and
pH of the samples were performed according to the
AOAC methods (AOAC 1990). The total phenolic
content was analyzed by the Folin– Ciocalteau phenol
reagent assay with slight modification (Spanos and
Wrolstad 1990). Folin– Ciocalteau phenol reagent for
the analysis of the polyphenols and Na
2
CO
3
were
supplied from Sigma-Aldrich Chemie (Schnelldorf ,
Germany). Then 5 ml Folin– Ciocalteau phenol
reagent (0.2 N) and 4 ml Na
2
CO
3
(7.5% w/v) were
added to 100 ml of each sample with 900 ml distilled
water and incubated at ambient temperature for 2 h.
The absorbance was measured at 765 nm using a UV –
VIS spectrophotometer (model UV-160A; Shimadzu,
Tokyo, Japan). Results were calculated and expressed
as grams of gallic acid equivalent per kilogram dry
weight.
D-Pinitol, sucrose, glucose and fructo se analyses were
carried out using a Shimadzu LC-20AD high-
performance liquid chromatography (HPLC) solvent
delivery system equipped with a guard column
(CARBOsep Coregel 87P, 4 £20 mm
2
;Transge-
nomic, Omaha, NE, USA) connected to an analytical
column (CARBOsep Coregel 87P, 7.8 £300 mm
2
;
Transgenomic) and a Shimadzu RID-10A refractive
index detector. The columns were heated to 858C with a
Varian Mistral column oven (Varian, Palo Alto, CA,
USA). MilliQ water as the mobile phase was allowed to
flow at the rate of 0.6 ml/min. The method used for
chromatographic analysis of the samples was offered by
the manufacturer of the analytical column (Transge-
nomic). The samples of 2 g were weighed approxi-
mately and diluted with MilliQ water (1:25), shaken
gently and diluted with MilliQ water (1:10) again.
MilliQ water was purified with a Millipore Milli-Q Plus
system (Millipore, Espoo, Finland). Diluted samples
were passed through a 0.45 mm membrane filter
(CHROMAFIL
w
PET-45/25; Macherey-Nagel,
Du
¨ren, Germany) before injection. The samples were
injected with a 20 ml injection volume using a Shimadzu
SIL-20A autosampler. D-Pinitol, sucrose, glucose and
fructose concentrations were calculated using standard
curves obtained from the injection of the mixed
standard solutions prepared in MilliQ water in the
range of 25 –200 mg/ml as the external standards. D-
Pinitol, sucrose, glucose and fructose standards were
purchased from Sigma-Aldrich Chemie (St Louis, MO,
USA).
Results and discussion
Total acidity of the carob syrup samples ranged from
9.9 to 13.4 g anhydrous citric acid/kg dry weight
while mean value was found to be 11.7 ^1.3 g
(Table I). The determination of the formol number in
fruit juices and concentrates represents a parameter
for the characterization of fruit juice and concentrates.
The lower formol numbers indicate that less fruits are
used for carob syrup production. Formol number values
ranged from 59.5 to 93.0 while the mean was
78.6 ^11.4. The pH ranged from 4.96 to 5.44 while
the mean was 5.15 ^0.15. The soluble solid content of
samples ranged from 66.6 to 73.78Brix while the mean
value was 71.7 ^2. 0 8Brix. The total phenolic content of
H
H
H
H
H
CC
CC
CC
H
CH3
OH
HO
OH
OH
Pinitol
Mono methyl
d
-inositol
OH
Figure 1. Chemical structure of D-pinitol.
Determination of D-pinitol in carob syrup 573
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samples ranged from 7.16 to 12.45 g gallic acid
equivalent/kg dry weight while the mean value was
8.86 ^2.07 g gallic acid equivalent/kg dry weight.
Ayaz et al. (2007) reported that total phenolic content
of carob pods was 13.51 g gallic acid equivalent/kg
dry weight.
Mean values of D-pinitol, sucrose, glucose and
fructose were 84.63, 385.90, 152.44 and 162.03 g/kg
dry weight, respectively (Table II). This indicated that
sucrose was the main sugar followed by glucose and
fructose in all samples. The fructose/glucose ratio was
found 1:1 in the present study, while other researchers
found the fructose/glucose ratio to be 3:1 (Biner et al.
2007) or 1:10 (Ayaz et al. 2007). These differences in
results may arise from the differences between HPLC
analysis methods. Sugars have classically been
analyzed by HPLC using an acetonitrile:water mixture
as a mobile phase and a NH
2
-bonded column as a
stationary phase. The sugar profile of carob products
has been analyzed with this classic HPLC method.
Ayaz et al. (2007) analyzed the sugar composition of
carob pods using a cation-exchange column, 0.0085
mol/l H
2
SO
4
as a mobile phase and a photodiode array
detector at 210 –300 nm scanning range. Previous
studies on the sugar profile of carob have not detected
the presence of D-pinitol using the other methods of
sugar determination by HPLC with a refractive index
detector (Karkacier et al. 2003, Biner et al. 2007).
In the present study, a different mobile phase (water)
and column (gel column) described in the sugar
analysis were used. In gel columns, D-pinitol and
sugars can be analyzed simultaneously (Figure 2).
The D-pinitol content of carob syrup samples
ranged from 63.7 to 95.7 g/kg dry weight while the
mean value was 84.6 ^10.7 g/kg dry weight. How-
ever, there has been no study to characterize the carob
syrup related to the content of D-pinitol. Kim et al.
(2005) reported that carob pods contain 40 g/kg total
chrio-inositol, which consists of 99% D-pinitol.
Soy bean and dried soy whey, both rich sources of
D-pinitol, contain approximately 5 g/kg and 20 g/kg of
this compound, respectively (Kim et al. 2005). The
study showed that carob syrup was the richest source
of D-pinitol compared with other food legumes.
Baumgartner et al. (1986) reported that D-pinitol in
cacao powder could be used as a marker to indicate
adulteration by addition of carob pulp powder. On the
contrary, carob syrup could be sometimes adulterated
with low-quality sugar-containing products. In this
case, the D-pinitol concentration may be considered to
Table I. Results from some descriptive analyses of carob syrup.
Sample Total acidity
a
Formol number pH value Soluble solids (8Brix) Total phenolic content (g/kg dry weight)
1 10.97 ^0.05 87.50 ^2.12 5.02 ^0.01 66.65 ^0.07 11.99 ^0.39
2 9.88 ^0.12 75.00 ^1.41 5.15 ^0.00 70.95 ^0.07 7.60 ^0.02
3 13.41 ^0.07 76.50 ^0.71 5.10 ^0.01 73.00 ^0.00 12.45 ^0.12
4 12.57 ^0.05 65.50 ^3.54 5.12 ^0.03 71.50 ^0.15 7.24 ^0.06
5 10.45 ^0.18 59.50 ^0.71 4.96 ^0.03 71.85 ^0.21 7.16 ^0.03
6 11.08 ^0.17 93.00 ^1.41 5.34 ^0.03 73.05 ^0.07 7.81 ^0.05
7 11.32 ^0.05 93.00 ^2.83 5.20 ^0.00 71.75 ^0.07 7.94 ^0.08
8 12.53 ^0.06 83.50 ^0.71 5.44 ^0.00 73.75 ^0.07 10.94 ^0.05
9 13.32 ^0.03 85.50 ^0.71 5.07 ^0.04 73.60 ^0.14 7.85 ^0.20
10 12.58 ^0.07 76.00 ^2.83 5.12 ^0.00 71.50 ^0.14 7.61 ^0.17
Minimum 9.88 59.50 4.96 66.65 7.16
Maximum 13.41 93.00 5.44 73.75 12.45
Mean 11.73 ^1.27 78.61 ^11.44 5.15 ^0.15 71.76 ^2.04 8.86 ^2.07
Data presented as mean ^standard deviation.
a
Anhydrous citric acid (g/kg dry weight).
Table II. D-Pinitol, sucrose, glucose and fructose contents of carob syrup determined by HPLC.
Component (g/kg dry weight)
Sample D-Pinitol Sucrose Glucose Fructose
1 90.16 ^5.79 418.07 ^0.83 123.76 ^3.68 135.55 ^1.51
2 90.04 ^0.25 398.89 ^0.92 155.93 ^0.35 160.35 ^0.24
3 70.99 ^6.01 311.32 ^7.64 156.90 ^4.85 188.81 ^2.21
4 95.71 ^0.69 431.36 ^2.3 130.32 ^0.66 143.22 ^0.48
5 63.75 ^2.01 333.91 ^3.29 197.51 ^3.51 190.44 ^1.46
6 83.14 ^2.12 359.52 ^9.53 147.03 ^3.44 160.79 ^4.99
7 94.16 ^0.25 456.13 ^2.49 125.89 ^0.37 128.43 ^0.74
8 94.78 ^0.28 381.96 ^0.49 164.73 ^0.07 172.51 ^0.44
9 82.34 ^0.03 362.61 ^2.22 155.25 ^0.32 179.60 ^0.22
10 90.99 ^0.50 409.59 ^1.38 150.74 ^0.60 165.75 ^0.06
Minimum 63.75 311.32 123.76 128.43
Maximum 95.71 456.13 197.51 190.44
Mean 84.63 ^10.73 385.90 ^45.07 152.44 ^21.72 162.03 ^21.45
Data presented as mean ^standard deviation.
N. Tetik et al.574
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be an indicator for the detection of adulteration in
carob syrup.
Conclusion
The present study showed that carob syrup is a rich
source of D-pinitol compared with other known
sources such as soybean and soy products. Owing to
the high D-pinitol content (about 85 g/kg dry weight),
approximately 10 g carob syrup consumption could be
enough for the suggested dose (10 mg D-pinitol/kg
body weight) (Narayanan et al. 1987) that lowers the
blood sugar level in type II diabetes. Interdisciplinary
investigations might also be done to elucidate the
mechanism of lowering blood sugar by carob
syrup consumption. Also, D-pinitol may be a natural
marker for the detection of adulteration in carob
syrup with other adulteration materials.
Acknowledgements
TheauthorsaregratefultoAssociateProfessor
Dr David Turner (School of Plant Biology, Faculty
of Natural and Agricultural Sciences, The University
of West Australia, Crawley, WA, Australia) and Mark
Bechara (Agricultural and Biological Engineering,
The Pennsylvania State University, State College, PA,
USA) for their reviews and comments on manuscript.
Declaration of interest: The present study was
supported by the Akdeniz University Research Fund.
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