Jordan Journal of Chemistry Vol. 5 No.4, 2010, pp. 389-400

 

389  

JJC

Determination of Ubiquinone,10 in Ten Different Sorts of Iraqi dates "Phoenix dactylefra" at Different Stages of Fruit Maturation

Ghadah Al-Faraji and Muthana Shanshal*

Department of Chemistry, College of Science, University of Baghdad, Jadiriya, Baghdad, Iraq.

Received on Feb. 2, 2010 Accepted on Aug. 22, 2010

Abstract The saponification extraction method was applied for the isolation and identification of

CoQ10 in 10 different sorts and at 4 different maturation stages of Iraqi dates. The purification of

the extract was done applying column chromatography (CC) and thin layer chromatography

(TLC) as well as final recrystallization from ethanol. The identification was done applying HPLC,

HPLC/MS, NMR and UV spectroscopy. It was found that, for different sorts different CoQ10

concentrations exist. The highest concentration was found for the Zahdi (most common and

cheapest) sort. The relative CoQ10 content increases from the initial “hababuk- kermi” stage to

the final and ripe date fruit.

Keywords: Ubiquinone; Coenzyme Q10; Iraqi dates; Phoenix dactylefra.

Introduction CoenzymeQ10 (Ubiquinone,10) was first isolated by Morton et al. from beef heart

mitochondria [1]. It was isolated simultaneously by Crane et al.[2] and Folkers et al.[3]

from the cells of other organisms. Its molecular structure was determined to be 2,3-

dimethoxy,5-methyl,6-polyisoprene parabenzoquinone [3].

Ubiquinone,10 (Coenzyme Q10 )

Ubiquinone,10 was found in different parts of the living cell besides the

mitochondrion of different organisms [4]. In its reduced form it acts as antioxidant [5, 6]

and is effective in the treatment of heart diseases [7]. In the mitochondrion it acts as

important electron carrier within the energy producing respiratory chain [8].

                                                            * Corresponding author: E-mail: mshanshal2003@yahoo.com

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The biochemical active part of the CoQn(n= 6, ….10) is the quinone ring, which

undergoes redox reactions faciliating the electron and proton transportation in the

mitochondria, via the so called Mitchells cycle [9]. The reduction process proceeds

through a ubiquinol semiradical leading to CoQH2.

The head-to-tail structure of CoQn is essential for its biological function; it is thus

similar to the vitamins E and K which include electron accepting quinone groups and

hydrophobic polyisoprenoid chain. Among these the CoQ10 possesses the longest side

chain. It is thus very lipophilic. The side chain is responsible for its fixation in the

mitochondrion, both in the plasmic membrane as well as in other phospholipids

containing parts of the cell [10].

CoQ10H2 is an effective antioxidant [14], it protects the mitochondrion membrane

and DNA from destruction. It acts as an activator of Vitamin E. It was found useful for

the treatment of various heart diseases. It helps blood circulation, increases the

immunity and oxygen level in human tissues. Although human body produces CoQ10, a

daily intake of approx. 100mg is required [15]. Different foods show different CoQ10

contents. The content varies from 157 mg/100g in Reindeer to 0.07 mg/100g in low fat

milk [16]. Chemical synthesis of CoQ10 requires highly stereoselective chemical

reactions [17]. More common are the biotechnological production methods. Such

microbiological methods, utilizing cultures of the type, Agrobacterium , Rhodobacter,

Paracoccus, Rhodotorula, Candida, are applied for the efficient industrial production of

the compound [18].

To extract the mitochondrion CoQ10 it is important to destruct the cell wall

through sonication, extract the content with adequate solvents and then remove the

nonsoluble materials [19]. Three prominent methods are known in the literature;

a- saponification method in which the phospholipids are removed through alkaline

hydrolysis followed by solvent extraction;

b- direct extraction with suitable organic solvents.

c- solid phase extraction [20].

Determination and chemical test of CoQ10 proceeds through different chemical

methods depending on the type of reagent used for the test such as Graven’s test,

Dam–Karrers’ test and Sullivan’s test [21]. For the identification various analytical and

spectrometric methods had been applied too [22], among these are the high

performance liquid chromatography, HPLC, and liquid chromatography–mass

spectrometry, LC-MS, combination [23-25]. Both methods gained increasing interest in

the last two decades.

Date is considered in many Afro- Asian countries, as one of the most important

food sources. Iraq (Mesopotamia) is one of the oldest date palm cultivating countries.

Date palms (Phoenix dactylifera) were reported in the old Babylonian lyrics 4000 years

ago [26]. The mature fruit is conformed of a hard kern surrounded by a fleshy mass

which forms the actual part to be consumed [27], figure 1.

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Date fruit passes through different and distinct stages of maturation up to its final

ripe form. The time period required for the total process is about 200 days. Four

different stages of maturation are noticed for all different types of Iraqi dates. These

are; a- the “Hababuk Stage” in which the fruit is green, round with a bitter taste and of

an olive type shape, its duration lasts for 5-6 weeks, b- “Khalal stage” of a yellow or

yellow-red colour tasting light bitter to sweet, (19 – 25 weeks) c- “Rutab stage”

coloured either yellow or red yellow depending on the type of date fruit (26 – 28 weeks)

and d- the ripe date fruit with complete red colour and sweet taste. Physically the date

fruit is classified as dry, semi-dry and soft. There are more than 531 sorts of Iraqi dates [28,29]. Some of these are more common than the others. The sorts considered in the

present work are ten, named according to the native language,” Khadrawy, Sayeer,

Zahdi, Barhi, Khastawi, Breem, Sukri, Saba’athraa, Brean and Barban”.

Figure 1: The Different maturation stages of Iraqi dates

In general the chemical composition of date fruit was found to include;

monosugars (44 – 88%) , water (84% at the early stages of maturation, 20% in the ripe

fruit), fibers (6.4 – 11.5% for the different sorts), tannin, polyphenols, rare earths (1.5 –

2.5%), proteins and amino acids (2.3 – 6.5%), fatty acids such as palmitic, capric,

caprilyc, linoleic, myristic, pelargonic, lauric, as well as flavones, flavonols, and

anthocyanins. It includes the vitamins B1, B2 and traces of vitamin C. Some sorts show

flavoxanthin ، lutein, lycoptrene, α-Carotene and Chlorophyll (at the early kemri or

khalal stages) [30].

The only known work in the literature dealing with the presence of CoQ,n in date

fruit in general, was our former work, limited to the Zahdi sort at the ripe stage alone

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[30a]. It remained open how does the ubiquinone content change from one date sort to

the other and along its maturation path. The present work deals with this problem.

Experimental All HPLC measurements were done with a Shimadzu SPD-10 AVP, LC-10A

type, instrument (Tokyo, Japan). The UV–VIS spectra were recorded with a Shimadzu

SPD-6AO (UV-VIS) spectrophotometer. The mass spectra were recorded with a

Shimadzu GC/ MS-QP 5050A, Japan. For the LC–Mass spectrometry measurements

was used Liquid Chromatography Tandam Mass Spectrometry, Agilent 1100, Series,

LC/ MSD Trap, USA. The NMR measurements were recorded applying BRUKER

AVANCE/ DPX 300 of Brucker AG, Karlsruhe, BRD. TLC was performed on Silica gel

F254 glass plates (20×20cm) (Merck); Coenzyme Q10 (HPLC grade was bought from

Fluka), Alumina (Acidic Brokman activity I), (BDH.) and Silica gel 60 (63-200µm)

(Sorbent), were used for column chromatography.

The date samples were collected from palm trees in the directorate of Baghdad

and Salah-Eldin, Iraq. For each sort of date, samples of all four stages of maturation

were taken from the same tree. The ten chosen sorts of date were classified according

to the Iraqi standards of date fruits, 1981, vs the market demanded sorts, Zahdi, Sayer,

Khadhrawi, Breem and Sukri and the locally consumed sorts; Berhi, Khastawi,

Barban, Tabarzel and Sabe’athraa. The collection of the date samples to be analyzed

started at the end of June when the first stage (hababuk) begins. The dates were

washed, the kern depleted and the fleshy mass used for digestion immediately. Similar

treatment was applied, at the end of July, for the second, yellow, maturation stage,

khelal. The third maturation stage, rutab, was collected at the mid of August till the

beginning of September. After washing the fruit and depleting the kern, the fleshy

mass was kept in the refrigerator at –20o C for further digestion and extraction

purposes. Similar treatment was done for the ripe date fruit that was collected at the

interval; beginning of October- mid November.

50 g of each of the different date species were cut , mixed with 50 g distilled

water and then homogenized using laboratory Waring blender of 17000 rpm for 10

minutes till a homogenized fluid is formed. The dry and ripe fruit, however, was kept

with water for 48 hours before cutting and homogenizing.

The Saponification Process.

The procedure used in this work was similar to that known in the literature to

extract mitochondrion contents [32 –39], with the difference of applying methanol rather

than ethanol as solvent. In a 500 mL, three neck flask, 100 g of the homogenized

mixture were combined with 2.5 g pyrogallol, dissolved in 70 mL methanol and 25 mL

(25% KOH in water) under continuous stirring. The flask was connected to a nitrogen

tube, immersed in the reaction solution, a stirring rode and a thermometer. The

reaction mixture was heated at 80 – 90o C for 20 – 25 minutes. After completion of the

reaction, the flask was coold down to room temperature using an ice bath.

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The cold reaction mixture was transferred to a separation funnel and mixed with

100 mL petroleum ether (40o – 60o C). The mixture was shaken then for 10 – 15

minutes and left for separation, where a brown organic layer was formed. This was

separated and the remaining yellow aqueous layer extracted with 25 mL petrol ether.

The extraction was repeated three times.

The combined organic extracts were washed with 1/3 of the mixture volume

water. The washing was repeated three times. The separated organic solution was

dried over anhydrous Na2SO4, filtered and the solid sulfate washed with cold petroleum

ether. The filtrate was then evaporated under reduced pressure applying a rotary

evaporator (40oC). 5 mL ethanol were added to the solid residue and the evaporation

repeated to remove any traces of water in the flask. The yellow–orange residue was

dissolved in absolute ethanol and kept in the dark at –20oC for analysis.

The formerly described extraction procedure was repeated for all 4 different

maturation species of 10 different sorts of Iraqi dates. The crude extraction solution

was used for the HPLC study. As standard authentic sample a 10 ppm solution of

CoQ10 (Fluka) in ethanol was applied for comparison purpose.

HPLC Quantitative Analysis

50 µL of CoQ10 solution were injected in the HPLC instrument and the analysis

done using: Waters-Sperisorp S5- ODS2 column (250 x 4.6 mm) with pebble mesh of

0.5 µm (35:65 v/v deionized water: acetonitrile) as mobile phase at a flow rate 1

ml/min. The UV-detector wave length was 275 nm.

Thin Layer Chromatography, TLC

For the separation process 16 different solvent mixtures were tested. The best

separation results were obtained with a 9:1 (v/v) chloroform: ethylacetate mixture.

During the separation the chromatographic jar was wrapped with Al foil.

Column chromatography

The separation and purification of CoQ10 from the saponification extract was

performed by chromatography over silica gel and alumina (50x1cm) columns. The

column was packed in heptane to remove all nonpolar compounds. After introducing

the extraction solution to the top of the column, it was washed successively with

mixtures of heptan + CHCl3 (2-20%) with increasing polarity. The eluted fractions were

tested with UV absorption (275 nm) and TLC plates for the presence of CoQ10.

The Alumina column (50x1cm), was packed with petroleum ether (40 – 60oC) [40-

42]. and eluted with a mixture of petroleum ether and ethylacetate of increasing polarity

in a similar manner as that described before for the silica column. The collected

fractions were tested for CoQ10 applying UV absorption (275nm) and TLC. In some

experiments n-hexane was used instead of petroleum ether.

Results and Discussion.

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The saponification method had been applied preferrably for the extraction of

hydrophobic natural products such as the carotens, vitamins, A, E, D and ubiquinones [31]. It was used in this work for CoQ10 extraction. The method is ideal for getting rid of

the phospholipids found in the walls of the mitochondria which show strong affinity

towards the CoQ10, and the presence of which hinders the purification of the

ubiquinone and its chromatographic separation.

Generally for each 1 g of the row material one applies 1 ml water, 1.5 ml

methanol, 0.05 g pyrogallol, and 0.5 mL (25%) aqueous KOH solution. Petroleum

ether, hexane or 1- propanol are the best suitable solvents for the extraction. In order

to obtain good separation and identification for CoQn it is important to work under

exclusion of light. This fact was indicated by Kishi [32] who showed that exposing the

coenzyme to sun light for 1 hour causes a photochemical degradation and a

considerable change in its UV absorption spectrum, Fig 2. Former work described in

the literature dealing with the CoQ10 separation showed that its reduced form CoQ10H2

gets oxidized during the separation process [32 – 42].

We found that applying ethanol caused a partial exchange of the methoxy

groups in the CoQ10 molecule by the ethoxy group. Both TLC and MS measurements

showed the presence of monoethoxy- and diethoxy- ubiquinones beside the normal

ubiquinone molecule in the reaction solution. To avoid this problem the reaction was

carried out in methanolic solution, as the substitution reaction (with the methoxy group)

does not change the chemical structure of the ubiquinone.

All operations were done under complete exclusion of light through wrapping of

the reaction vessels with aluminum foils.

Figure 2: Change in the UV absorption spectrum of CoQ10 due to exposure to sunlight

for 1 hour [32].

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HPLC Analysis.

The HPLC method was found efficient for the identification of ubiquinones (Q10,

Q9, Q8) [43– 49]. As separation column, usually ODS column had been used. In the

present study Spherisorb, ODS column was used for the identification of the CoQ10

Under the present measurement conditions, the oxidized CoQ10 (10 ppm), shows an Rt

value of 3.89 minutes and the relative band area 31008. These values were applied for

the quantitative HPLC identification of the coenzyme according to the standard relation [50 – 51].

Table 1 shows the HPLC measured concentrations of CoQ10 for the crude

extraction solutions of all the ten sorts of Iraqi dates at their various maturation stages.

The concentrations are reported in mg/100g units as common in the literature. It is

seen that the Zahdi sort records the highest coenzyme concentration in all the different

maturation stages as compared with the other date sorts. Comparison with the

determined concentration in the Sukri sort, which showed the lowest coenzyme

concentration, one finds the following figures, at the kemri stage (0.432 mg/100g

Zahdi; 0.216 mg/100g Sukri ), at the khelal stage (1.366 mg/100g Zahdi; 0.322

mg/100g Sukri ), at the rutab stage (2.25mg/100g Zahdi; 1.560 mg/100g Sukri) and at

the ripe date stage(6.743 mg/100g Zahdi; 0.832 mg/100g Sukri).

Inspecting the values in the table, one finds that the presence of CoQ10

increases on going from the initial kemri (hababuk) phase to khelal and then rutab.

This increase however does not hold on going from rutab to the ripe and dry date fruit,

with the exception of the dry sorts Breem, Zahdi and Barhi. An explanation to that

might be the decrease in the water content of the growing fruit, which is usually about

83% in kemri and 66% in khelal and 43% in rutab phase [52]. This decrease in the water

content is the probable cause for the increase in the relative quantity of other

components. It should be mentioned that the ripe Breem and Zahdi dates are quite dry

when compared with the fruit of the other sorts. Table 1: HPLC determined CoQ10 concentration (mg /100g) in 10 different sorts of

Iraqi dates at their 4 maturaion stages.

Kemri phaseKhilalal phaseRutab phaseDry date fruit Date sort

0.392 0.629 1.21 5.092 Breem

0.336 0.459 1.015 0.903 Tabarzel

0.216 0.322 1.560 0.8316 Sukri

0.384 0.402 1.054 0.885 Khadhrawi

0.432 1.366 2.250 6.743 Zahdi

0.328 0.664 1.159 1.140 Sayer

o.256 0.618 1.632 1.443 Barban

0.336 0.522 1.286 1.166 Sab,ithraa

0.360 1.012 1.141 1.125 Khestawi

0.240 1.063 1.024 1.730 Berhi

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TLC purification of the crude CoQ10 extract.

Figure 3 shows the UV absorption spectrum measured for the crude saponification

extraction solution of CoQ10. The spectrum shows no resemblance to that of a pure

CoQ10.

Figure 3: UV absorption spectrum of the saponification extract of zahdi sort of Iraqi

Date.

Thin layer chromatography was applied to inspect the saponification extraction

solution [30a]. As stated before 16 elution solvents were tested. The best results were

obtained utilizing the elution solvent (9:1 v/v) chloroform: ethylacetate, where the

separated (CoQ10) spot showed considerable similarity in its UV spectrum and Rf value

with that of the authentic sample, figure 4. The sample, dissolved in ethanol showed a

positive Graven’s test and a correct behavior on reduction with NaBH4.

Figure 4; UV absorption spectrum of the saponification extract of the ripe Tabarzel sort

after purification applying TLC; a- before reduction, b- after reduction with NaBH4.

Column chromatographic purification of the crude CoQ10 extract .

Column chromatography was applied too for the purification of the saponification

extract. Applying the silica gel column, the best elution solvent was a mixture of

chloroform (4-8%) in heptane. This was proofed through tests applying UV absorption

and TLC. The TLC gave two spots, the second of them, Rf = 0.25 (similar to the

authentic spot) was extracted with ethanol subjected to sonication, filtered and the

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solution tested with UV absorption, in the oxidized and reduced forms. It behaved

similar to authentic CoQ10. In the mass spectrometric measurement of the same

solution, the mother peak of 863.7 appeared as well as the other signals specific for

the coenzyme. LC/MS measurement yielded, among others a signal, the mass

spectrum of which was identical to that of the coenzyme too.

Applying the alumina, Brockman-activity 1, column, the elution liquid was a

mixture of petroleum ether and ethyl acetate. The most effective separation was

obtained with 4-8% ethylacetate in petroleum ether. It gave the highest detectable

concentration of the coenzyme, according to the UV measurements and TLC tests.

The resulting solution was dried with a rotary evaporator then dissolved in ethanol.

LC/MS measurements showed a presence of CoQ10 in the solution which corresponds

to 60% of the extracted material

Figure 5: HPLC chromatogram of CoQ10 isolated from the tabarzel sort of Iraqi dates.

Preparative TLC following the alumina column treatment of the total extraction

solution, allowed the isolation of the major amount of the extracted coenzyme.

Extraction of the band containing the coenzyme with ethanol, and then leaving the

ethanol solution for 7- 10 days at -20oC lead to the precipitation of yellow crystals. After

drying in a desiccator under vacuum 10 mg of ubiquinone were obtained which

showed all physical properties of the coenzyme, i.e. MS, UV (oxidized and reduced),

Graven’s test etc. Figure 5 shows the HPLC chromatogram of CoQ10 isolated from the

Tabarzel sort of Iraqi ripe date. The LC/MS test showed a dominating peak and MS

spectrum corresponding to 97% of the resulting solid material, Figure 6. The ethanol

filtrate showed no signals for CoQ10.

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Figure 6: LC/MS spectrum of the alumina column and TLC purified CoQ10 extracted

from Zahdi ripe fruit. Peak No 2 in the LC belongs to the coenzyme.

To conclude the physical identification of the isolated CoQ10, 1H- and 13C- NMR

spectra were recorded for the sample. In both spectra the signals of all expected nuclei

appeared correctly, and similar to those of an authentic sample. The assignments are

listed in Tables 2 and 3, confirming the structure of the isolated ubiquinone.

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Table 2: 1H NMR signals measured for the CoQ10 isolated from the zahdi sort of Iraqi

date, in DCCl3.

δ Type of SignalNumb of ProtonsFunctional Group

1.57 S 24H CH3

1.66 S 3H CH3 )cis (terminal

1.72 S 3H CH3 terminal (trans)

1.93-2.0 m 20H -CH2- CH=C(CH3)-CH2-

2.0-2.09 m 19H -CH2- CH=C(CH3)-CH2-

3.95 S 3H -OCH3

3.97 S 3H -OCH3

5.11 m 9H -CH2-CH=C-

.

Table 3: 13C NMR signals measured for the CoQ10 isolated from the zahdi sort of Iraqi

dates (DCCl3).

δ Functional group

16.07 C=C(CH3)

25.30 C=C(CH3) terminal CH3

26.75 CH2-CH2-CH=C

39.78 CH2-CH2-CH=C

61.19 -OCH3

124.30 C=C(CH3)

134.99 C=C(CH3)

138.91 aromatic C connected to –CH3

141.72 aromatic C connected to –isoprene group

144.41 aromatic C connected to –OCH3

Acknowledgement The authors are thankful to Mu’tah University, Jordan for providing laboratory

facilities during 2005. The authors thank Prof. Khalid Tarawnah and Prof. Mouayad

Abood, for the their valuable assistance.

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