Effect of Different Solvents, Methods and Extraction Conditions on Determination of Some Chemical Compounds of Carrot

Introduction

Phenolic compounds constitute one of the most numerous groups of plant metabolites. Different types of phenolic compounds (phenolic acids) are almost ubiquitous in plant foods such as vegetables and fruits. Phenolic compounds have been showed to have antioxidants effects and protecting against harmful cell damage from oxidation (Herrmann, 1988;Ohkawa et al.,1991; Gensler et al., 1994;  Bravo, 1998and Noroozi et al., 1998).

Natural phenolic compounds range from simple molecules, such as  phenolic acids, to high polymerized compounds such as tannins .  Phenolic acids  may be divided into two groups; benzoic acids, such as (p-hydroxybenzoic, protocatechuic, vanillic and gallic acids) which usually present as free acids, and cinnamic acids which include (ferulic, caffeic, p-coumaric, chlorogenic acids). Harborne ,1973; Misaghi, 1982;  Bravo, 1998 and King and Young 1999.

Phenolic compounds can be extracted using different solvents such as water, methanol, aqueous aceton, etc.Some compounds are more soluble in water and another's more soluble in methanol.Extraction efficiency could thus depend on the water/methanol ratio.( Henning,1980; Bravo et al., 1994 & Saura-Calixto and Bravo, 1996).

In a study on carrot, (Nagahashi et al. 1996; and Regnier and Macheix, 1996) mentioned that phenolic acids such as ferulic and p-coumaric acids  can be extracted by alkaline hydrolysis. On the other hand  according to Ibrahim (1996) total soluble phenolic compounds of carrot were extracted with 80% methanol at 0 °C for 72 hours.

Bajaj and Arora (1979) reported that caffeic acid level in carrot roots was 83.3mg/kg. On the other hand Nagahashi et al. (1996) noticed that two phenolic acids unique to carrot root cell were identified as p-hydroxybenzoic acid and vanillic acid. 

            Alasalvar et al. (2001) illustrated that,10 phenolic compounds were determined  in yellow  carrot. chlorogenic acid was the most predominant phenolic compound in  carrot .  

Escarpa et al. (2000)reported that at fast HPLC method used for the analysis of polyphenols in vegetables.

        This investigation was designed to choosen carrot known as source of phenolic acids also extraction and determination of this chemical compounds from carrot and clear the effect of different methods of extraction on the determination of this phenolics .

Materials And Methods

In the present study yellow carrot was purchased from Cairo local markets. Carrot was washed by water several times, then cut into cubics, equal in volume then, classified into three groups as follows: 

1 - Macerated using meat grinder.

2 - Dried using dry oven at 50°C.

3 - Freeze dried at (-49°C) for 12hours.

Phenolic compound standards include p-hydroxybenzoic, vanillin, chlorogenic,   caffeic, p-coumaric and ferulic  were obtained from "Sigma Chemical Company, St Louis, MO, USA".

Phenolic compounds of carrot were extracted using different solvents such as, water/methanol ratios, Hcl, NaoH by different methods and extraction conditions. The combined extractions were transferred into aqueous phase and evaporated the solvent to a known volume.

            Separation was achieved by gradient elution using an initial composition of 95% water, with pH adjusted to 2 with phosphoric acid and 5% acetonitrile. Concentration of the latter solvent was increased to 25% in20 min and up to 50% in another 20 min. (Brenes- Balbuena et al., 1992). The separation conditions were as following: flow rate(1ml/min); wave length at 280nm ( Brenes- Balbuena et al. , 1992 and Donner et al. , 1997 ) ; volume of injection (10ml); temperature (room temperature); the mobile phase composition was (gradient system) of buffer ( Schuly et al. , 1999 and Escarpa et al. , 2000 )  and initial pH was 2.

According to  Escarpa et al. (2000) phenolic compounds were determined by using high performance liquid chromatography (HPLC) . Phenolic compounds were identified by standard phenolics through retention times as shown in Table (1). The quantities of identified phenolic compounds were calculated by comparing relative area percent of standard of calibration curves. Phenolic compound standards were chromatographed singly and in the mixture. The samples were chromatographed under the same conditions.

Table (1): Retention times of phenolic acids detected

with  ultraviolet detectors.

Results And Discussion

      The effect of water / methanol ratio on extraction of phenolic acids  on carrot were illustrated in Table(2). The results revealed that some phenolic acids such as p-hydroxybenzoic  , vanillin, caffeic, p- coumaric and ferulic were not found in fresh carrot .These results were near to those obtained by( Babic et al.,1993; Aubert et al., 1994 and Alasalvar et al.,2001) .

Table (2): Effect of water / methanol ratio on extraction

of some phenolic  acids on carrot ( mg/100g.) .

* F.W = Fresh matter.

** D.W = Dry matter.

On the other hand chlorogenic acid was the predominant compound , it was increased from 0.46 to1.09 and 2.03 mg/100g. by using 20,50 and 80% methanol respectively. This increments may be due to chlorogenic acid was more soluble in methanol. This results are in accordance with those reported by (Saura-Calixto &Bravo,1996 and Alasalvare et al., 2001).

It is worthy to mentioned that drying showed an increased and found some phenolics such as vanillin,chlorogenic,caffeic and ferulic acids. It was fond that vanillin was raised by 133%while ferulic acid was decreased by 24%  (on dry matter) by using 50 and 80% methanol respectively , may be due to vanillin produced from ferulic (Lozovaya et al.,1996 and Talcott et al.,2000).    

Data presented in Table (3) show the using of methanol (80%) at different temperature and  extraction times and its affect on determination of  some phenolic acids on carrot. There were six identified phenolic acids were separated from fresh carrot after 24, 48 and 72 hours at 0°C and room temperature, chlorogenic acid was the predominant compound . The phenolic acids were raised after 2 or3 days compared with the first day. These results go in parallel with found by Merida et al., (1991) and Alasalvar et al .,(2001) who noticed that five phenolic acids were not found in fresh carrot and their content increased after the first day .

Generally, the highest value of phenolic acids were with stored at room temperature compared with 0°C. These results may be due to the enzymes witch are most effective on release some esterase-bound phenolic acids as mentioned by (Bartolome and Gomes-Cordoves,1999) who noted that some enzymes released about 33% to 70% of extractable bound ferulic acid and about 0.8 to 8 % of extractable bound p- coumaric acid .It worthy to mentioned that, Although most phenolic acids were raised after 2 and 3 days at different temperature, chlorogenic acid was decreased from 6.91 mg/100g.after 24 hours to 2.15 mg/100g.after 72 hours. This result may be due to chlorogenic acid had degraded to caffeic acid as noticed by Rodriguez et al.(1994a)       

Table (3): Using methanol (80%) at different temperature

and extraction times and its affect on determination

of some phenolic acids on carrot ( mg/100g. fresh weight).

Data concerning the effect of solvent with different pH values on extraction  of some phenolic  acids after 16 hours  of  incubating  freeze - dried carrot  samples were illustrated in Table (4).The results clearly indicated that p- hydroxybenzoic ,vanillin ,chlorogenic ,caffeic ,p- coumaric and ferulic acids were determined in all incubating freeze - dried carrot  samples. This extracted phenolic acids at 2°C ranged from 0.07 to 1.87, 0.13 to 3.60 and 0.18 to 4.31 mg/100g. by using extraction solvents A,B and C respectively.

On the other hand extracted phenolic acids at 35°C ranged from 0.11 to 2.08, 0.18 to 4.17 and 0.24 to 4.57 mg/100g. by using extraction solvents A,B and C respectively.Chlorogenic was the highest phenolic acid value , it was 4.31 and 4.57 mg/100g. at 2°C and 35°C respectively in extraction solvent (methanol 50% +1.5 M Hcl ).It worthy to mentioned that total phenolic acids at 2°C and 35°C were 2.59,2.97& 4.51,5.48 and 6.17,6.94 mg/100g. in extraction solvents A,B and C respectively. It was concluded

Table (4): Effect of solvent with different pH values on extraction

of some phenolic  acids after 16 hours

of  incubating freeze - dried carrot  samples  (mg/100g.). 

A = Methanol (50%) + 0.5 M Hcl .   

B = Methanol (50%) +1 M Hcl  .  

C = Methanol (50%) +1.5 M Hcl  .  

that the optimal extraction of phenolic acids were obtained by incubating freeze - dried carrot  samples for 16 hours at 35°C in 50 % methanol +1.5 M Hcl. The same trend of results was observed by Hakkinen et al. (1998).

     Table (5) summarized the effect of extraction solvent (methanol 80%) with different alkaline media on determination of phenolic acids on fresh  carrot . It was demonstrated that , there were six identified phenolic acids were separated from fresh carrot by using methanol 80% with different alkaline concentration compared to chlorogenic acid which was found only by using methanol 80% before . It is obvious from the results thatphenolic acids on fresh carrot can be extracted by alkaline hydrolysis and this method showed the best results. These results are in accordance with found by Wallace et al.(1991) ; Nagahashi et al.(1996) and Regnier and Macheix (1996).  

Table (5): Effect of extraction solvent (methanol 80%) with different  alkaline media on determination of phenolic acids on fresh carrot (mg/100g.).

It is clear from the results that chlorogenic acid was the main compound when using NaoH (0, 5%) meanwhile caffeic acid was the highest value when using NaoH by 1,1.5 and 2%. Also it was found that ferulic acid was decreased when using NaoH by 1.5and 2%. This results my be due to chlorogenic acid could be degraded to caffeic acid and ferulic acid may be produced vanillin. These results are in agreement with those obtained by Rodriguez et al.(1994a) and Lozovaya et al.(1996).

It worthy to mentioned that total phenolic acid on fresh carrot were 3.1, 3.72, 4.12 and 4.56 mg/100g. fresh carrot by using 0.5, 1, 1.5 and 2% of NaoH respectively.This increment may attributed to liberation of some phenolic acids conjugated with polysaccharides in cell wall especially ester linked cinnamics (  chlorogenic, caffeic, p-coumaric,  ferulic ) . This results confirmed by Desphande and Shalunke(1982) ; Ya et al.(1989) and Beveridge et al.(2000).