Int J Pharm Journal Sci Nanotech Vol 5; Issue 3 • October−December 2012 International of Pharmaceutical Sciences and Nanotechnology Volume 5 •  Issue 3 • October – December 2012 MS ID: IJPSN-6-5-12-ZAM

1808 Research Paper

Separation and Purification of Proanthocyanidins Extracted from Pomegranate’s Peels (Punica Granatum) W. Zam1*, G. Bashour1, W. Abdelwahed2 and W. Khayata1 1

Department of Analytical and Food Chemistry and

2

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Aleppo, Syrian Arab Republic.

Received June 5 2012; accepted September 14, 2012 ABSTRACT This study was designed to find the best method to separate proanthocyanidins from other phenolic compounds extracted from pomegranate’s peel and to investigate their radical scavenging capacity. Water is used as an effective solvent for extracting antioxidant compounds from pomegranate’s peel (Punica granatum). The final extract contained closely related compounds not readily separable. Proanthocyanidins are the most potent antioxidant found in this extract and the purification and separation

from other phenolic compounds is an important challenge. A comparison of two SPE cartridges, including silica-based C18 and aminopropyl for the isolation and purification of proanthocyanidins extracted from pomegranate’s peel, showed that the proposed SPE method with aminopropyl cartridge using alkalized aqueous acetone as eluent was more effective for the purification and the separation of extractable proanthocyanidins.

KEYWORDS: Punica granatum; total phenolics; proanthocyanidins; purification; SPE cartridge.

Introduction Proanthocyanidins are potent free radical scavengers and are believed to be contributors to the health benefits of fruits and vegetables (Prior and Gu, 2005). Studies showed that proanthocyanidin antioxidant capabilities are 20 times more powerful than vitamin C and 50 times more potent than vitamin E (Ishtiaq, 2007). These complex flavonoids have been linked to concentrationdependent anti carcinogenic activity (Seiler et al., 2006) and inhibition of bacterial growth (Shan et al., 2007). Proanthocyanidin oligomers demonstrated immunomodulatory effects (Foo et al., 2000), whereas A-type proanthocyanidins from cranberry and blueberry have been shown to improve urinary tract health by impeding uropathogenic bacterial attachment (Kenny et al., 2007). Several investigations showed improved vascular health after short- or long-term consumption of proanthocyanidins or foods and supplements that contained them (SantosBuelga and Scalbert, 2000). These effects included vasodilation (presumably as a result of increased NO production), decreased platelet aggregation (Vitseva et al., 2005), reduced sensitivity of low-density lipoproteins (LDL) to oxidization (Shafiee, et al., 2003) and modulation of several reactions associated with inflammation (Gary, 2004). Many investigations are still being done as there is a difficulty in isolating these substances in pure form from natural sources, e.g., plant extracts. These sources generally produce mixtures of closely related compounds, not readily separable even with the aid of modern

chromatographic and analytical methods. To concentrate and obtain polyphenol rich fractions before analysis, strategies including sequential extraction or liquid-liquid partitioning or solid phase extraction (SPE) based on polarity and acidity have been commonly used. In general, elimination of lipids can be achieved by washing the crude extract with non-polar solvents such as hexane (Ramirez-Coronel et al., 2004), dichloromethane (Neergheen et al., 2006), or chloroform (Zhang et al., 2008). To remove polar non-phenolic compounds such as sugars, organic acids, and other water-soluble constituents, an SPE process is usually carried out. The polyphenols are then eluted with absolute methanol (Thimothe et al., 2007), ethyl acetate (Rodriguez-Saona and Wrolstad, 2001), ethanol (Dai and Mumper, 2010) or aqueous acetone (Ramirez-Coronel et al., 2004). The separation of proanthocyanidins poses some difficulties and many cartridges and solutions have been proposed to separate them according to their degree of polymerization. Column chromatographies on Sephadex LH-20 (Qa’dan et al., 2010; Gad et al., 2006; Wei et al., 2011), Toyopearl HW-40F (Jayma et al., 2009), C18 SepPak cartridge (Nunez et al., 2006; Sun et al., 1998) and NH2 cartridge (Xia et al., 2011) were employed to separate proanthocyanidins. This study was designed to find the best method to separate proanthocyanidins from other phenolic compounds extracted from pomegranate’s peel and to investigate their radical scavenging capacity. For this reason, two column support matrices were comparatively evaluated: HyperSep C18 and HyperSep NH2 (amino1808

Zam et al: Separation and Purification of Proanthocyanidins Extracted from Pomegranate’s Peels (Punica Granatum)

propyl) using different solvents (ethyl acetate, aqueous acetone, acidified aqueous acetone and alkalized aqueous acetone) and a DPPH radical–scavenging activity test was used to evaluate the antioxidant capacity of the purified proanthocyanidins.

Materials and Methods

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minutes at 40°C temperature (water bath), the absorbance was measured at 734 nm. The total polyphenol content was calculated and expressed as gallic acid equivalents (GAE; g/100 g dry mass) using a gallic acid (0-120 mg/L) standard curve. All samples were prepared in triplicates.

Proanthocyanidins Content

Folin-Ciocalteu reagent 2 N (Sigma-Aldrich, Switzerland), gallic acid (Sigma-Aldrich, China), vanillin (SigmaAldrich, Belgium) and 2,2-Diphenyl-1- picrylhydrazyl (DPPH) and +(-) catechin were purchased from SigmaAldrich (USA), sodium carbonate anhydrous (Surechem, England), ferric ammonium sulphate (Carl Roth, Germany), methanol and ethanol (Sharlau, Spain), ethyl acetate, acetone, 1-butanol, hydrochloric acid and acetic acid were purchased from Surechem (England).

Proanthocyanidins content was measured by the vanillin/HCl assay according to the procedure reported by Sun et al. (1998). Aliquots of 1 mL of samples were mixed with 2.5 mL of 1% vanillin-methanol solution and 2.5 mL of 8% HCl-methanol solution. Then, the reaction mixture was incubated in a water bath for 20 minutes at 30ºC. The absorbance was measure at 500nm and blanked with methanol. Total proanthocyanidin content were expressed as catechin equivalents (mg/g) using a catechin (0.06-0.3 mg/1 mL) standard curve. All samples were prepared in triplicates.

Equipment

DPPH Radical–Scavenging Activity

Micropipette 100-1000 µL (Iso lab, Germany), sensitive balance (Sartorius, Germany), ultra sonic bath, electric stirrer and heater, moisture analyzer balance (Precisa, Switzerland), centrifuge (Shanghai surgical instruments factory, China), pH-meter (Crison, Spain), spectrophotometer (Jasco V-530, USA), lyophilizer (Martin Christ, Epsilon 2-6 D, Germany), HyperSep C18 (50 mg, 1 mL) and HyperSep NH2 (aminopropyl) (500 mg, 6 mL) were obtained from Thermo, England.

The antioxidant activity was measured in term of hydrogen donating or radical scavenging ability using the stable DPPH method according to the method proposed by Brand-Williams et al. (1995). 250 µL of the extract was diluted with distilled water to 10 mL. Aliquots of 200 μL of sample was mixed with 2 mL of 100 μM DPPH methanolic solution. The mixture was placed in the dark at room temperature for 60 minutes. The absorbance of the resulting solution was then read at 520 nm. The antiradical activity was expressed in terms of the percentage reduction of the DPPH. The ability to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging effect (%) = [(A0-A1)/A0]*100 …..(i) Where A0 is the absorbance of the control at 60 minutes, and A1 is the absorbance of the sample at 60 minutes. All samples were analyzed in triplicates.

Chemical and Reagents

Sample Preparation Fresh pomegranates were cleaned with water and dried with a cloth. The peels were manually separated, dried for a few days in an open air shade. The dried samples were then powdered in a blender. They were stored at – 18°C until analysis. The moisture content was determined by using a moisture analyzer balance.

Extraction Procedure 200 mg of dried and ground peel were placed in a thermostatic water bath shaker with 10 mL of deionized (DI) water at 50°C for 20 minutes. The liquid extract was separated from solids by centrifugation at 2000 rpm for 10 minutes. The supernatant was transferred to a 10 mL flask, and DI water was added to make the final volume 10 mL. Elimination of lipids was achieved by washing the crude extract with hexane, as reported by RamirezCoronel et al. (2004).

Total Polyphenol Content The total polyphenol content was determined by the Folin-Ciocalteu method according to the method described by the International Organization for Standardization (ISO 14502-1: 2005). Aliquots of 1 mL of diluted samples were mixed with 5 mL of 10-fold-diluted Folin-Ciocalteu reagent. After 3 minutes, 4 mL of 7.5% sodium carbonate was added. The mixtures were allowed to stand for 30

 

Affinity Many solvents have been used for the elution of polyphenols from solid cartridges. The most usual ones are ethanol, methanol and acetone and their water mixtures. In particular acetone:water mixture was widely used for the elution of proanthocyanidins from the column, as proceeded by Min et al. (2008) and Caili Fu et al. (2007). In order to determine the best solvent for the elution of polyphenols from the solid cartridge, four different solvents were tested. Water, acetone-water (70:30, v/v), ethanol-water (50:50, v/v) and ethyl acetate were used for the extraction of total antioxidants from pomegranate’s peel.

Purification Procedure Hypersep C18 A 1 mL sample of aqueous pomegranate’s peel extract was purified using C18 column that had previously been equilibrated with 3 column volumes of methanol and

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washed with 3 column volumes of distilled water at a flow rate of 1.0 mL/minute, as described by RodriguezSaona et al. (2001). The cartridge was washed with two column volumes water to elute most of organic acids and sugar, as described by Shui and Leong (2004). Proanthocyanidins were then eluted from the SPE column with 1 mL of acetone:water (70:30, v/v) at different pH 3.5, 7.0, 9.5. Hypersep NH2 A 3 mL sample of aqueous pomegranate’s peel extract was purified using NH2 column that had previously been equilibrated with 3 column volumes of methanol:water (1:1) at a flow rate of 1.0 mL/minute, as reported by Caili Fu et al. (2007). The cartridge was washed with methanol:water (1:1) as a mobile phase until the eluent was colorless to separate carbohydrates and phenolic acids, as described by Min, Pinchak, Merkel, Walker, Tomita & Anderson (2008). Proanthocyanidins were eluted from the SPE column with 3 mL of acetone:water (70:30, v/v) with different pH 3.5, 7.0, 9.5.

Purification procedure Hypersep C18 The use of Hypersep C18 as a solid cartridge with acetone 70% at different pH values for the purification of proanthocyanidins wasn’t an effective method. The proanthocyanidins were eluted from the cartridge but were not separated from the other phenolic compounds (Table 2). TABLE 2 Purification of crude aqueous extract by HyperSep C18. Fraction

Crude aqueous pomegranate peel’s extract acetone/water (70:30, v/v) at pH 3.5 acetone/water (70:30, v/v) at pH 7.0 acetone/water (70:30, v/v) at pH 9.5 a

Lyophilization Lyophilization was carried out in order to calculate the degree of purification and the recovery of proanthocyanidins after purification. The purified filtrate was transferred to a roundbottom flask and acetone was removed using a rotary evaporator under vacuum at 40°C. The residual aqueous phase and the crude aqueous extract, before purification, were transferred to a lyophilizer. Freezing was carried out at – 40°C for 2 hours. Temperature of the primary freeze-drying phase was kept at – 10°C for 6 hours at a pressure of 10 Pascal. Secondary drying phase was carried out at a temperature of + 20°C for 3 hours at a pressure of 5 Pascal.

Results and Discussion Affinity The yield of total extractable phenolic compounds and proanthocyanidins were measured and the results are shown in Table 1. The acetone:water (70:30, v/v) was the most effective solvent for extracting total phenolic compounds and proanthocyanidins, and was further used to purify and isolate proanthocyanidins. TABLE 1 The yield of total phenolic compounds and proanthocyanidins extracted from pomegranate’s peel using different solvents. Fraction

Crude aqueous pomegranate peel’s extract Acetone/Water (70:30, v/v) Ethanol/Water (50:50, v/v) Ethyl acetate a Values

Total phenolic compounds (mg/10 mL) a

Total proanthocyanidins (mg/10 mL) a

29.67±0.048

4.34±0.031

28.47±0.35

5.82±0.013

26.94±0.26

3.85±0.075

4.55±0.12

0.32±0.013

are mean ± SDs, (n = 3)

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Total phenolic compounds (mg/10 mL) a

Total proanthocyanidins (mg/10 mL) a

29.67±0.048

4.34±0.031

23.92±0.061

4.11±0.041

24.32±0.066

3.45±0.051

27.48±0.035

3.91±0.043

Values are mean ± SDs, (n = 3)

Hypersep NH2 Results showed that the use of Hypersep NH2 with acetone water mixture (70:30, v/v) at an alkaline pH was the most effective method for the elution and separation of proanthocyanidins from other phenolic compounds (Table 3). TABLE 3 Purification of crude aqueous extract by HyperSep NH2. Fraction

Crude aqueous pomegranate peel’s extract acetone/water (70:30, v/v) at pH 3.5 acetone/water (70:30, v/v) at pH 7.0 acetone/water (70:30, v/v) at pH 9.5 a

Total phenolic compounds (mg/10 mL) a

Total proanthocyanidins (mg/10 mL) a

29.67±0.048

4.34±0.031

22.79±0.040

3.45±0.044

0.28±0.014

0.60±0.018

2.12±0.021

2.94±0.037

Values are mean ± SDs, (n = 3)

Further experiments were done to find out the best alkaline pH which will give the best yield of proanthocyanidins. For this reason acetone 70% was prepared at four different pH values (8.0, 9.5, 10.5, 11.5) and all the eluents were used separately to purify the same aqueous crude extract using HyperSep NH2. The total proanthocyanidins content was measured (Table 4). TABLE 4 Purification of crude aqueous extract by HyperSep NH2 using acetone 70% in different pH. Fraction

Total proanthocyanidins (mg/10 mL) a

acetone/water (70:30, v/v) at pH 8.0 acetone/water (70:30, v/v) at pH 9.5 acetone/water (70:30, v/v) at pH 10.5 acetone/water (70:30, v/v) at pH 11.5

0.81±0.031 2.95±0.037 2.00±0.042 1.00±0.047

a

Values are mean ± SDs, (n = 3)

Zam et al: Separation and Purification of Proanthocyanidins Extracted from Pomegranate’s Peels (Punica Granatum)

Acetone 70% at pH 9.5 was the best eluent which gave the highest yield of proanthocyanidins. Therefore, this eluent was chosen to isolate proanthocyanidins from other phenolic compounds. But one time elution with acetone 70% at pH 9.5 only eluted 67.97% of the total proanthocyanidins extracted from pomegranate’s peel. For this reason, elution was repeated 4 times using the same eluent and the results are shown in Table 5. TABLE 5

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The freeze-dried cakes were dissolved in 10 mL distilled water in order to measure the total phenolic compounds and proanthocyanidins. Results indicated that the final freeze-dried cake, before purification, contained 67.59% non-phenolic compounds, 4.63% of proanthocyanidins and 27.77% phenolic compounds (67.59% + 4.63% + 27.77% = 100%). Whereas the final freeze-dried cake after purification contained 64.26% proanthocyanidins, 29.83% other phenolic compounds and 5.90% non-phenolic compounds (64.26% + 29.83% + 5.90% = 100%) (Figures 3 and 4).

Purification of crude aqueous extract by HyperSep NH2 using acetone 70% at pH 9.5. Fraction

Crude aqueous pomegranate peel’s extract 1st time elution 2nd time elution 3rd time elution 4th time elution a

Total phenolic compounds (mg/10 mL) a

Total proanthocyanidins (mg/10 mL) a

29.67±0.048

4.34±0.031

2.12±0.021 0.79±0.012 0.48±0.005 0.04±0.005

2.95±0.037 0.77±0.035 0.40±0.014 0.10±0.015

Values are mean ± SDs, (n = 3)

Four times elution with acetone 70% at pH 9.5 allowed the purification of 97.23% of the total extracted proanthocyanidins and only 11.56% of the total extracted phenolic compounds were present in the final cumulative filtrate.

Fig. 3. Composition of the final freeze-dried cake of aqueous extract before purification.

Lyophilization The final freeze-dried cake had a light pink color and weighed 93 mg for the crude aqueous extract before purification (Figure 1) and had a light brown color and weighed 6.1 mg for the purified extract (Figure 2).

Fig. 4. Composition of the final freeze-dried cake of aqueous extract after purification.

Fig. 1. Final freeze-dried cake of aqueous extract before purification.

Only 10.13% of proanthocyanidins content of the crude aqueous pomegranate’s peel extract was lost in the final freeze-dried cake, but they were clearly separated from the other compounds. As shown in Table 6, this method allowed the elimination of 81.08% of the total extractable phenolic compounds which didn’t elute with acetone water mixture (pH 9.5) from the cartridge. TABLE 6 Content of total phenolic compounds and proantho-cyanidins in the crude aqueous extract and in the final freeze-dried cake.

Fig. 2. Final freeze-dried cake of aqueous extract after purification.

 

Fraction

Total phenolic compounds (mg)

Total proanthocyanidins (mg)

Non-phenolic compounds (mg)

Crude aqueous pomegranate peel’s extract

30.35

4.34

63.31

Purified and lyophilized extract

5.74

3.92

0.36

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Antioxidant activity was measured for the crude aqueous extract and for the final freeze-dried cake. The final freeze-dried cake accounted for 38.22% of the total antioxidant activity of the crude aqueous peel extract as seen in Table 7. TABLE 7 Antioxidant activity of the crude aqueous extract and of the final freeze-dried cake. Fraction

Crude aqueous pomegranate peel’s extract Purified and lyophilized extract a

Antioxidant activity expressed by DPPH scavenging activity (%)a

Percentage of antioxidant activity (%)a

91.26±0.036

100

34.88±0.064

38.22

Values are mean ± SDs, (n = 3)

Conclusions Acetone:water mixture gave the highest yield of total extractable proanthocyanidins and was therefore used as an eluent solvent for the purification and separation of those compounds using two different SPE cartridges (C18 based-silica and aminopropyl-based cartridges). The pH of the eluent is an important factor in the isolation of proanthocyanidins. Results showed that alkaline mixtures at pH 9.5 were more effective in the isolation of those compounds. By comparison of the two SPE cartridges, aminopropylbased cartridges allows the isolation and purification of proanthocyanidins extracted from pomegranate’s peel. The proposed method is economic, simple and easy to use. It allows the purification of about 90% of the total extractable proanthocyanidins and allows its separation from other phenolic compounds. Whereas only about 18% of total extractable phenolic compounds were present in the final freeze-dried cake. Acknowledgements The authors acknowledge the financial support received from the University of Aleppo. They would also like to thank Daher Wardy for linguistic help. References Brand-Williams W, Cuvelier ME, Berset C (1995). Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss Technol. 25-30. Caili Fu, Alvin Eng Kiat Loo, Fiona Ping Ping Chia, Dejian Huang (2007). Oligomeric Proanthocyanidins from Mangosteen Pericarps. J Agric Food Chem. 55: 7689-7694. Dai Jin, Mumper, Russell J. (2010). Plant Phenolics: Extraction, Analysis and Their Antioxidant and Anticancer Properties. Molecules 15: 7313-7352. Foo LY, Lu Y, Howell AB, Vorsa N (2000). The structure of cranberry proanthocyanidins which inhibit adherence of uropathogenic P-fimbriated Escherichia coli in vitro. Phytochemistry 54: 173-181. Gad GY, Mary HG, Diana, MC, Igor VB, Ilya Raskin, Mary Ann Lila (2006). Comparative phytochemical characterization of three Rhodiola species. Phytochemistry 67: 2380-2391.

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Determination of Polyphenols in Tobacco by UPLC/MS/MS and Multivariate Analysis Chem Res. Chinese Universities 27(4): 550-556. Zhang Y, Seeram NP, Lee R, Feng L, Heber D (2008). Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties. J Agric Food Chem. 56: 670-675. Address correspondence to: Wissam Zam, Faculty of pharmacy-university of Aleppo Department of Analytical and Food Chemistry Syria Tel: +963-932-724703 E-mail: [email protected]