|Year : 2019 | Volume
| Issue : 1 | Page : 67-71
Determination and quantification of p-coumaric acid in pineapples (Ananas comosus) extracts using gradient mode RP-HPLC
Wan Mazlina Md Saad1, Rasdin Ridwan1, Nur Syamimi Md Lasim1, Nur Liyana Mohd Rapi1, Fatimah Salim2
1 Centre of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA Selangor, Puncak Alam Campus, Malaysia
2 Department of Chemistry, Centre of Foundation Studies, Universiti Teknologi MARA, Dengkil Campus, Dengkil; Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA Selangor, Puncak Alam Campus, Selangor, Malaysia
|Date of Web Publication||20-Feb-2019|
Dr. Wan Mazlina Md Saad
Centre of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA Selangor, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Pineapple (Ananas comosus) is a good source of phenolic compounds such as p-coumaric acid. P-Coumaric acid is a hydroxycinnamic acid known to possess antioxidant, antimicrobial, antimutagenic, and immunoregulatory activities. Objective: The present study aims to determine and quantify the p-coumaric acid in the juice extract and methanol extract of unripe and ripe pineapples by gradient mode reverse-phase high-performance liquid chromatography (RP-HPLC). Materials and Methods: The analysis was carried out using 0.5% phosphoric acid and 100% acetonitrile (can) as mobile phase A and B at gradient elution conditions of 0–30 min from 5% B to 80% B, 30–33 min at 80% B, 33–35 min from 80% B to 5% B, and 35–40 min at 5% B. The chromatography separation was performed using Gemini C18 column at flow rate of 0.8 mL/min and detection was made at 280 nm. Results: P-Coumaric acid was determined at retention time of 16.16 min. Standard curves were linear at R2 = 0.9973 with low limit of detection and limit of quantification being 0.0208 and 0.0694, respectively. P-Coumaric acid showed the highest concentration in juice extract of ripe pineapple, 11.76 μg/mL compared to the methanol extract, 0.03 μg/mL, respectively. P-Coumaric acid concentration in juice extract of unripe pineapple was 0.41 μg/mL. Conclusion: The gradient mode RP-HPLC proved to be efficient for determination and quantification of p-coumaric acid in pineapples extracts.
Abbreviations Used: RP-HPLC: Reverse-phase high-performance liquid chromatography; ACN: Acetonitrile; H3PO4: Phosphoric acid; PLPs: Pineapple leaf phenols; HepG2: Hepatoma G2; PLEs: Pineapple leaf extracts; TC: Total cholesterol; TG: Triglyceride; DAD: Diode array detector; UV-Vis: Ultraviolet-visible; dH2O: Distilled water; LOD: Limit of detection; LOQ: Limit of quantification.
Keywords: Ananas comosus, gradient mode, p-coumaric acid, pineapple, reverse-phase high-performance liquid chromatography
|How to cite this article:|
Md Saad WM, Ridwan R, Lasim NS, Rapi NL, Salim F. Determination and quantification of p-coumaric acid in pineapples (Ananas comosus) extracts using gradient mode RP-HPLC. Phcog Res 2019;11:67-71
|How to cite this URL:|
Md Saad WM, Ridwan R, Lasim NS, Rapi NL, Salim F. Determination and quantification of p-coumaric acid in pineapples (Ananas comosus) extracts using gradient mode RP-HPLC. Phcog Res [serial online] 2019 [cited 2020 Jan 23];11:67-71. Available from: http://www.phcogres.com/text.asp?2019/11/1/67/252566
- P-Coumaric acid was successfully determined and quantified in unripe and ripe pineapple extracts using gradient-mode reverse-phase high-performance liquid chromatography. P-Coumaric acid showed the highest concentration in juice extract of ripe pineapple compared to methanol extract.
| Introduction|| |
Pineapple (Ananas comosus) belongs to Bromeliaceae family and one of the popular tropical and subtropical fruits. Pineapple is a native to South America and is a nonseasonal fruit which available throughout the year. Pineapple has sweet taste, acidic flavor, and consumed around the world as juice, jelly, jam, and dried product. Good quality of pineapple fruit is indicated by the dense and juicy yellow-colored pulp with medium acidity and high sugar content.
Pineapple has high content of phytonutrients and dietary antioxidants such as phenolics, flavonoids, minerals, and vitamins. P-Coumaric acid, C9H8O3 [Figure 1], is one of the phenolic compounds present in flesh and leaves of pineapple. P-Coumaric acid is a natural hydroxycinnamic acid that exists in an esterified form with tartaric acid, the tartaric p-coumaroyl ester. This compound is known to possess antioxidant, antimicrobial, antimutagenic, analgesic, sedative, and immunoregulatory activities., The previous study found that p-coumaric acid from pineapple leaf phenols significantly prevent fat accumulation in oleic acid-induced hepatoma (HepG2) cells. Cho et al. and Chai et al. demonstrated that pineapple leaf extracts significantly reduce plasma total cholesterol and triglyceride in obese rats owing to high content of phenolic acids including p-coumaric acid.
Several studies have demonstrated the biological effect of pineapple (A. comosus).,, According to Romano et al., pineapple exhibits antiproliferative and chemopreventive effects in vitro and in vivo study. The study demonstrated that 1 μg/mL of bromelain derived from pineapple stem reduced cell proliferation and promoted apoptosis through caspase 3/7 activation in human colorectal carcinoma (Caco-2) cells. However, an optimal dose of 1–3 mg/kg prevent preneoplastic lesions, polyps, and tumor formation induced in mouse colon administered with carcinogenic agent, azoxymethane. In another study, Yantih et al. demonstrated hepatoprotective effect of pineapple juice in rats. Supplementation of 2 mL/kg body weight of pineapple juice for 4 weeks decreased ALT and AST levels in rats induced with 27 mg/kg body weight of isoniazid. The authors suggested that the hepatoprotective effect may be due to antioxidant activity of phenolic compounds in the juice. However, recent study by El-Shazly et al. demonstrated antiobesity effect of pineapple juice in rats supplemented with high-fat diet (HFD). The results of the study showed that pineapple juice suppressed HFD-induced obesity in rats through reduction of body weight gain, serum lipid, hepatic lipid accumulation, and decreased the number and size of adipocytes after 8 weeks juice supplementation. The study suggested that consumption of pineapple juice that contains a whole synergistic interaction of phytonutrients, particularly phenolic compounds, may contribute to the antiobesity effect. Overall, the findings from these previous studies suggest that pineapple (A. comosus) may provide beneficial effects for treatment and prevention of health-related diseases.,,
Analyses of phenolic compounds were commonly conducted using Folin–Ciocalteu; however, the method is qualitative and lack of sensitivity. Karthikeyan et al. suggested that the efficient technique for qualitative and quantitative analysis of p-coumaric acid is by using chromatographic method owing to high selectivity and less of background interferences. To date, study on phenolic content particularly p-coumaric acid in Malaysia pineapple is still lacking. Thus, the present study aims for determination and quantification of p-coumaric acid in unripe and ripe of pineapples extracts using gradient mode reverse-phase high-performance liquid chromatography (RP-HPLC).
| Materials and Methods|| |
Chemical and Reagents
P-Coumaric acid standard (purity ≥99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN) and methanol of HPLC grade were purchased from Merck (Germany). Phosphoric acid (H3 PO4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was prepared using ultrapure water purifier system (Elgastat, Bucks, UK).
The gradient-mode RP-HPLC was carried out using Thermo Scientific™ Dionex-UltiMate™ 3000 HPLC system equipped with solvent reservoirs, pump, autosampler injector, column oven, and ultraviolet-visible diode array detector module operated at four wavelengths per analysis. Chromeleon data software (Version 7, Thermo Fisher Scientific Inc., MA, USA) was used for data analysis.
Unripe and ripe pineapples (A. comosus) were purchased from local market, Meru, Selangor. The pineapples peels were removed. Edible part of the flesh pineapples were cut into cubes, juiced, and filtered using sieve. The juices were filtered again using cotton wool placed into neck of the funnel to remove any debris or unnecessary matter. The juice extract was directly analyzed. For methanol extract, the unripe and ripe pineapple juices were frozen at −80°C. The frozen juices were the put in freeze-drier (Labconco, USA) for 4 days until completely dried. A known quantity of dried juice powders was extracted using methanol. The methanol extracts then evaporated using rotary evaporator and stored at 4°C until analysis.
Reverse-phase high-performance liquid chromatography analysis for p-coumaric acid
Standard preparation procedure
Stock solution of p-coumaric acid was prepared in methanol at 0.5 mg/mL and filtered through a 0.45-μm syringe filter. A series of working standard solution was prepared by diluting stock solution with methanol in the range of 0.1–400 μg/mL.
Sample preparation procedure
500 μL was taken directly from unripe juice extract and diluted with 500 μL distilled water (dH2O). For methanol extract, freeze-dried unripe pineapple was dissolved in methanol at 5 mg/mL. For ripe pineapple juice extract, 300 μL of juice was diluted with 700 μL dH2O while methanol extract was dissolved in methanol at 5 mg/mL. Samples were filtered through 0.45-μm filter prior injected to RP-HPLC.
Gradient mode reverse-phase high-performance liquid chromatography analysis
Determination and quantification of p-coumaric acid were performed according to the study conducted by Jakobek et al. and method by Ridwan et al., with slightly modification. The RP-HPLC analysis was performed using Gemini C18 column, 250 mm × 4.6 mm, 110 Å, 3 μm (Phenomenex, Torrance, CA, USA) with a flow rate of 0.8 mL/min. Column temperature was maintained at 20°C while detection was performed at 280 nm. 10 μL of sample was injected into Gemini C18 column by using gradient elution program. Two solvents used were 0.5% H3 PO4 (solvent A) and 100% ACN (solvent B). The elution conditions were as follows: 0–30 min from 5% B to 80% B, 30–33 min 80% B, 33–35 min from 80% B to 5% B, and 35–40 min 5% B. The concentrations of p-coumaric acid in samples were quantified based on calibration curve of standard. Concentration of p-coumaric acid in pineapple extracts was calculated using the formula:
Concentration of analyte = A/S
Where A is the peak area of analyte and S is the slope of calibration curve.
The validation of gradient-mode RP-HPLC method was achieved by determined the linearity of calibration curve, limit of detection (LOD) and limit of quantification (LOQ). The linearity of gradient-mode RP-HPLC method for quantification of compound was determined by injecting 5 μL of six different concentrations of p-coumaric acid standard at 0.1, 1, 100, 200, 300, and 400 μg/mL. Linear regression analysis was constructed by plotting a graph of peak area against p-coumaric acid standard concentrations. The regression equation was calculated in the form of y = mx, where y was the peak area of compound and x was concentration. Regression coefficient (R2) was calculated from the calibration curve. The LOD is the lowest amount of analyte in the samples that can be detected but not necessarily quantitated under the stated experimental conditions, while LOQ is the lowest amount of analyte in sample which can be quantitatively determined with suitable precision and accuracy. LOD and LOQ can be expressed as LOD = 3Sa/b and LOQ = 10Sa/b where Sa is the standard deviation of the response which can be estimated by standard deviation of Y-intercept of regression line and b is the slope of calibration curve.
| Results and Discussion|| |
Determination of p-coumaric acid in standard and pineapples extracts
The present gradient mode RP-HPLC for analysis of p-coumaric acid in unripe and ripe pineapples utilized 0.5% H3 PO4 and 100% ACN as mobile phase A and B, respectively. 0.5% H3 PO4 was used as mobile phase A to suppressed ionization of phenolic and carboxylic groups, leading to improvement of retention time and resolution of the target compound. To produce optimum chromatographic separation, organic modifier (ACN) was chosen as mobile phase B to alter compound's retention time by lowering polarity of the mobile phase. Chen et al. supported that mobile phase combinations containing modifier such as H3 PO4 with ACN were efficient for phenolic compounds analysis. The chosen gradient elution (0–30 min from 5% B to 80% B; 30–33 min 80% B; 33–35 min from 80%, B to 5% B) at flow rate of 0.8 mL/min resulted that most compounds were eluted including p-coumaric acid. The used of reverse-phase column Gemini C18 as the stationary phase resulted in good separation of p-coumaric acid. Ferreira et al. supported that C18 column demonstrated good retention time and peak symmetry of p-coumaric acid. The wavelength was set at 280 nm in accordance to the previous study conducted by Häkkinen  that showed 280 nm provide excellent detection of p-coumaric acid in fruit samples.
The peak of p-coumaric acid standard was detected at retention time of 16.16 min. The retention time of p-coumaric acid in samples was compared to the standard peak [Figure 2] and [Figure 3]. The peak of p-coumaric acid in juice extract of unripe and ripe pineapple and methanol extract of ripe pineapple was detected at 16.16 min. However, p-coumaric acid was not detected in methanol extract of unripe pineapple. The retention time (min), height, and peak area of standard and samples are tabulated in [Table 1]. Longer retention time for p-coumaric acid in the present study may be due to higher affinity of the compound towards the stationary phase. Xu and Howard  supported that p-coumaric acid is a less polar compound compared to other phenolic acids such as gallic acid, chlorogenic acid, and caffeic acid, thus leading to increased time for the compound to be eluted.
|Figure 2: Comparative chromatograms of p–Coumaric acid. (A) Standard of p-Coumaric acid, (B) Juice extract of unripe pineapple and (B) Methanol extract of unripe pineapple. p-Coumaric acid was detected at 16.16 min in standard and juice extract of unripe pineapple under 280 nm wavelength using gradient elution method of 0.5% H3PO4 and 100% ACN as mobile phases and flow rate at 0.8 min/mL|
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|Figure 3: Comparative chromatograms of p–Coumaric acid. (a) Standard of p-Coumaric acid, (b) Juice extract of ripe pineapple and (c) Methanol extract of ripe pineapple. p-coumaric acid was detected at 16.16 min in standard and juice extract of ripe pineapple under 280 nm wavelength using gradient elution method of 0.5% H3PO4 and 100% ACN as mobile phases and flow rate at 0.8 min/mL|
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|Table 1: Retention time (min), height (mAU), and peak area (mAU*s) of samples and standard|
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Linear regression was obtained from the calibration curve of p-coumaric acid [Table 2], and good correlation was achieved between the concentration of p-coumaric acid standard and peak area. The linear regression equation was y = 0.8643x with regression coefficient, R2 = 0.9973. The result of regression coefficient value in the present study was comparable with Bataglion et al. study with R2 = 0.998 for determination of phenolic compounds including p-coumaric acid in fruit samples. The LOD and LOQ were conducted for sensitivity assessment of the gradient-mode RP-HPLC. LOD is the lowest amount of analyte in sample that can be detected but not necessarily quantitated under stated experimental conditions. LOQ is the lowest amount of analyte in sample that can be quantitatively determined with suitable precision and accuracy. The LOD and LOQ were 0.0208 and 0.0694 μg/mL, respectively. The linearity, LOD, and LOQ showed that the gradient mode RP-HPLC method was accurate and sensitive for quantification of p-coumaric acid.
|Table 2: Calibration data form gradient mode reverse-phase high-performance liquid chromatography|
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Quantification of p-coumaric acid in pineapple extracts
Consumption of pineapples extracts rich in p-coumaric acid is beneficial for diseases prevention. Thus, an efficient gradient-mode RP-HPLC is essential for quantification of p-coumaric acid in juice extracts and methanol extracts. The concentration of p-coumaric acid was calculated based on calibration curve achieved with good correlation coefficients and linear regression equations, R2 = 0.9973, y = 0.8643x. The results are tabulated in [Table 3].
|Table 3: P-coumaric acid concentration (μg/mL) in juices extract and methanol extract of unripe and ripe of pineapples|
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Ripe pineapple juice extract showed the highest concentration of p-coumaric acid, 11.75 μg/mL when compared to methanol extract, 0.03 μg/mL. The concentration of p-coumaric acid in ripe pineapple juice extract was in the range of Wen and Wrolstad's  study reported that p-coumaric acid content range from 1 to 21 μg/mL in pineapple juice samples. The concentration of p-coumaric acid in juice extract of unripe pineapple was 0.41 μg/mL while no p-coumaric acid detected in the methanol extract. Shofian et al. stated that extraction method is highly influence the concentration of compound quantified in certain fruits. Ridwan et al. supported that methanol extract may diminish the solubility of bioactive compounds in fruit result in lower yield compared to fruit juice extract. The present result demonstrated juice extract of ripe pineapple most effectively quantified higher yield of p-coumaric acid, thus suggesting that consumption of pineapple directly from juice is essential for the prevention of health-related diseases.
| Conclusion|| |
The gradient-mode RP-HPLC has been successfully determined and quantified of p-coumaric acid concentration in both extracts of unripe and ripe pineapples. The validated method is accurate and sensitive with good linearity (≥0.99), low values of LOD and LOQ. Juice extract effectively yielded higher p-coumaric acid, thus potentially used for quantitative routine analysis in fruit juices. The high content of p-coumaric acid in juice extract of ripe pineapple suggested pineapple as a good source of nutraceutical and health benefit ingredients. However, further researches are necessary to explore the biological activities of p-coumaric acid in pineapple to support its potential for disease prevention.
We acknowledge the Ministry of Higher Education, Malaysia, through Fundamental Research Grant Scheme (FRGS Grant No: FRGS/1/2016/WAB01/UITM/02/3) and Institute of Research Management and Innovation (IRMI), Universiti Teknologi MARA (UiTM) for funding the study and Atta-ur-Rahman Institute, UiTM Puncak Alam, Centre of Medical Laboratory Technology, and Centre of Postgraduate Study, Faculty of Health Sciences, UiTM Selangor, Puncak Alam Campus, for providing facilities throughout this study.
Financial support and sponsorship
The study is funded by the Ministry of Higher Education, Malaysia, through Fundamental Research Grant Scheme (FRGS Grant No: FRGS/1/2016/WAB01/UITM/02/3) and IRMI, UiTM.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]