Studies on the contents changes of punicalagin and its synthetic related substance in pomegranate leaf during development

【Objective】This study was to determine the content changes of punicalagin and its synthetic related substance shuch as gallic acid, ellagic acid, pentagalloylglucose, shikimic acid, 3-dehydroshikimicacid in pomegranate leaf during leaf development, and explore the correlation relationship between punicalagin and its synthetic related substance. The content changes and correlation relationship between leaf length and width, total flavonoids, total anthocyanin and DPPH radical scavenging capacity were determined. The correlations of all above indexes also were analyzed.【Methods】The developing leaves of‘Taishanhong'pomegranate from April 25 thto July 24 thwere as the experiment materials. The length and width of leaves were measured with vernier caliper. The content of total flavonoids was determined with AlCl3 colorimetry. Pomegranate leaves(0.1 g) were extracted with 10 m L mixture solutions of 1.5 mol·L- 1HCl and 95% ethanol(V/V=15∶85), then total anthocyanin was determined by colorimetric analysis at 535 nm. The content of punicalagin, gallic acid and ellagic acid were determined with high-performance liquid chromatography(HPLC) method with absorption wavelength at 280 nm. The mobile phase was consisted of acetonitrile and 2% acetic acid solution with proportion 20:80 under isocratic elution conditions.The Folin-Ciocalteu method was used for total polyphenol concentration determination. 2.0 m L of extract sample of pomegranate leaves was mixed with 2.0 m L of DPPH-ethanolic solution. 2.0 m L of diluted extract sample was mixed with 2.0 m L of ethanolic solution. An ethanol solution was used as the blank. Absorbance at 517 nm was used to determine DPPH radical scavenging capacity. The contents of shikimic acid, 3-dehydroshikimic acid and pentagalloylglucose were determined with HPLC method. The mobile phase was methanol and 1% phosphoric acid(V/V=5∶95) under isocratic elution conditions, and the maximum absorption wavelength for both was at 214 nm. The mobile phase for pentagalloylglucose detemination was acetonitrile and 0.5% phosphoric acid(V/V=20∶80) under isocratic elution conditions, and the maximum absorption wavelength was at 275 nm. The determination of punicalagin, gallic acid, ellagic acid, shikimic acid, 3- dehydroshikimic acid and pentagalloylglucose were done by Agilent 1260 series high-performance liquid chromatography(HPLC) system equipped with an autosampler and diode array detector(DAD). Separations were performed on a Zorbax SB-C18 column(150 mm×4.6 mm ID, 5 μm).Column temperature was set at 30 ℃. The mobile phase flowed at a speed of 0.8 m L·min-1.The injection volume of standard or sample solution was 10 μL.【Results】The optimized HPLC determination method of punicalagin, gallic acid, ellagic acid, pentagalloylglucose, shikimic acid and 3-dehydroshikimic acid was established. The retention time of α-punicalagin, β-punicalagin, gallic acid, ellagic acid, shikimic acid,3-dehydroshikimic acid and pentagalloylglucose was 2.768 min, 2.926 min, 2.453 min, 4.325 min, 3.045 min, 3.971 min and 5.594 min, respectively. The separation effective and peak shape of above substances were well. According to the standard curve, the content of above seven components was calculated. The pomegranate leaves took about fifty days into functional leaves. The length and width of functional leaves respectively were 10.2 cm and 2.99 cm in July 24 th. The contents of gallic acid, shikimic acid, total phenols and DPPH radical scavenging activity decreased first and then increased during pomegranate leaf development with peak value in April 25 thand July 24 th. The highest content of gallic acid, shikimic acid, total phenols and DPPH radical scavenging activity was 3.624 mg·g-1, 3.388 mg·g-1, 171.238 mg·g-1and91.033% respectively in April 25 th. The lowest content of gallic acid, total phenols and DPPH radical scavenging activity was 0.709 mg·g-1, 38.039 mg·g-1and 35.983% in June 24 th. The lowest content of shikimic acid was 0.826 mg·g-1in June 14 th. The contents of punicalagin and anthocyanin decreased gradually with the leaf development, and reached the highest 0.553 mg·g- 1and 0.339 mg·g- 1respectively in April 25 th and the lowest 0.167 mg·g-1and 0.161 mg·g-1in July 24 th. The contents of pentagalloylglucose and 3-dehydroshikimic acid increased first and then decreased with the highest 5.62 mg·g-1and 6.442 mg·g-1respectively in June 24 th, and had significant differences between development stages. The lowest content ofpentagalloylglucose and 3-dehydroshikimic acid was 3.14 mg·g-1and 1.914 mg·g-1in April 25 th, respectively. The change trend of ellagic acid was down → up →d own →up during leaf development, with peak value in April 25 th, June 14 thand July 24 threspectively, and the contents were 0.394 mg·g-1, 0.357 mg·g-1and 0.622 mg·g- 1respectively. The lowest content of ellagic acid was 0.132 mg·g- 1in May 25 th. The change trend of total flavonoids was down → up →down with peak value 3.792 mg·g- 1in April 25thand2.552 mg·g-1in June 24 th, and was significant different from other development stages. The lowest content of total flavonoids was 1.264 mg·g-1in May 15 th. Correlation analysis indicated that leaf length was significant positive correlated with leaf width. Both leaf length and width was significant positive correlated with3-dehydroshikimic acid, significant negative correlated with shikimic acid, gallic acid, punicalagin, anthocyanin, total flavonoids, total phenols and DPPH radical scavenging activity, respectively. Gallic acid was significant positive correlated with shikimic acid, punicalagin, anthocyanin, total phenols and DPPH radical scavenging activity respectively, and significant negative correlated with pentagalloylglucose and 3-dehydroshikimic acid. There was significant positive correlation between punicalagin and anthocyanin, total phenols, shikimic acid, DPPH radical scavenging activity, and significant negative correlation between punicalagin and ellagic acid. Pentagalloylglucose had significant positive correlated with 3-dehydroshikimic acid, and significant negative correlated with DPPH radical scavenging activity and shikimic acid.The relationships beyween anthocyanin, total flavonoids, total phenols and DPPH radical scavenging activity were significant and positive. The correlation of shikimic acid and 3-dehydroshikimic acid were significantly negative.【Conclusions】There were differences in the content and correlation relationship between punicalagin and its synthetic related substance during leaf development. Shikimic acid and 3-dehydroshikimic acid were closely related to the synthesis of gallic acid. The metabolism of gallic acid could produce pentagalloylglucose. Pentagalloylglucose might be the precursor substance in the synthesis of punicalagin.Ellagic acid was the degradation product of punicalagin. The contents of punicalagin, anthocyanin, total flavonoids and total phenols were closely related to the antioxidant capacity of pomegranate leaf.