Comparing pH differential and methanol‐based methods for anthocyanin assessments of strawberries

Abstract

Anthocyanins are a group of water‐soluble polyphenolic pigments found primarily in flowers, vegetables, and fruits. These pigments play critical roles in plant and human health. Spectrophotometric methods are a simple and inexpensive way to quantify anthocyanins in plant tissues. Two main spectrophotometric methods have been developed, organic solvent‐based, and pH differential methods. Both of these methods are subject to interference from light‐absorbing impurities and need to be optimized for different matrixes of different plant materials. Eight methods have been tested in this experiment to quantify anthocyanins in strawberry fruits. Six organic solvent‐based methods tested methanol, chloroform‐methanol, and MgO in different ratios. The other two methods were pH differential method and a combination of organic solvent‐based and pH differential method. Two methods used organic solvents (methanol and chloroform‐methanol) were the best in extracting anthocyanin from strawberry fruits. Adding MgO increased the pH of the extract and was less efficient in anthocyanin extraction. All other methods had lower anthocyanin yield compared with methanol and chloroform‐methanol methods and are not recommended for strawberry fruit anthocyanin extraction.

Anthocyanin Extraction Method and Sample Preparation Affect Anthocyanin Yield of Strawberries

“…Taghavi et al 27 have shown that the methanol:water method was better than the chloroform:methanol method for frozen strawberry samples, which is the opposite of what has been shown here, because water is a better solvent than chloroform for anthocyanins. With freeze-dried samples, there is no water in either the extraction buffer or the samples, the solubility of anthocyanins was limited to methanol, but a few anthocyanins dissolve in chloroform ( ie pelargonidin), and therefore, the anthocyanin yield was reduced.…”

“…The low anthocyanin content of the pH differential method was also seen in frozen strawberry samples. 27 The other downside of this method is the hazy anthocyanin solution produced that could not be completely clarified by either increasing the centrifugation time or even filtering. This method is extensively used by food scientists and horticulturists to assess the quality of fresh and processed fruits and vegetables.…”

“…Solovchenko et al 6 claimed that chloroform reduced the light-absorbing impurities, although such an effect was not seen in frozen strawberry samples. 27 The fact that the lack of water in freeze-dried strawberry samples could make a difference is probable. Another reason for higher anthocyanin yield in the presence of chloroform is the solubility of pelargonidin, for which strawberry is a rich source .…”

“…The result is opposite to what the authors experienced using frozen strawberry samples. 27 The extraction buffer of the Neff and Chory 10 method had a higher chloroform to methanol ratio than the chloroform-methanol method (Solovchenko et al 6 ). Neff and Chory 10 used it for Arabidopsis leaves, which have a higher chlorophyll content.…”

“…Taghavi et al 27 have shown that the methanol method (Lindoo and Caldwell 7 ) was the most efficient for anthocyanin extraction of frozen strawberry fruits. However, in this experiment with freeze-dried strawberry fruits, this trend has not been seen, and this method gave the lowest anthocyanin content of all the methods used.…”