Comparison of the effect of time and extraction methods on the phenolic compounds and antioxidant properties of walnut green husk of different regions of northern Iran

In recent years, much attention has been focused on agricultural wastes especially those containing phenolic compounds with natural antioxidant properties. One of these sources is the green husks of walnuts. The contents of phenolic compounds of plants are influenced under various factors including genetic factors, agricultural varieties and the species, climate and extraction procedures. Extraction is the first step for researchers who work on plants. Traditional extraction techniques with solvent such as water have disadvantages such as time consuming process, solvent consumption and high labor work. So the need to develop and use new extraction techniques, including combination of ultrasound bath and microwave devices, has provided valuable results. This method increases the mass transfer rate of the cell wall and intracellular compounds more rapidly transported out of the cell. In the ultrasound method to create cavitation in the extraction process and with collapse of bubbles and mechanical effects on the cell wall and most influential into the cell solvent improves the mass transfer. Extraction process performed by ultrasound process was used to facilitate the release of the intracellular content. These two methods can increase extraction efficiency within a shorter time and using less solvent, increasing the amount of extracted compounds are less damaging to the environment. Three different weather conditions for walnut production in northern Iran and different methods for extraction and measurement of phenolic compounds were adapted to find the best area and the most efficient extraction method.
Three areas were selected from north parts of Iran including Bandar Gaz with mild climate, Shahrood with semi-arid climate and Hezarjerib region with mountainous climate located in the Alborz Mountain chains in which walnut is one of their major products. Walnuts randomly were picked by hand without any damage to their husks from a few walnut trees in a random garden in each region. The selected walnut trees were almost similar and were about 35 to 40 years old. In the selected areas, it is not conventional to use pesticides or chemicals and toppings for walnut trees. Walnuts collected were transported to the lab in cold conditions (8 to 10° C) and after cleaning the walnuts, green husks were separated and kept at -18 °C. Solvent extraction of phenolic compounds was carried out by choosing ethanol - water in a ratio of (1-1) by soaking, ultrasound and microwave methods at different times. The amount of phenolic compounds was measured using a spectrophotometer device with the aid of using the Folin Ciocalteau reagent, and antioxidant properties were measured using DPPH free radical scavenging and regenerative power of Iron III.
This research was carried out on a factorial design (334) with three replications with Duncan's test for comparison of averages at the 95% confidence level. Comparing the extraction methods (soaking, microwave and ultrasound) showed that the most efficient method was ultrasound method at 30 minutes and the highest phenolic compounds in walnut green husk was for Hezarjerib area. In addition to the savings in time of extraction and safety in this method, the extraction of phenolic compounds is preferred to the other two methods. The results showed that the antioxidant properties were increased with increase in the amount of phenolic compounds. The results of this study also revealed that the walnuts green husks from Hezarjerib had the highest antioxidant properties followed by temperate region (Bandargaz) and semi-arid region (Shahrood).