# Identification and Isolation Techniques for Plant Growth Inhibitors in Rice
## 6 Current Status, Existing Limitations, and Future Perspectives
### Current Status and Existing Limitations  
#### 6.1.1 Extraction and Separation Techniques  
Extraction is a key initial step in the analysis of rice PGIs. For many years, rice PGIs have been extracted and isolated by using conventional extraction methods such as sonification, percolation, Soxhlet extraction, and maceration [164]. However, the limitations of conventional techniques include their ineffectiveness, time and money consumption, and pollution caused by a large amount of used organic solvents. Due to these reasons, more sophisticated green approach techniques have been studied and introduced, such as ultrasonication-assisted extraction (UAE), microwave-assisted extraction (MAE), solid-phase microextraction (SPME), and pressurized liquid extraction (PLE or PSE) [165]. Some of these methods are referred to as \"green methods\" because they adhere to the requirements established by the United States Environmental Protection Agency, such as a reduction in organic solvent consumption, especially the dangerous chemicals used for synthesis, a designation for energy conservation, the elimination of derivatives, catalysis, improvement in extraction efficiency and selectivity, a design to prevent sample degradation, as well as naturally safer chemistry for accident prevention [164]. Pressurized liquid extraction (PLE) is performed at elevated temperatures and pressures to enable the extraction solvent to quickly penetrate the plant cells while preventing the compounds from degrading. This technique is a promising candidate method for extracting compounds which are stable under high temperatures [166]. However, it was not possible to optimize the extraction process by implementing a greater PLE temperature, since applying too much heat might destroy the phenolic compound. [PERSON] [167] discovered that phenolic compounds became less stable and showed mild degradation at 150 \({}^{\circ}\)C and severe degradation at 200 \({}^{\circ}\)C. By testing the stability of individual phenolic compounds, various PLE temperatures are considered, and the ideal extraction temperature was determined. The validated method was successfully applied for the analysis of a wide variety of rice grains [133]. On the other hand, various groups of compounds with various characteristics require different ideal extraction temperatures, which need further comprehensive investigation. Similar to PLE, microwave-assisted extraction (MAE) efficiency depends on extraction duration, temperature, and the type and ratio of the solvent. In terms of extraction efficiency, the amount of time, and solvent used, MAE and PLE outperformed other extraction techniques including Soxhlet, reflux, and ultrasonic extraction. A study on the extraction of a total of fifteen phenolic contents in various rice grains proved that MAE is a powerful tool for the determination of phenolic compounds from a wide variety of rice grains [134]. The advantages of MAE were introduced by [168], such as higher extract yield, minimal equipment size, and rapid heating for the extraction of bioactive compounds from plant sources. MAE is also considered a green technique because of its reduced use of organic solvents. However, the biggest limitation of MAE, unfortunately, is also the probability of the degradation of temperature-sensitive compounds under higher temperatures. The use of UAE, which breaks down the cell structure with sonic energy and quickly releases bioactive chemicals, might prevent these issues. In addition, small samples can be extracted by using ultrasound-assisted extraction, which would provide an increase in yield while significantly reducing extraction time and solvent usage. However, it is challenging to replicate this procedure, because using a lot of energy might cause the phytochemical to degrade by creating free radicals. In addition, a solvent-free modern technique was introduced recently which is advanced in many aspects.