*4.2.2 Ultrasonic-assisted extraction*

*Recent Advances in Rice Research*

**4.1 Conventional solvent extraction bioactive compounds**

Conventional extraction is being used at a small-scale level to extract bioactive components from several plant materials. This technique is usually based on the extraction efficiency of different solvents, which are being used for this purpose. The manual solvent extraction at ambient temperature is the most commonly used method in extracting bioactive compounds from grains. The solvents included acidified methanol with 1.0 N HCl (85:15, v/v), acidified methanol with 1 M phosphoric acid (95:5 v/v), acidified methanol with trifluoro acetic acid (99.8:0.2, v/v), acidified methanol with glacial acetic acid (95:5, v/v), and acetone/water (80:20, v/v). The extraction ratio was a material to solvent at 1:10 (w/v) [30]. In addition, in cold conditions, methanol (85%) and HCl (1 mol/L) was found to be an appropriate extraction solvent for anthocyanins, along with 85% methanol or

70:29.5:0.5, v/v acetone:water:acetic acid for free proanthocyanidins [17].

was eluted with a unit volume of 80% (v/v) acetonitrile in water.

The longer extraction time, costly and high purity solvent, evaporation of the huge amount of solvent, low extraction selectivity, and thermal decomposition of thermolabile compounds are major challenges of conventional extraction. These limitations of conventional extraction methods can be improved by introducing the promising techniques or non-conventional extraction techniques, for example, ultrasound-assisted extraction (UAE) and microwave-

**Microwave-assisted extraction** has been implemented as an alternative technique for extracting anthocyanins from pigmented rice because of its ability to reduce both consumption time and solvent volume. For the MAE method, a combination of 70°C, 300 W, with 10 min in MAE was the most effective in extracting

**4.2 Non-conventional extraction techniques**

assisted extraction (MAE) [31].

*4.2.1 Microwave-assisted extraction (MAE)*

According to Shao et al., [12] soluble-free, soluble-conjugated and insolublebound phenolics of white, red and black rice were extracted by using 80% methanol. The soluble phenolics mixture was extracted and concentrated to obtain soluble phenolics. In order to get soluble-free phenolics, the concentrated soluble phenolics were further extracted by ethyl acetate three times, and then dried by a rotary evaporator, and dissolved in 5 mL of 50% methanol. To get soluble-conjugated phenolics, the concentrated soluble phenolics were hydrolyzed using 4 M NaOH for 2 h followed by adjusting pH to 1.5–2.0, extraction with ethyl acetate, drying using a vacuum evaporator, and then dissolving in 5 mL of 50% methanol. After the extraction of soluble phenolics, the residues were used to extract insoluble-bound phenolics. Similarly, the soluble-conjugated phenolics could be prepared from the concentrated soluble phenolics extracts by using 4 M NaOH and ethyl acetate. In addition, our group [13] also used solid phase extraction (SPE) techniques to purify and prepare soluble-free (unbound fraction) and soluble-conjugated (polyphenol-rich bound fraction) phenolic compounds of pigmented rice. The crude extracts of colored rice were purified by applied to C18 solid phase extraction unit. The solid phase cartridge was pre-washed in 0.2% (v/v) formic acid in acetonitrile and then pre-equilibrated in 0.2% (v/v) formic acid in water. The unbound materials including free sugars, organic acids and vitamin C were collected. The SPE unit was then washed with a unit volume of 0.2% (v/v) aqueous formic acid and then with 2 volumes of ultra-pure water. The polyphenol-rich bound fraction

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Ultrasound-assisted extraction (UAE) has been used in applications of foodprocessing technology to extract bioactive compounds from plant materials. Ultrasound at levels greater than 20 kHz is used to disrupt plant cell walls. It helps to improve the solvent's ability to penetrate the cells and obtain a higher extraction yield. The UAE operates at a low operating temperature through processing and maintain a high extract quality for compounds. Recently, Setyaningsiha et al. (2019) reported the optimization of the UAE conditions for individual phenolic compounds extraction from rice grains using 80% methanol in water for 25 min at 45°C with amplitude 47%, cycle 0.4 s − 1, pH 4.25 and sample-to solvent ratio of 1:5 [32]. The developed method presented the acceptable value for linearity and precision (RSD). Therefore, the proposed UAE method is an effective technique for the determination of individual phenolic compounds including caffeic, *p*-coumaric, syringic, chlorogenic, isovanillic, isoferulic ferulic, *p*-hydroxybenzoic, sinapic, *p*-hydroxybenzaldehyde, protocatechuic, vanillic acids, protocatechuic aldehyde and quercetin in rice samples. However, the UAE has two main negative properties mainly related to experimental repeatability and reproducibility [31].
