What Is Black Rice Extract Anthocyanin?

Black rice is a special type of rice that is rich in nutrients and has certain health benefits. It is considered a delicacy that nourishes the body and has the effect of “appetizing the stomach, invigorating the spleen, warming the liver, improving eyesight and blood circulation, and nourishing the essence by relieving dryness”. It has long been loved by people in East Asia. Studies have confirmed that the physiological health benefits of black rice are mainly due not to nutrients such as dietary fibre, vitamins and minerals in black rice, but to the anthocyanin pigment rich in black rice bran [1-4].

Anthocyanin is a plant polyphenol widely distributed in nature, and is present to varying degrees in the petals and seed coats of most plants. During the ripening process of black rice, a large amount of anthocyanin pigments accumulate in the seed coat, which gives the brown rice a brownish red, purplish red, purplish black or even black color. Recent studies have found that in addition to giving plants rich colors, anthocyanins also have physiological functions such as anti-oxidation, anti-inflammation, lowering blood lipids, and inhibiting tumor growth. At the same time, as a relatively safe natural pigment, anthocyanin also shows broad application prospects in the food industry [5]. Black rice is abundant in resources, and anthocyanin pigments are characteristic substances of black rice. Understanding the research status of black rice anthocyanins is of certain guiding significance for the development and comprehensive utilization of black rice. This paper provides a review of the latest research progress on black rice anthocyanin at home and abroad.

1 Extraction and detection methods of black rice anthocyanin

At present, for the extraction of anthocyanin-like substances from plant materials, an alcohol (mostly 50% to 80% methanol or ethanol) solution hydrochloric acid is generally selected as the extracting agent. After extraction, filtration and concentration, a pigment sample is obtained for physicochemical property research. In addition, some researchers have tried to use microbial cell wall disruption, enzymatic methods, alkaline acid precipitation, and supercritical extraction to extract anthocyanins[6]. It has also been reported in the literature that acidified aqueous solutions also have a fairly good extraction capacity for anthocyanins in black rice, and that the purity of the pigment can be increased by adding a recrystallization step[7].

Zhang Mingwei optimized the best extraction conditions for black rice pigments through orthogonal experiments: 60% ethanol as the solvent, a black rice husk liquid ratio of 1:4, 60°C, and extraction for 4 hours [8]. Cacace believes that although increasing the extraction temperature has no significant effect on the antioxidant capacity of blackcurrant alcohol extracts,   but when the temperature rises above 35 °C, it will lead to an increase in anthocyanin degradation and thus reduce the anthocyanin content in the extract [9]. Our laboratory selected 60% edible alcohol (containing 0.1% HCl), which is safer for the human body, as the extracting agent. The liquid-to-material ratio was 1:1, and the mixture was extracted at room temperature for 3 × 12 h. concentrated under vacuum at ≤42 °C to prevent the destruction of anthocyanin by high temperatures, and then adsorbed onto acidified Amberlite XAD-7HP macroporous resin. The yield of anthocyanin extract from black rice can be stabilized at about 5.5% [4].

Anthocyanin is a glycoside compound formed by the combination of anthocyanidin and various monosaccharides in their natural state. Currently, 17 anthocyanidins have been discovered, of which the most common are six (Figure 1). Anthocyanins that occur in higher plants are mostly derivatives of these six anthocyanidins [5]. Due to the large variety of anthocyanins and the fact that they usually coexist, the total anthocyanin content is usually used to evaluate the amount of anthocyanins in a substance. However, the biological activity of individual anthocyanin monomers varies due to differences in the chemical structure of the B ring. This requires a clear understanding of the composition and content of anthocyanins in black rice.

Black rice extract is mainly composed of anthocyanins. Xia Xiadong et al. used high-performance liquid chromatography (HPLC) to isolate anthocyanin extracts from black rice husks and determined that the dominant anthocyanins in black rice were cyanidin 3-glucoside (Cy-3-G) and peonidin 3-glucoside (Pn-3-G) based on retention time. Cy-3-G and peonidin 3-glucoside (Pn-3-G), and further liquid chromatography-mass spectrometry analysis showed that the contents of Cy-3-G and Pn-3-G were 38.0% and 5.2%, respectively [4, 10]. This is consistent with the reports of Chiang and Kaneda [11-12]. However, Hyun also isolated the malvidin pigment from the Heugjinjubyeo black rice variety [13]. The above studies show that the anthocyanin composition may differ in different black rice varieties, but the dominant type is cyanidin, and the sugar base is glucose.

2. The physiological functions of black rice anthocyanins

Anthocyanins are often referred to as flavonoids because their 2-phenylbenzopyran skeleton is very similar to the 2-phenylchromone structure of flavonoids. However, compared to flavonoids, anthocyanins still have some unique chemical properties, such as the anthocyanin carbon ring with a positive oxygen ion, strong polarity, rarely in the form of a glycoside, and changes in chemical structure under different pH conditions [14-15]. These characteristics suggest that anthocyanins may have different biological effects and functions from those of flavonoids. Flavonoids and isoflavonoids have been studied extensively and are already used in clinical disease prevention and treatment. Some studies have reported that the daily intake of anthocyanins is significantly higher than that of flavonoids, but the physiological functions of anthocyanins have not been studied in depth [14]. However, studies in recent years have gradually proven that anthocyanins have a series of physiological health benefits, including antioxidant, blood lipid regulation, anti-inflammatory and anti-tumor effects, which have begun to attract widespread attention from researchers.

2.1 Antioxidant

Overnutrition, especially the intake of large amounts of simple sugars and saturated fats, can lead to the production of large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) free radicals in the body. ROS and RNS can oxidize and damage biological macromolecules such as DNA, proteins and lipids, and trigger various diseases, including diabetes, tumors and atherosclerosis [16]. Because anthocyanins have multiple phenolic hydroxyl groups in their molecular structure, they can bind to transient free radicals to form stable free radicals, chelate metal ions that initiate lipid peroxidation, participate in a synergistic antioxidant effect, and reduce or eliminate the damaging effects of free radicals on body tissues and organs by terminating free radical chain reactions, chelating metal ions, and scavenging active oxygen.   thereby reducing or eliminating the damaging effects of free radicals on body tissues and organs. Ling's research group fed high-fat diets containing 5% black rice bran to rabbits and Apolipoprotein-E (Apo-E) gene-deficient mice, and found that black rice bran can effectively remove active oxygen free radicals in these two experimental animals and inhibit the oxidation of low-density lipoprotein (LDL) [1, 3].

Adding 5 g of black rice anthocyanin extract per kilogram of feed can significantly reduce the levels of oxidized glutathione (GSSG) and malondialdehyde (MDA) in the serum of fructose-fed rats [17]. Kaneda's analysis concluded that Cy-3-G is the main antioxidant component in black rice bran,   which exhibits stronger free radical scavenging and protects HaCaT epidermal cells from ultraviolet damage than cyanidin (Cy)[12]. The research group led by Tsuda has successively demonstrated that anthocyanin Cy-3-G can significantly reduce the production of lipid peroxides in rat serum, reduce free radical damage caused by rat liver ischemia-reperfusion, and protect vitamin C in the blood from oxidation [18-19]. In addition to its strong free radical scavenging ability, black rice anthocyanins have also been shown to significantly increase the activity of superoxide dismutase (SOD) and catalase (CAT) in the liver of mice, which may be another mechanism by which anthocyanins exert their antioxidant effect in the body [11].

2.2 Regulating blood lipids

Among the various biological activities of anthocyanins, their protective effect on cardiovascular disease has attracted the most attention. The French consume a lot of saturated fatty acids and cholesterol in their daily diet, yet their incidence and mortality rates of cardiovascular disease are very low. Epidemiological studies have suggested that this may be related to the anthocyanins in the red wine they regularly drink. A feeding trial on ApoE gene-deficient mice showed that black rice anthocyanins can significantly reduce the levels of serum triglycerides (TG) and total cholesterol (TC) in mice, and reduced the area of unstable atherosclerotic plaques. The effect was similar to that of simvastatin, a commonly used lipid-lowering drug in clinical practice [4]. Tsuda used the purple corn pigment Cy-3-G, which is also a dominant anthocyanin, as the test subject, and confirmed that anthocyanins can effectively prevent obesity in mice caused by a high-fat diet,   by reducing TG and the expression of enzymes related to fatty acid synthesis, and reducing lipid deposition in the mouse liver [20]. Xia et al. showed that anthocyanin Cy-3-G may reduce the deposition of cholesterol in atherosclerotic plaques by activating the adenosine triphosphate-binding cassette transporter to promote the efflux of cholesterol from macrophage foam cells [21].

2.3 Anti-inflammatory

Anthocyanins have demonstrated good anti-inflammatory properties in a number of in vitro and in vivo studies. Wang et al. found that a six-month dietary intervention with black rice bran significantly reduced the levels of inflammatory factors such as soluble vascular cell adhesion factor, soluble CD40 ligand and high-sensitivity C-reactive protein in patients with coronary heart disease [22]. Purple corn pigment rich in Cy-3-G can normalize the high expression of TNF-α in obese mice induced by a high-fat diet [20].

Anthocyanins can also reduce inflammation by regulating capillary permeability. Feeding rats anthocyanins extracted from European bilberry (V. myrtillus L.) can effectively inhibit the swelling of their paw pads, The effect was dose-dependent. Anthocyanins can act as cyclooxygenase-2 (COX-2) inhibitors and reduce the levels of prostaglandin E2 and, in particular, PGE-2 when joints and connective tissue are inflamed. Unlike conventional anti-inflammatory drugs, anthocyanins promote the synthesis of type II collagen [23]. Anthocyanin can inhibit the activity of inducible nitric oxide synthase (iNOS) and thereby inhibit the NO level in mouse macrophage RAW264.7 cells [2]. Recent research in our laboratory has found that anthocyanin Cy-3-G can inhibit the inflammatory response of CD40-induced endothelial cells by regulating the intracellular distribution of cholesterol [24].

2.4 Other functions

Nam et al. compared the various biological activities of black rice bran and white rice bran alcohol extracts and found that black rice bran extract can significantly inhibit the gene mutation of mammalian cells caused by 4-nitroquinoline 1-oxide (4NQO) and the tumorigenesis induced by TPA [25]. Hagiwara et al. confirmed that 4NQO) caused by mammalian cell gene mutation, as well as tumorigenesis induced by TPA [25]. Hagiwara et al. confirmed that purple corn pigment can effectively inhibit 1,2-dimethylhydrazine-induced rat rectal cancer [26]. Chen et al. found that purified black rice anthocyanin can inhibit the invasion of SKHep-1 tumor cells by reducing the expression of matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (u-PA). MMP- 9) and urokinase- type plasminogen activator ( u-PA) expression to inhibit SKHep- 1 tumor cell invasion and metastasis [27]. In addition, black rice bran extract has been shown to have tyrosinase inhibitor activity, which can inhibit the biosynthesis of melanin and reduce the deposition of pigments in the skin [28].

3 Conclusion

Black rice anthocyanin is a natural food coloring agent that is safe, non-toxic, colorful and abundant. It also has a wide range of disease prevention and health promotion effects, and has great application potential in the fields of food and medicine. At present, research on black rice anthocyanin is still in its infancy. There are certain differences in the content and composition of anthocyanin between different varieties, and its health effects and mechanisms are not yet fully understood. Therefore, future research should focus on screening and selecting black rice varieties with high anthocyanin content, improving the extraction process to obtain anthocyanin pigments with high purity and good stability, the dose-effect relationship of anthocyanin intervention in animal and human diseases, and the regulation of gene expression.

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