## 2 Results and Discussion
### PCA, PLS-DA, and OPLS-DA
In addition, inositol was also identified as an important contributor to the separation of BR from others in the PLS-DA loading plots (Table S1). Based on a previous study, BR contains more inositol than RR and WR [23]. These previously reported observations were consistent with our findings in the present study (Table S2). Figure 1: Partial least squares-discriminant analysis (PLS-DA) score (**A**) and loading plots (**B**) derived from 110 metabolites of black, red, and non-pigmented (white) rice cultivars. The ellipse represents the Hotelling T2 with 95% confidence in the score plot. Plot annotation 1, C20-ol (Eicosanol); 2, C21-ol (Heneicosanol); 3, C22-ol (Docosanol); 4, C24-ol (Tetracosanol); 5, C26-ol (Hexacosanol); 6, \(\beta\)-Tocopherol; 7, \(\gamma\)-Tocopherol; 8, C27-ol (Heptacosanol); 9, C28-ol (Octacosanol); 10, \(\gamma\)-Tocotrienol; 11, \(\alpha\)-Tocopherol; 12, Cholesterol; 13, \(\alpha\)-Tocotrienol; 14, Campesterol; 15, C30-ol (Triacontanol); 16, Stigmasterol; 17, \(\beta\)-Sitosterolol; 18, Lutein; 19, Zexaanthin; 20, \(\beta\)-Carotene; 21, Cyanidin-3-O-glucose; 22, Peonidin-3-O-glucose; 23, Catechin; 24, Epicatechin; 25, C16:0 (Palmitic acid); 26, C18:0 (Stearic acid); 27, C18:1 (Oleic acid); 28, C18:2 (Linoleic acid); 29, C18:3 (\(\alpha\)-Linolenic acid); 30, C20:0 (Arachidonic acid); 31, Pyruvic acid; 32, Lactic acid; 33, Alanine; 34, Oxalic acid; 35, Valine; 36, Ethanolamine; 37, Leucine; 38, Glycerol; 39, Phosphoric acid; 40, Isooleucine; 41, Proline; 42, Glycine; 43, Succinic acid; 44, Fumaric acid; 45, Serine; 46, Threonine; 47, \(\beta\)-Alanine; 48, Malic acid; 49, Salicylic acid; 50, Methionine; 51, Aspartic acid; 52, Pyroglutamic acid; 53, \(\gamma\)-Aminobutyric acid; 54, Cysteine; 55, Threonine acid; 56, Glutamic acid; 57, \(p\)-Hydroxy benzoic acid; 58, Phenylalanine; 59, Asparagine; 60, Xylose; 61, Vanillic acid; 62, Glutamine; 63, Protocatechic acid; 64, Shikimic acid; 65, Citric acid; 66, Fructose; 67, Galactose; 68, Lysine; 69, Glucose; 70, \(p\)-Cumaric acid; 71, Tyrosine; 72, Mannitol; 73, Ferulic acid; 74, Inositol; 75, Caffe acid; 76, Tryptophan; 77, Sinapinic acid; 78, Sucrose; 79, Raffinose 80, 1-Butanol; 81, Pentanal; 82, 1-Pentanol; 83, Toluene; 84, Hexanal; 85, 1-Hexanol; 86, Ethylbenzene; 87, \(p\)-Xylene; 88, 2-Heptanone; 89, 2-Butyl furan; 90, Styrene; 91, Heptanal; 92, 2-Acetyl-1-pyrroline; 93, \(\alpha\)-Pinene; 94, 1-Heptanol; 95, Benzaldehyde; 96, 1-Octen-3-ol; 97, 2-Pentyl furan; 98, Octanal; 99, \(p\)-Cymene; 100, D-Limonene; 101, 3-Octen-2-one; 102, \(\gamma\)-Terpinene; 103, 1-Octanol; 104, Linalool; 105, o-Cymene; 106, Nonanal; 107, 1-Nonanol; 108, Naphthalene; 109, Decanal; 110, 1H-Indole. The important metabolites of PLS 2 were C18:0, sinapinic acid, C20:0, ferulic acid, \(\beta\)-tocopherol, 1-butanol, catechchin, epicatechin, malic acid, and shikimic acid, for which the eigenvector values were 0.234828, 0.209490, 0.189271, 0.164575, 0.200056, \(-\)0.26382, \(-\)0.19669, \(-\)0.18505, \(-\)0.16048, and \(-\)0.13863, respectively (Table S1). These metabolites significantly contributed to the separation of non-pigmented rice (WR) from pigmented rice. In addition, the analysis revealed that WR contained more fatty acids than other rice samples. OPLS-DA was next performed to delineate the differences in metabolism in rice samples by maximum separation (Figure 2). OPLS-DA is an extension of a supervised PLS method. In this approach, the X-variables separate the systematic variation into two parts, one that models the correlation between X and Y (prediction), and one that models the orthogonal components. Thus, OPLS-DA yields maximum separation by classes of observations based on their variables. Consequently, OPLS-DA outcomes are more easily interpreted than PLS-DA outcomes. At first, data for non-pigmented (WR) and pigmented rice were compared to construct the OPLS-DA model for identifying the metabolic differences determined by the formation of rice pigment. Non-pigmented rice (WR) and pigmented rice groups were compared in OPLS-DA models (Figure 2A).