Analysis of Quality Involving in Minerals, Amylose, Protein, Polyphenols and Antioxidant Capacity in Different Coloured Rice Varieties

Xiaoqiong Chen; Xia Zhou; Zhiyuan Yang; Chaojian Gu; Yu Tao; Qiaoling Guo; Daiming Guo; Hongyu Zhang; Peizhou Xu; Yongxiang Liao; Yuping Wang; Qilong Duan; Xiuhua Ran; Liqiang Wang; Yang Li; Xianjun Wu

doi:10.3136/fstr.25.141

Abstract

In this study, we investigated six minerals, amylose, soluble protein and total protein, total polyphenols contents, and antioxidant capacity for three black rice, two red rice and three white rice cultivars. The results showed that coloured rice varieties contained the higher minerals and polyphenols content than those of white rice. Besides, the study indicated that polyphenols contributed to accumulation of divalent and trivalent minerals, but little effect to monovalent minerals. Black- and red rice possessed the stronger antioxidant capacity than that of white rice, and red rice presented the highest among three different coloured rice varieties. Then the study showed that protein content slightly influenced accumulation of amylose. Besides, protein and amylose content both existed hardly in significant difference among different coloured rice varieties. Hence, this suggests that red- and black rice has higher levels of nutrition and stronger antioxidant capacity for one's health than white rice.

Introduction

Rice (Orazy sativa L.) is consumed as stable food by over half the world's population, and the main cultivation regions include South Asia, China, Korea and Japan. Rice as food provides all kinds of nutrition element and physiological function for human, such as mineral, amylose and protein and bioavailability. Besides, different coloured rice varieties contain different nutrition components, for example, black rice possesses rich ferulic acid, vanillic; and red rice contain rich ρ-coumaric, γ-oryzanol (Abdel-Aal and Hucl, 1999; Sompong et al., 2011). Moreover, red- and black rice contain rich anthocyanin pigment and prothanocyanin, which belong to a group of phenolic compound that play an important biological role, such as, strong antioxidant capacity, cardiovascular disease, anticancer, anti-inflammatory, hypocholesterolemic and antidiabetic properties (Acquaviva et al., 2003; Chen et al., 2012; Harborne and Williams, 2000; Kazemzadeh et al., 2014; Lee et al., 2010; Russo et al., 2005).

It is known that a considerable number of studies mainly focused on identifying anthocyanin pigment for different coloured rice varieties, and examining the antioxidant activity in the last a few decades (Chen et al., 2012; Koh and Mitchell, 2009; Hu et al., 2011; Ling et al., 2001; Toyokuni et al., 2002). Besides, there were also a few studies for nutrition distribution in kernel characteristic for rice milling technical. For example, Itani et al. (2002) checked in distribution of amylose content, nitrogen and minerals in rice kernels for rice milling. And mean time, there were studies for correlation in between mineral and physiological function components, for example, Lee et al. (2015) studied correlation in between mineral Ca, Fe, Zinc and phytic acid. However, there have been not enough studies on whether anthocyanin pigment and polyphenols affecting accumulation of Cu and Mn and the other microelements. Besides, there was no study involved in whether protein content influencing synthesis of amylose in different coloured rice varieties.

The first objective of this study was to analyse whether coloured rice contribute to accumulation of Fe, Zn, Cu, Mn, Mg and Ca. The second objective was to compare antioxidant capacity to different coloured rice varieties. The third objective was to analyze whether protein content affect synthesis of amylose. Therefore, the study checked polyphenols, amylose, protein content, minerals for different coloured rice varieties. Besides, the study also estimated polyphenols and antioxidant capacity in order to provide good proof for rice breeding and selecting bio-functional rice varieties in the near future.

Materials and Methods

Plant Materials and growth conditions The red rice varieties Akasenmemochi (glutinous) and Kasalath (non-glutinous), black rice cultivars Blackrice756 (non-glutinous), Asamurasaki (glutinous) and Yunanheixiangnuo (glutinous), and white rice varieties Nakateshinsenbon (non-glutinous), Qingyou (non-glutinous) and DV123 (non-glutinous) were assessed in this study. Rice plants were varieties in a paddy field from April to September, 2016 in Wenjiang District (latitude 30°429N, longitude 103°509E, altitude 539.3 m), Chengdu City, Sichuan Province. Seeds were stored at 5 °C and dehulled just before use.

Chemicals Bovine Serum Albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). AAPH (2,2′-azobis 2-amidino-propane di-hydrochloride), Folin-Ciocalteu's reagent, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonsaure) (Louis, MO, USA), Gallic acid was from Biomedicals (Illkirch, France). Standards zinc (Zn), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn) and copper (Cu) were given from Ferrous Metals and Electronic Materials Analysis and Testing Center, China (Beijing, China). The other chemicals used in this study were of the highest grade available and purchased from Sichuan Xilong Chemical Industries Ltd. (Chengdu, China).

Sample preparation Randomly selected hulled grains (caryopsis) in each variety were milled with an ultra centrifugal mill (Jiuyang, China). Milled rice powder (0.2 g) was soaked in 1 % HCl methanol, and sequentially extracted for 24h at room temperature with shaking. The total extract volume was adjusted to 10 mL with the same extraction solvent. The extracts were used for the measurement of phenolic content and the oxygen radical absorbing capacity ORAC assay. All extracts were stored at –20 °C until use.

Measurement of total phenolic content (TPC) TPC was determined by the Folin–Ciocalteu method as described previously (Zhang et al., 2010). A standard curve was constructed using a stock concentration of 1 mg/mL of gallic acid dissolved in 80 % methanol. The aliquots were prepared at a volume range of 0.0, 0.025, 0.05, 0.10, 0.20, 0.4 and 0.6 mL of the stock solution and were mixed with 1.25 mL of Folin–Ciocalteu's reagent and 3.75 mL of 20 % Na2CO3 solution. The resulting solution was transferred to cuvettes and was incubated at 30 °C for 2 h in the dark. The absorbance of each sample was measured at 760 nm (Jasco U best-30 spectrophotometer, Tokyo, Japan) for the determination of the total phenolic content.

Determination of minerals The total contents of zinc (Zn), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn) and copper (Cu), were determined in extracts obtained upon mineralization in nitric acid (HNO₃ p.a.) with a concentration of 1.40 g·cm⁻¹ in a MARS 5 microwave oven (CEM Corporation, USA), in HP500 teflon vessels (the parameters of the process, i.e., weight of analytical samples, volume of nitric acid, and temperature of the mineralization process complied with the US-EPA3051 Protocol). Total concentrations of the six analyzed metals were determined by using an inductive coupled plasma (ICP) atomic emission spectrometer (ICP-AES) (IRISIntrepidXSP II, Thermo, USA). The analysis by use of an ICP-AES was conducted after preparing the standard calibration curves corresponding to each element. Mean and SD were calculated.

Determination of P and K content For P content, rice flour was digested with 18.4N sulfuric acid and potassium sulfate by the Kjeldahl method and analyzed by the sodium salicylate method procedure. For K content, extracts from rice flour dissolved in 0.34N HCl were analyzed by Polarized-Zeeman Atomic Absorption Spectrophotometry (Z8000, Hitachi Co.Ltd., Japan).

Measurement methods of amylose and protein content For amylose analysis, rice flour was digested in 1 N KOH for a night at low temperature, and its amylose-iodine was analyzed by automated colormetric procedure (Auto-analyzer 2, method No.185-72A, technico Co. Ltd., USA).

Total protein content was measured using Kjeldahl method. Soluble protein content was determined using Coomassie Brilliant Blue G-250 staining as Grintzails et al. described, and was assessed by absorbance at 595 nm using UV-1750 spectrophotometer (Shimadzu,Tokyo, Japan) (Grintzails et al., 2015).

ORAC method The assessment of the ORAC assay was carried out using a Lab systems Fluoroskan Ascent FL plate reader (Sigma Chemical Co.), according to a procedure described by Wu et al. (Wu et al., 2004). The compound 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was used as the peroxyl radical generator, the compound Trolox as the standard and the compound fluorescein as the fluorescent probe. An excitation wavelength of 485 nm was selected using appropriate filters, and the fluorescence emission was measured at 520 nm every 2 min over a 90 min period. The measurements were carried out in triplicate. The relative Trolox equivalent ORAC value was calculated as follows: relative ORAC value = (Area under the curve (AUC) sample - AUC blank) micromoles of Trolox equivalent per gram of dry weight/(AUC Trolox-AUC blank) micromoles of Trolox equivalent per gram of dry weight.

Statistical analysis All measurements from the same extract were carried out in triplicate in order to determine reproducibility. Statistical significance was accepted at P < 0.05, and determined using a t-test (SPSS, USA). To ensure the reliability of mean value for repeatability measurements, we performed One-Sample Kolmogorov-Smirnov Test to prove the reliability of the data (Table 1).

Table 1. Normal distribution test by One-Sample Kolmogorov-Smirnov Test

	N	Mean	Asymp. Sig. (2-tailed)
Starch	24	0.406	0.341
ORAC	24	106.4929	0.239
Total protein	24	83.7083	0.985
Soluble protein	24	16.8922	0.431
Polyphenol	24	448.2069	0.211
Ca	24	33.2838	0.154
Fe	24	6.2038	0.394
Zn	24	4.5929	0.066
Mg	24	135.1273	0.998
Cu	24	0.6092	0.428
Mn	24	2.4617	0.598
K	24	160.4608	0.201
P	24	197.5708	0.066

Note: N represents Number of samples.

Results and Discussion

Total phenolic content (TPC) The total phenolic content (TPC) of samples, prepared in 1 % HCl methanol, was evaluated by UV spectrophotometry (Fig. 1A). The results showed that the TPC of white rice (122.94 – 197.13 mg/100 g) was significantly lower than black rice (429.61 – 606.71 mg/100 g). Red rice (660.00 – 836.09 mg/100 g) was the highest among three different coloured rice varieties. In the comparison of different coloured rice varieties for TPC, Yao et al. (2010) reported that black rice (8.58 g/100 g) possessed the highest TPC, and was 86-fold higher than that of red rice (0.1 g/100 g). On the contrary, our data showed that red rice possessed the highest TPC. Similar to our study, Sompong et al. (2011) found that in studies of ten red rice cultivars and three black rice cultivars, the highest TPC was found in red Thai rice (691 mg/100 g). As Sompong et al. reported, Chen et al. (2012) also found that the highest TPC was red rice Tohboshi 725.69 mg/100 g. Besides, some studies have reported that TPC presented varied markedly in the same coloured rice strain (Sompong et al., 2011 and Chen et al., 2012). Therefore, we conclude that coloured rice cultivars contain more TPC than the ordinary white rice, but exist significant difference among different coloured rice varieties and different cultivars of rice of the same color differ significantly as a result of differences in genetic background.

Fig. 1.

Total phenol content and Oxygen radical absorbing capacity (ORAC) in different coloured rice varieties. A, Total phenol content. B, Oxygen radical absorbing capacity (ORAC). Data are expressed as mean values (n = 3). Identical superscript letters denote TPCs and ORAC that are not significantly different at P < 0.05.

Comparison of minerals in different coloured rice varieties Contents of six minerals in different coloured rice varieties were analyzed using the ICP-AES (Table 2). The results showed that Mg content (112.08 – 161.80 mg/ 100 g) was the highest than the other minerals content in rice, followed by Ca (21.86 – 59.91 mg/ 100 g) > Fe (3.08 – 13.37 mg/ 100 g) > Zn (2.65 – 9.22 mg/ 100 g) > Mn (2.13 – 2.73 mg/ 100 g) > Cu (0.50 – 0.75 mg/ 100 g). Furthermore, significant difference in different rice varieties were observed for Mg, Ca, Fe, Zn and Cu, and were higher in coloured rice varieties than white rice varieties as result Suliburska and Krejpcio reported (2014). To further analyse, Zn content existed in significant difference between red– and black rice, for example, that in red rice was almost 3-fold than black rice as Yang et al. reported. By contrast, Suliburska and Krejpcio reported that Zn content in brown rice (1.8 mg/100 g) hardly existed in significant difference than in white rice (1.6 mg/100 g). It is possible that the genetic makeup of rice, as well as environment factors during cultivation, led to the observed differences in Zn content. Significant difference for Ca content was observed between individual cultivars within the same coloured rice other than different coloured rice varieties. For example, Blackrice756 (599.12 mg/100 g) was almost 2-fold than Yunanheixiangnuo (233.94 mg/100 g) within black rice varieties. Suliburska and Krejpcio (2014) reported that Ca content was richer in coloured rice than white rice. However, we just only partly agree to conclusion reported by Sulibrska and Krejpcio, for example, white rice (qingyou 35.57 mg/100 g) was higher than black rice (Yunanheixiangnuo 23.39 mg/100 g), although, Blackrice756 possessed the highest Ca content in 8 cultivars checked. It is reasonable to presume that anthocyanin pigment hardly influence accumulation of Ca in rice at least in our study. Fe content showed significant difference between coloured rice and white rice varieties, for example, black rice (Yunanheixiangnuo 13.37 mg/100 g) was 4-fold than white rice (DV123 3.08 mg/ 100 g). Besides, Fe content also presented significant difference within coloured rice varieties. But there were little difference within white rice varieties. Similar to result of Fe, content of Mn and Cu hardly existed in difference between black- and red rice, but significant difference were observed between coloured rice and white rice varieties. In a comparison of red, black, and white rice cultivars, Suliburska and Krejpcio (2014) reported that content of minerals in brown rice were higher than in white rice. Similar difference in the content of minerals in black rice and white rice were reported by Grembecka and shefer (2009) who found higher amounts of Mg in coloured rice (above 100 mg/100 g) than in white rice (7–29 mg/100 g). In brief, coloured rice has high levels of minerals and good for one's health than white rice.

Table 2. Content of six elements in different colored rice varieties

Cultivars	Ca (mg/100 g)	Fe (mg/100 g)	Zn (mg/100 g)	Mg (mg/100 g)	Mn (mg/100 g)	Cu (mg/100g)	K (mg/100g)	P (mg/100g)
Black rice
Blackrice756	59.91±5.34^h	6.97±0.69^d	2.65±0.40^a	138.09±2.32^e	2.13±0.24^a	0.60±0.23^ab	162.04±0.38^f	155.78±34.64^b
Yunanheixiangnuo	23.39±2.86^c	13.37±1.62^f	3.21±0.28^b	140.33±5.22^f	2.53±0.14^b	0.66±0.17^ab	150.00±0.61	353.04±25.66^f
Asamurasaki	48.67±4.36^g	5.68±0.45^c	3.09±0.33^b	151.29±4.97^g	3.34±0.17^d	0.65±0.07^ab	231.72±6.40^h	162.46±16.18^c
Red rice
Akasenmemochi	22.54±2.72^b	4.19±0.33^b	9.12±1.00^d	117.85±2.71^b	2.31±0.26^a	0.75±0.11^b	108.78±6.05^a	175.68±8.83^d
Kasalath	21.86±1.79^a	9.96±0.67^e	9.22±2.18^d	161.80±2.78^h	2.73±0.04^c	0.65±0.11^ab	152.84±6.12^d	266.58±31.43^e
White rice
Qingyou	35.57±2.69^f	3.24±0.14^a	3.27±1.57^bc	127.84±5.37^c	2.28±0.34^a	0.53±0.02^a	179.04±6.24^g	157.23±7.32^b
Nakateshinsenbon	29.99±3.22^e	3.14±0.14^a	3.14±0.72^b	112.08±1.89^a	2.19±0.14^a	0.54±0.06^a	157.94±5.71^e	175.65±11.46^d
DV123	24.33±0.95^d	3.08±0.15^a	3.39±0.81^c	131.73±2.67^d	2.19±0.05^a	0.50±0.02^a	141.33±6.15^b	134.15±39.36^a

Note: A Values are expressed as means ± SD (n = 3). Values within each column with the identical superscript letter are not significantly different at P < 0.05.

Comparison of P and K in different coloured rice varieties P and K were checked in different coloured rice varieties (Table 2). The results showed that they all existed among significant difference in different rice cultivars rather than different rice varieties.

Analysis of protein and amylose in different coloured rice varieties Total proteins are composed of soluble protein and insoluble protein. The present study analyzed soluble protein content by coomassie brilliant blue G-250 staining, and total protein content using Kjeldahl method (Table 3). The results showed that protein of rice was mainly composed of insoluble protein, ranged from 76.5 to 84.9 % among the different rice varieties. Besides, the study showed that soluble protein hardly influenced accumulation of insoluble protein, for example, Asamurasaki contained 91.31 and 13.76 g/ Kg for total protein content and insoluble, respectively. By contrast, DV123 just only contained total protein 82.87 g/ Kg, but soluble protein attained 17.67 g/ Kg. We presume that accumulation of soluble protein hardly affect accumulation of insoluble protein. Significant difference was observed among the different cultivars of the same coloured rice rather than different coloured rice varieties (Table 3). Amylose content was checked using automated colormetric procedure (Fig. 2). The results showed that amylose content presented significant difference among the different rice cultivars, and difference mainly derived from different cultivar of the same coloured rice varieties. Therefore, we conclude that amylose and protein content are mainly decided by genetic factor.

Table 3. Soluble protein, total protein, and soluble protein/total protein ration in different colored rice varieties

Cultivars	Soluble protein (g/Kg)	Total protein (g/Kg)	Soluble protein/total protein
Black rice
Blackrice756	12.93±0.37^a	84.77±1.06^d	15.30%
Yunanheixiangnuo	20.81±0.62^d	88.79±0.64^e	23.40%
Asamurasaki	13.76±0.57^a	91.31±0.45^f	15.10%
Red rice
Akasenmemochi	17.69±0.58b^c	81.01±0.79^b	21.80%
Kasalath	16.02±0.28^b	86.33±2.98^d	18.66%
White rice
Qingyou	18.87±0.86^c	80.66±1.25^b	23.40%
Nakateshinsenbom	17.38±0.02^bc	73.92±1.58^a	23.50%
DV123	17.67±0.33^bc	82.87±1.27^c	21.30%

Note: Values are expressed as means ± SD (n = 3). Values within each column with the identical superscript letter are not significantly different at P < 0.05.

Fig. 2.

Amylose content in different coloured rice varieties. Data are expressed as mean values (n = 3). Identical superscript letters denote amylose content that are not significantly different at P < 0.05.

Antioxidant capacity The antioxidant capacity of three white, three black and two red rice cultivars was evaluated using the ORAC assay (Fig. 1B). The results showed that the antioxidant capacity of rice followed the rank order: Red rice cultivars (177.09 – 177.85 1 mol Trolox / g) > black rice cultivars (112.88 – 136.71 mol Trolox /g) > white rice cultivars (38.46 – 47.56 3 mol Trolox/ g). Furthermore, significant differences were observed in different coloured rice varieties, for example, black rice cultivars were almost 3-fold higher in ORAC than white cultivars. However, antioxidant capacity of black rice cultivars were lower than that of red rice cultivars. In a comparison of red, black and purple rice cultivars, Yao et al. (2010) reported that the 1,1-diphenyl-2-picrylhyazyl (DPPH) radical scavenging activity of black rice was greater than that of red rice; similar result was reported by Laokuldilok et al. (2011). In contrast, Oki et al. (2002) found that antioxidant capacity of red rice was higher than black and white rice using DPPH. Similar to result Oki et al., Chen et al. (2012) reported that red rice was the highest, followed by black rice, the lowest was white rice by ORAC. It is possible that genetic makeup of rice, as well as environment factors during cultivation, led to the observation difference in antioxidant capacity. Previous researches showed that polyphenols contribute to free-radical scavenging capacity in vivo and vitro because of their ideal antioxidant structure (Rice-Evans et al., 1997). However, Fig. 1 showed that the highest polyphenol content did not present the strongest antioxidant capacity in a comparison of different polyphenol content of different rice cultivars as Chen et al. (2012) reported. It is perhaps due to exists in different types and ration of polyphenols in different coloured rice varieties, and different polyphenols possess different antioxidant capacity. For example, some research reported that most abundant phenolic acid found in red rice was ferulic acid, followed by c-coumaric acid and vanillic acids (Sompong et al., 2011). Abdel-Aal and Hucl (1999) found that the antioxidant capacity of -oryzanol was almost 10-fold higher than that of tocopherols. Han et al. (2004) reported that 2-arylbenzofuran showed stronger antioxidant capacity among black rice. However, further studies are needed to evaluate the antioxidant capacity of different phenolic compound and determine how the structure of these affects their antioxidant capacity.

Correlation analysis for between two different components involved in six minerals, total phenol, antioxidant capacity, protein and amylose To find relationships among 6 mineral element content, pearson correlation analysis were performed for the accession (Table 4). Among 6 mineral elements, there were negative correlation between Ca and Fe, or Zn or Cu. By contrast, there were positive correlation between Cu and Zn, and between Mg and Mn or Fe. Besides, the other mineral element hardly existed in correlation. These results suggested that high Mg content might be accompanied with high Fe and Mn content, and high Zn content contributed to assimilation of Cu. However, Jiang et al. (2007) reported positive association in milled rice were recognized between the content of Fe and Zn or Mn, between the contents Zn and Mn or Cu, and between Cu and Mn, while there was no visible correlation between Cu and Fe contents. Our results partly report by Jiang et al. (2007). The difference of results perhaps derive from different genetic background of rice cultivars and varieties circumstance. Then this study checked correlation between mineral elements and total phenol (Table 4). The results showed that phenol content contributed to assimilation of microelement. For example, correlation between Cu and total phenol contents presented significant difference (0.901, p< 0.002). The study of Karamac et al. (2007) showed that tannins present in buckwheat groats were strong chelators of Cu, Fe and Zn. Suliburska and Krejpcio (2014) reported that the content of divalent and trivalent minerals was higher in the outer layer of grains (bran), where they were bound with phytates, polypheonls and fibres. Chen et al. (2012) reported that red and black rice both contained higher total phenols content than white rice. Therefore, it is reasonable to presume that the higher amounts of minerals in red and black rice derive from phenolic compound that promote accumulation of divalent and trivalent minerals, such as Cu, Fe, Mg and Zn.

Table 4. Correlation among six mineral elements and between mineral element and total phenol

	Ca	Fe	Zn	Mg	Mn	Cu	Total phenol
Ca	1
Fe	−0.13	1
Zn	−0.54	0.13	1
Mg	0.15	0.62	0.17	1
Mn	0.14	0.31	0.09	0.68	1
Cu	−0.13	0.45	0.64	0.22	0.4	1
Total phenol	0.1	0.37	0.65	0.4	0.39	0.90**	1

Note: Correlation is significant at the 0.01 level (2-tailed).

About correlation analysis between total phenol contents and P (r²=0.18) or K (r²=0.01), the results showed that both of them had no visible correlation. We presume that polyphenol hardly affect accumulation of monovalent minerals, and P and K are mainly decided by genetic factor as Huang et al. (2016) reported.

Lastly, we also analyzed correlation between two different components involved in total phenol, antioxidant capacity, protein and amylose. The results showed that there was low correlation (r²=0.27) between amylose and protein. Synthesis of amylose affect slightly accumulation of protein as Ge et al. (2007) reported that protein content influenced accumulation of amylose. But we do not understand the interference mechanism each other. Therefore, further research for correlation between protein and amylose is required to expand our understanding of this area.

Besides, the results also showed that there were little correlation (r²=0.51, r²=0.22) between polyphenol and protein or amylose of rice, respectively. Therefore, we conclude that protein and amylose do not affect synthesis of polyphenol. Then we checked correlation (r²=0.75) involved in phenol and antioxidant, and the results showed that phenol contributed to antioxidant capacity of rice, but they did not existed in close correlation. It is perhaps different cultivars contain different polyphenol component, in addition, different phenol components possess different antioxidant capacity. For example, Abdel-Aal and Hucl (1999) found that the antioxidant capacity of -oryzanol was almost 10-fold higher than that of tocopherols. Han et al. (2004) reported that 2-arylbenzofuran showed stronger antioxidant capacity among black rice.

Summary and Conclusion

In conclusion, black- and red rice varieties possess good quality and rich nutrition components than those of white rice. Therefore, it is rather important to continue to study coloured rice, and it can provide good proofs for breeding good pigmented rice cultivars in the near future.

Abbreviations

AAPH

2,2′-azobis 2-amidino-propane di-hydrochloride

ORAC

Oxygen radical absorbance capacity

TPC

Total phenolic content

BSA

Bovine Serum Albumin

Zinc

Calcium

Magnesium

Iron

Manganese

Copper

Funding

the project of the Chengdu City Science and Technology Bureau, and the project 14Z0011 of the Education Department of Sichuan Province, China

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