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山茶花(Camellias)是我国传统名花,花色为其重要观赏性状[1]。花青苷为山茶花花色形成的物质基础,总花青苷及主要花青苷含量决定其花色[2-4]。研究还表明,花青苷具有抑制肿瘤[5-6]、降血糖[7]和抗氧化[8-9]等作用,可用于医药、保健和化妆等产品的开发。Li等[10-11]利用核磁共振、质谱和紫外可见光谱等技术研究了山茶花花青苷,分别在滇山茶(Camellia reticulata Lindl.)中鉴定出5种含2-O-β-木糖基及其相应不含2-O-β-木糖基的花青苷,滇山茶园艺品种‘大理茶’中鉴定出10种含2-O-β-木糖基花青苷。李辛雷等[2,4]应用高效液相色谱-光电二极管阵列检测和超高效液相色谱-四极杆-飞行时间质谱联用技术,从山茶(C. japonica L.)不同花色品种中鉴定了出7种花青苷,且其花色随总花青苷及主要花青苷含量增加而加深。
杜鹃红山茶(C. azalea Wei)四季开花,是培育四季茶花的优良亲本[12],广州棕科园艺开发有限公司通过杜鹃红山茶与山茶‘媚丽’杂交,已培育出新品种50余个[13]。已有杜鹃红山茶研究主要集中于种群结构 [14]、生理特性[15]、生物学特性[16]和繁殖[17-18]等方面,李辛雷等[19]对其色素成分进行了初步研究,但其花青苷从亲本到杂交后代的变异特征尚不清楚。鉴于此,本试验应用高效液相色谱-光电二极管阵列检测(HPLC-DAD)和超高效液相色谱-四极杆-飞行时间质谱联用技术(UPLC-Q-TOF-MS),对杜鹃红山茶、山茶‘媚丽’及其杂交后代花青苷成分与含量进行研究,明确其花青苷变异特征,以期为高花青苷含量山茶花新品种选育提供科学依据,进一步为其花青苷功能产品的开发利用提供物质基础。
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根据UPLC-Q-TOF-MS图谱,对杜鹃红山茶、山茶‘媚丽’及其杂交后代花瓣中花青苷成分进行鉴定,共检测到14种花青苷(图2),其紫外-可见光谱及质谱数据见表1。根据Cy糖苷在513~520 nm有特征吸收峰及碎片离子m/z 287,推定14种花青苷均为Cy型花青苷[24];花青苷在440 nm与λvis-max两处吸收值之比A440/Avis-max变化范围为32%~34%,确定14种花青苷均为3-O-糖苷类型[25-26]。根据290~340 nm波长下肩峰的出现推定化合物被芳香酸酰化[27],峰P5~P14在310~316 nm波长下肩峰的出现推定其花青苷被芳香酸酰化。
图 2 杜鹃红山茶与‘媚丽’杂交品种花青苷成分的HPLC图谱
Figure 2. HPLC chromatogram of anthocyanin components in hybrids of C. azalea and C. japonica 'Meili'
表 1 杜鹃红山茶与山茶‘媚丽’杂交后代花青苷的紫外–可见吸收光谱与质谱数据
Table 1. Chromatographic and spectral data of anthocyanins in hybrids of C. azalea and C. japonica 'Meili'
色谱峰
Peak
No.保留时间
Retention time
/min吸收波长
λmax
/nmA440/
Avis-max
/%分子离子
Molecular ions
(m/z)碎片离子
Fragment ions
(m/z)推定结果
Tentative
identification参考文献
ReferenceP1 8.25 281,516 32 581 449,287 Cy3GaX [10-11] P2 8.78 282,516 32 449 287 Cy3Ga [2-4] P3 9.98 282,516 33 581 449,287 Cy3GX [10-11] P4 10.54 282,514 32 449 287 Cy3G [2-4] P5 25.64 281,315,516 33 611 449,287 Cy3GaECaf [2-4] P6 25.89 282,315,517 33 743 611,449,287 Cy3GaECafX [10-11] P7 26.76 284,311,515 34 727 595,449,287 Gy3GaZpCX [10-11] P8 27.19 285,310,516 34 595 449,287 Gy3GaZpC [10-11] P9 28.52 283,315,515 33 743 611,449,287 Cy3GECafX [10-11] P10 28.77 283,316,515 33 611 449,287 Cy3GECaf [2-4] P11 29.68 283,313,516 34 595 449,287 Cy3GaEpC [2-4] P12 30.07 282,312,516 33 727 595,449,287 Cy3GaEpCX [10-11] P13 31.89 284,314,514 34 727 595,449,287 Cy3GEpCX [10-11] P14 32.24 283,313,515 34 595 449,287 Cy3GEpC [2-4] 注:Cy3GaX: 矢车菊素-3-O-(2-O-β-木糖基)-β-半乳糖苷; Cy3Ga: 矢车菊素-3-O-β-半乳糖苷; Cy3GX: 矢车菊素-3-O-(2-O-β-木糖基)-β-葡萄糖苷; Cy3G: 矢车菊素-3-O-β-葡萄糖苷; Cy3GaECaf: 矢车菊素-3-O-[6-O-(E)–咖啡酰]-β-半乳糖苷; Cy3GaECafX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-咖啡酰]-β-半乳糖苷; Cy3GaZpCX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(Z)-p-香豆酰]-β-半乳糖苷; Cy3GaZpC: 矢车菊素-3-O-[6-O-(Z)-p-香豆酰]-β-半乳糖苷; Cy3GECafX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-咖啡酰]-β-葡萄糖苷; Cy3GECaf: 矢车菊素-3-O-[6-O-(E)-咖啡酰]-β-葡萄糖苷; Cy3GaEpC: 矢车菊素-3-O-[6-O-(E)-p-香豆酰]-β-半乳糖苷; Cy3GaEpCX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-p-香豆酰]-β-半乳糖苷; Cy3GEpCX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-p-香豆酰]-β-葡萄糖苷; Cy3GEpC: 矢车菊素-3-O-[6-O-(E)-p-香豆酰]-β-葡萄糖苷。下同。
Notes: Cy3GaX: Cyanidin-3-O-(2-O-β-xylosyl)-β-galactoside; Cy3Ga: Cyanidin-3-O-β-galactoside; Cy3GX: Cyanidin- 3-O-(2-O-β-xylosyl)-β-glucoside; Cy3G: Cyanidin-3-O-β-glucoside; Cy3GaECaf: Cyanidin- 3-O-(6-O-(E)-caffeoyl)-β-galactoside; Cy3GaECafX: Cyanidin-3-O-(2-O-β-xylosyl-6-O-(E)-caffeoyl)-β-galactoside; Cy3GaZpCX: Cyanidin-3-O-(2-O-β-xylosyl-6-O-(Z)-p-coumaroyl)-β-galactoside; Cy3GaZpC: Cyanidin- 3-O-(6-O-(Z)-p-coumaroyl)-β-galactoside; Cy3GECafX: Cyanidin- 3-O-(2-O-β-xylosyl-6-O-(E)-caffeoyl)-β-glucoside; Cy3GECaf: Cyanidin-3-O-(6-O-(E)-caffeoyl)-β-glucoside; Cy3GaEpC: Cyanidin-3-O-(6-O-(E)-p-coumaroyl)-β-galactoside; Cy3GaEpCX: Cyanidin- 3-O-(2-O-β-xylosyl-6-O-(E)-p-coumaroyl)-β-galactoside; Cy3GEpCX: Cyanidin-3-O-(2-O-β-xylosyl-6-O-(E)-p-coumaroyl)-β-glucoside; Cy3GEpC: Cyanidin- 3-O-(6-O-(E)-p-coumaroyl)-β-glucoside. The same as below.峰P2和P4质谱数据为分子离子m/z 449,碎片离子m/z 287,其中,m/z 287为Cy苷元特征质荷比,其裂解释放162 u对应一分子己糖;根据峰P2和峰P4与标准品Cy3Ga和Cy3G共洗脱特性以及花青素半乳糖洗脱时间小于花青素葡萄糖苷特性[24],确定峰P2为矢车菊素-3-O-β-半乳糖苷,峰P4为矢车菊素-3-O-β-葡萄糖苷[28-29]。峰P1和P3质谱数据为分子离子m/z 581,碎片离子m/z 449、287,m/z 581到m/z 449丢失132 u,m/z 449到m/z 287丢失162 u,参考Li等[10-11]文献,推定其分别为矢车菊素-3-O-(2-O-β-木糖基)-β-半乳糖苷(Cy3GaX)、矢车菊素-3-O-(2-O-β-木糖基)-β-葡萄糖苷(Cy3GX)。
峰P5和P10质谱数据为分子离子m/z 611,碎片离子m/z 449、287,m/z 611到m/z 449丢失162 u,m/z 449到m/z 287丢失162 u,判定其为矢车菊素-3-O-[6-O-(E)-咖啡酰]-β-半乳糖苷(Cy3GaECaf)和矢车菊素-3-O-[6-O-(E)-咖啡酰]-β-葡萄糖苷(Cy3GECaf)[2-4]。峰P6和P9质谱数据为分子离子m/z 743,碎片离子m/z 611、449、287,m/z 743到m/z 611丢失132 u,m/z 611到m/z 449丢失162 u,m/z 449到m/z 287丢失162 u,推定其为矢车菊素-3-O-(2-O-β-木糖基-6-O-(E)-咖啡酰)-β-半乳糖苷(Cy3GaECafX)和矢车菊素-3-O-(2-O-β-木糖基-6-O-(E)-咖啡酰)-β-葡萄糖苷(Cy3GECafX)。
峰P8、P11和P14质谱数据为分子离子m/z 595,碎片离子m/z 449、287,m/z 595到m/z 449丢失146 u,m/z 449到m/z 287丢失162 u;根据其A440/Avis-max(34%)及在310、313 nm波长下肩峰的出现,推定其为Cy-3-O-芳香酸酰化型糖苷而排除鼠李糖苷[30];根据顺式花青苷洗脱时间小于反式花青苷[31-32]及花青素半乳糖苷洗脱时间小于花青素葡萄糖苷的特性,判定峰P8、P11和P14分别为矢车菊素-3-O-[6-O-(Z)-p-香豆酰]-β-半乳糖苷(Cy3GaZpC)、矢车菊素-3-O-[6-O-(E)-p-香豆酰]-β-半乳糖苷(Cy3GaEpC)和矢车菊素-3-O-[6-O-(E)-p-香豆酰]-β-葡萄糖苷(Cy3GEpC)。
峰P7、P12和P13质谱数据为分子离子m/z 727,碎片离子m/z 595、449、287,m/z 727到m/z 595丢失132 u,m/z 595到m/z 449丢失146 u,m/z 449到m/z 287丢失162 u,推定其分别为矢车菊素-3-O-[2-O-β-木糖基-6-O-(Z)-p-香豆酰]-β-半乳糖苷(Cy3GaZpCX)、矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-p-香豆酰]-β-半乳糖苷(Cy3GaEpCX)和矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-p-香豆酰]-β-葡萄糖苷(Cy3GEpCX)[10-11]。
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杜鹃红山茶、山茶‘媚丽’及其35个杂交后代中,‘媚丽’检测到7种花青苷,其中Cy3G、Cy3Ga、Cy3GEpC和Cy3GaEpC含量较高,Cy3GaECaf、Cy3GaZpC和Cy3GECaf含量均低于10 μg·g−1。杜鹃红山茶中检测到14种花青苷,除与山茶‘媚丽’相同的7种外,还包括其相应的含2-O-β-木糖基的花青苷;含量较高的花青苷有8种(表2),分别为Cy3G、Cy3Ga、Cy3GEpC和Cy3GaEpC以及相应的含2-O-β-木糖基的Cy3GX、Cy3GaX、Cy3GEpCX和Cy3GaEpCX,其余成分均低于1 μg·g−1。杜鹃红山茶与山茶‘媚丽’35个杂交后代除MD78外,均检测到8种主要相关花青苷,其余成分含量均较低或未检测到。
表 2 杜鹃红山茶与山茶‘媚丽’杂交后代主要花青苷含量
Table 2. Content of main anthocyanins in hybrids of C. azalea and C. japonica 'Meili'
μg·g−1 样品 Samples Cy3GaX Cy3Ga Cy3GX Cy3G Cy3GaEpCX Cy3GaEpC Cy3GEpCX Cy3GEpC 合计 Total M — 28.23±2.15 — 215.43±15.70 — 43.75±2.55 — 239.21±12.68 526.61±30.67 D 141.54±0.47 5.75±0.00 466.23±1.57 44.88±0.16 23.38±0.07 2.07±0.01 40.05±0.12 11.90±0.03 735.81±2.43 MD2 8.38±0.43 1.70±0.02 120.51±0.78 25.76±0.20 24.35±0.74 21.04±0.52 125.89±4.31 34.18±1.42 361.80±7.28 MD4 5.56±0.46 6.81±0.33 85.48±4.70 163.12±9.15 57.52±2.64 10.50±0.47 108.55±5.92 293.89±17.35 731.42±40.29 MD6 17.82±0.10 29.37±0.96 189.85±4.36 561.41±16.24 78.56±2.88 6.62±0.34 136.90±4.33 385.62±11.67 1406.15±40.60 MD10 29.25±0.58 2.54±0.06 392.53±10.24 29.80±0.96 32.91±1.01 20.20±0.52 171.04±5.41 34.88±0.64 713.15±18.34 MD11 35.13±0.80 10.78±0.50 366.20±10.17 105.87±3.84 31.48±1.00 10.96±0.28 115.86±3.46 31.16±0.93 707.45±20.81 MD13 47.85±1.37 6.99±0.30 200.59±6.58 31.68±1.06 47.06±1.92 6.54±0.19 63.09±2.22 21.28±1.20 425.08±14.58 MD14 5.89±0.23 3.81±0.20 61.03±1.81 46.92±28.29 39.04±1.90 6.72±0.25 63.89±2.29 137.81±7.90 365.11±17.43 MD16 27.63±0.67 9.84±0.39 141.62±3.56 64.23±1.86 88.17±3.66 14.15±0.47 132.94±4.70 75.58±2.29 554.15±17.33 MD17 4.53±0.19 3.20±0.20 44.43±0.93 45.16±1.94 46.88±2.01 10.22±0.65 55.05±2.13 151.13±5.51 360.60±10.59 MD19 11.26±0.19 3.78±0.08 213.63±5.02 82.64±2.20 25.56±0.84 19.46±0.58 119.21±4.19 59.73±2.04 535.26±14.87 MD25 71.58±1.22 25.06±0.85 296.79±7.71 144.74±4.26 53.97±1.86 9.52±0.26 62.47±1.88 40.19±1.41 704.32±19.41 MD26 15.67±0.42 5.16±0.21 121.23±2.96 90.56±2.84 26.51±1.04 4.87±0.12 60.60±1.87 76.01±3.90 400.62±12.73 MD28 16.92±0.33 26.95±0.93 117.73±2.28 404.54±11.30 42.36±1.23 5.75±0.17 52.93±1.31 134.92±3.63 802.10±20.82 MD29 75.06±2.68 10.31±0.56 247.46±9.47 52.06±2.27 50.62±2.05 11.12±0.37 79.52±3.00 18.59±0.60 544.74±20.75 MD36 25.58±0.02 5.11±0.13 297.94±5.12 61.06±1.57 51.02±1.07 17.45±0.29 245.78±5.83 42.02±0.62 745.96±13.88 MD51 32.13±0.12 11.11±0.42 202.10±4.49 65.13±2.00 49.53±1.67 14.86±0.39 115.04±3.31 36.94±1.19 526.84±13.49 MD52 20.28±0.86 6.24±0.42 64.94±2.97 30.45±1.95 19.40±0.98 6.73±0.17 38.10±1.64 19.35±1.75 205.49±10.60 MD55 12.72±0.18 8.59±0.38 147.86±4.38 159.45±6.94 33.86±1.43 9.44±0.34 63.87±2.05 91.74±5.41 527.53±20.67 MD58 5.43±0.43 1.38±0.12 148.40±6.29 48.22±2.32 25.57±0.62 5.40±0.18 231.67±6.20 133.23±4.19 599.30±19.03 MD61 13.01±0.48 4.40±0.05 183.65±2.61 64.41±0.41 40.28±0.67 17.82±0.38 139.72±2.68 66.94±0.29 530.23±6.65 MD71 7.69±0.20 0.54±0.06 151.50±5.97 12.13±0.49 12.81±0.32 13.42±0.22 99.70±3.21 11.99±0.53 309.78±10.13 MD72 20.99±0.42 2.50±0.09 403.17±8.23 72.35±1.94 17.82±0.50 17.10±0.30 107.89±2.82 22.98±0.70 664.79±14.82 MD73 8.58±0.33 7.46±0.12 83.90±0.49 122.28±0.99 66.67±1.25 12.96±0.33 113.60±1.51 198.84±2.32 614.28±4.71 MD75 18.34±0.35 16.63±0.73 166.68±4.85 250.27±8.99 42.84±1.63 9.11±0.30 71.60±2.35 126.47±4.01 701.94±23.12 MD76 3.06±0.15 1.62±0.10 66.26±1.13 71.27±1.55 27.10±0.21 7.80±0.05 79.77±0.78 123.34±0.76 380.23±4.41 MD77 122.71±2.05 7.85±0.04 409.23±8.97 42.62±0.62 78.89±2.43 11.22±0.30 97.25±2.64 17.84±0.32 787.61±17.30 MD78 1.29±0.01 — 38.99±0.15 7.36±0.05 6.85±0.03 11.03±0.05 51.73±0.09 17.07±0.03 134.32±0.31 DM3 15.95±0.59 5.24±0.25 196.31±4.31 87.41±2.76 36.88±1.39 17.38±0.56 215.33±7.68 77.81±2.09 652.32±18.26 DM4 7.20±0.11 2.39±0.03 85.17±0.28 28.77±0.06 33.49±1.10 21.63±0.57 149.18±4.98 64.55±1.45 392.38±8.30 DM5 12.10±0.33 3.06±0.12 198.38±6.15 67.83±3.02 32.01±1.47 14.22±0.58 217.57±11.09 61.49±3.53 606.66±25.62 DM7 15.40±0.27 3.66±0.18 174.09±6.82 43.46±2.21 31.28±1.54 11.85±0.48 160.62±7.20 27.73±0.89 468.08±18.76 DM8 14.56±0.25 3.84±0.17 193.72±6.54 56.72±2.41 32.83±1.66 11.03±0.48 197.11±9.37 46.40±1.57 556.21±22.35 DM9 22.60±0.65 33.44±0.95 72.24±1.98 270.50±8.07 49.54±1.07 5.49±0.07 36.62±0.86 123.99±3.08 614.41±16.57 DM10 9.06±0.20 2.09±0.06 134.33±8.01 38.70±2.11 22.95±1.02 12.46±0.65 155.33±8.08 37.94±1.81 412.86±21.63 DM11 12.67±0.51 2.55±0.13 177.88±4.03 40.91±1.00 28.88±0.96 11.49±0.29 182.83±6.24 35.34±0.81 492.54±13.81 注: “-”表示未鉴定出。下同。
Notes: “—” : Not identified. The same below.杜鹃红山茶主要花青苷含量为735.81 μg·g−1,山茶‘媚丽’主要花青苷含量为526.61 μg·g−1,杜鹃红山茶主要花青苷含量为‘媚丽’的1.40倍;杂交后代中主要花青苷含量高于杜鹃红山茶的占11.43%,低于山茶‘媚丽’的占37.14%,双亲之间的占51.43%。杂交后代中Cy3GX、Cy3GaX、Cy3GEpCX和Cy3GaEpCX均来源于杜鹃红山茶,其中Cy3GX、Cy3GaX含量低于杜鹃红山茶,而Cy3GEpCX、Cy3GaEpCX含量大部分高于杜鹃红山茶;Cy3G、Cy3Ga、Cy3GEpC和Cy3GaEpC 主要来源于山茶‘媚丽’,含量大部分介于双亲之间。山茶‘媚丽’中含葡萄糖苷的花青苷含量远高于相应含半乳糖苷的花青苷;杜鹃红山茶及杂交后代中含2-O-β-木糖基的花青苷含量基本上高于相应不含2-O-β-木糖基的花青苷,含葡萄糖苷的花青苷(除个别杂交后代外)含量也高于相应含半乳糖苷的花青苷。
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杜鹃红山茶与山茶‘媚丽’杂交后代主要花青苷比例见表3。杜鹃红山茶花瓣中主要花青苷Cy3GX、Cy3GaX分别占63.14%、19.17%,合计82.30%;其次为Cy3G、Cy3GEpCX和Cy3GaEpCX,分别占6.08%、5.42%和3.17%。山茶‘媚丽’主要成分Cy3GEpC、Cy3G分别占44.76%、40.26%,合计85.02%;其次为Cy3GaEpC、Cy3Ga,分别占8.18%、5.27%。杂交后代中,Cy3GX、Cy3GaX所占比例均低于杜鹃红山茶,而Cy3GaEpCX(除MD72外)所占比例均高于杜鹃红山茶;Cy3G、Cy3Ga、Cy3GEpC和Cy3GaEpC所占比例大部分介于双亲之间,其中,Cy3GX、Cy3GEpCX平均比例分别为31.24%、22.18%,合计53.42%;Cy3G、Cy3GEpC平均比例分别为15.37%和14.20%,合计29.57%。可见,杜鹃红山茶花青苷主要成分为Cy3GX和Cy3GaX,山茶‘媚丽’为Cy3GEpC和Cy3G,杂交后代主要成分为Cy3GX和Cy3GEpCX,其次为Cy3G和Cy3GEpC。
表 3 杜鹃红山茶与山茶‘媚丽’杂交后代主要花青苷比例
Table 3. Proportion of main anthocyanins in hybrids of C. azalea and C. japonica 'Meili'
% 样品Samples Cy3GaX Cy3Ga Cy3GX Cy3G Cy3GaEpC Cy3GaEpCX Cy3GEpCX Cy3GEpC M — 5.27 — 40.26 8.18 — — 44.76 D 19.17 0.78 63.14 6.08 0.28 3.17 5.42 1.61 MD2 2.24 0.45 32.12 6.87 5.61 6.49 33.53 9.10 MD4 0.75 0.92 11.56 22.06 1.42 7.78 14.68 39.73 MD6 1.25 2.06 13.35 39.47 0.47 5.52 9.62 27.11 MD10 4.00 0.35 53.64 4.07 2.76 4.50 23.37 4.77 MD11 4.90 1.50 51.12 14.77 1.53 4.39 16.17 4.35 MD13 10.98 1.60 46.05 7.27 1.50 10.80 14.48 4.88 MD14 1.54 0.99 15.93 11.95 1.76 10.20 16.68 35.98 MD16 4.86 1.73 24.89 11.29 2.49 15.49 23.36 13.28 MD17 1.21 0.86 11.90 12.11 2.73 12.55 14.74 40.47 MD19 2.05 0.69 38.79 15.00 3.53 4.64 21.64 10.84 MD25 9.92 3.47 41.11 20.04 1.32 7.47 8.65 5.56 MD26 3.85 1.27 29.82 22.27 1.20 6.52 14.90 18.68 MD28 2.08 3.31 14.48 49.74 0.71 5.21 6.51 16.59 MD29 13.46 1.85 44.36 9.33 1.99 9.07 14.26 3.33 MD36 3.32 0.66 38.69 7.93 2.27 6.62 31.91 5.46 MD51 5.91 2.04 37.17 11.98 2.73 9.11 21.16 6.79 MD52 9.44 2.90 30.21 14.15 3.13 9.02 17.72 8.98 MD55 2.37 1.60 27.49 29.62 1.75 6.29 11.87 17.03 MD58 0.88 0.22 24.14 7.84 0.88 4.16 37.72 21.69 MD61 2.38 0.80 33.53 11.76 3.25 7.36 25.51 12.22 MD71 2.43 0.17 47.88 3.83 4.25 4.05 31.52 3.79 MD72 3.11 0.37 59.82 10.73 2.54 2.64 16.01 3.41 MD73 1.36 1.18 13.30 19.38 2.05 10.56 18.00 31.51 MD75 2.56 2.32 23.23 34.87 1.27 5.97 9.98 17.62 MD76 0.79 0.42 17.09 18.38 2.01 6.99 20.57 31.81 MD77 15.33 0.98 51.13 5.33 1.40 9.85 12.15 2.23 MD78 0.94 — 28.29 5.34 8.00 4.97 37.54 12.39 DM3 2.41 0.79 29.63 13.19 2.62 5.56 32.49 11.75 DM4 1.77 0.59 20.91 7.06 5.31 8.22 36.60 15.84 DM5 1.96 0.50 32.18 11.00 2.31 5.19 35.26 9.97 DM7 3.24 0.77 36.62 9.14 2.49 6.58 33.78 5.83 DM8 2.57 0.68 34.19 10.01 1.95 5.79 34.77 8.19 DM9 3.59 5.31 11.47 42.95 0.87 7.87 5.82 19.69 DM10 2.15 0.50 31.83 9.17 2.95 5.44 36.82 8.99 DM11 2.53 0.51 35.53 8.17 2.29 5.77 36.50 7.06
杜鹃红山茶与山茶‘媚丽’杂交后代花青苷变异特征
Variation Characteristics of Anthocyanin in Hybrids between Camellia azalea and Camellia japonica 'Meili'
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摘要:
目的 研究杜鹃红山茶、山茶‘媚丽’及其杂交后代花瓣中花青苷成分与含量,揭示其主要花青苷成分与含量的变异特征,为高花青苷含量山茶花新品种选育及其开发利用提供科学依据。 方法 应用高效液相色谱-光电二极管阵列检测(HPLC-DAD)和超高效液相色谱-四极杆-飞行时间质谱联用技术(UPLC-Q-TOF-MS)定性定量分析杜鹃红山茶、山茶‘媚丽’及其35个杂交后代花瓣中花青苷成分与含量。 结果 杜鹃红山茶及其杂交后代花瓣中共检测到14种花青苷,其中含量较高的主要花青苷有8种,包括矢车菊素-3-O-(2-O-β-木糖基)-β-半乳糖苷(Cy3GaX)、矢车菊素-3-O-β-半乳糖苷(Cy3Ga)、矢车菊素-3-O-(2-O-β-木糖基)-β-葡萄糖苷(Cy3GX)、矢车菊素-3-O-β-葡萄糖苷(Cy3G)、矢车菊素-3-O-[6-O-(E)-p-香豆酰]-β-半乳糖苷(Cy3GaEpC)、矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-p-香豆酰]-β-半乳糖苷(Cy3GaEpCX)、矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-p-香豆酰]-β-葡萄糖苷(Cy3GEpCX)和矢车菊素-3-O-[6-O-(E)-p-香豆酰]-β-葡萄糖苷(Cy3GEpC)。杜鹃红山茶主要花青苷总量高于山茶‘媚丽’,杂交后代花青苷总量存在较大的变异;杂交后代中Cy3GX、Cy3GaX含量均低于杜鹃红山茶,Cy3GEpCX、Cy3GaEpCX含量总体上高于杜鹃红山茶,Cy3G、Cy3Ga、Cy3GEpC和Cy3GaEpC含量基本上介于双亲之间。 结论 杜鹃红山茶及其杂交后代中含2-O-β-木糖基的花青苷含量高于相应不含2-O-β-木糖基的花青苷,含葡萄糖苷的花青苷高于相应含半乳糖苷的花青苷。杜鹃红山茶主要花青苷为Cy3GX和Cy3GaX, 山茶‘媚丽’为Cy3GEpC和Cy3G,杂交后代主要为Cy3GX和Cy3GEpCX,其次为Cy3G和Cy3GEpC;杂交后代中含2-O-β-木糖基的花青苷来源于杜鹃红山茶,其所占比例高于相应不含2-O-β-木糖基的花青苷,表明含2-O-β-木糖基的花青苷具较强的遗传能力。 -
关键词:
- 杜鹃红山茶
- / 杂交
- / 花青苷
- / 变异
- / 超高效液相色谱-四极杆-飞行时间质谱
Abstract:Objective The object of this study was to determine anthocyanin components and contents in petals from Camellia azalea, C. japonica 'Meili' and their hybrids, and reveal variation regulation of main anthocyanin components and content, that will provide the scientific basis for breeding and utilization of camellias with high anthocyanin content. Method Anthocyanin components and contents of C. azalea, C. japonica 'Meili' and thirty-five hybrids were measured by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) and aultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Result Fourteen anthocyanin components were detected in C. azalea and the hybrids, eight of which were main anthocyanin components with high content, including cyanidin-3-O-(2-O-β-xylosyl)-β-galactoside (Cy3GaX), cyanidin-3-O-β-galactoside (Cy3Ga), cyanidin-3-O-(2-O-β-xylosyl)-β-glucoside (Cy3GX), cyanidin-3-O-β-glucoside(Cy3G), cyanidin-3-O-(6-O-(E)-p-coumaroyl)-β-galactoside(Cy3GaEpC), cyanidin-3-O-(2-O-β-xylosyl-6-O-(E)-p-coumaroyl)-β-galactoside (Cy3GaEpCX), cyanidin-3-O-(2-O-β-xylosyl-6-O-(E)-p-coumaroyl)-β-glucoside (Cy3GEpCX) and cyanidin-3-O-(6-O-(E)-p-coumaroyl)-β-glucoside (Cy3GEpC). The total amount of eight main anthocyanin of C.azalea was higher than that of C. japonica 'Meili', and that of hybrids have larger variation. The contents of Cy3GX and Cy3GaX in hybrids were lower than that of C. azalea, and contents of Cy3GEpCX and Cy3GaEpCX in hybrids were higher than that of C. azalea. The contents of Cy3G, Cy3Ga, Cy3GEpC and Cy3GaEpC in hybrids were basically in the middle of the parents. Conclusion The contents of anthocyanins with 2-O-β-xylosyl is higher than that of corresponding anthocyanins without 2-O-β-xylosyl, and the contents of anthocyanins with glucoside is higher than that of corresponding anthocyanins with galactoside. The main anthocyanin components of C. azalea are Cy3GX and Cy3GaX, and those of C. japonica 'Meili' are Cy3GEpC and Cy3G. The main components of hybrids are Cy3GX and Cy3GEpCX, followed by Cy3G and Cy3GEpC. Anthocyanins with 2-O-β-xylosyl in hybrids originate from C. azalea, and the proportion of anthocyanins with 2-O-β-xylosyl is higher than that of corresponding anthocyanins without 2-O-β-xylosyl, that indicates anthocyanins with 2-O-β-xylosyl has stronger genetic ability. -
表 1 杜鹃红山茶与山茶‘媚丽’杂交后代花青苷的紫外–可见吸收光谱与质谱数据
Table 1. Chromatographic and spectral data of anthocyanins in hybrids of C. azalea and C. japonica 'Meili'
色谱峰
Peak
No.保留时间
Retention time
/min吸收波长
λmax
/nmA440/
Avis-max
/%分子离子
Molecular ions
(m/z)碎片离子
Fragment ions
(m/z)推定结果
Tentative
identification参考文献
ReferenceP1 8.25 281,516 32 581 449,287 Cy3GaX [10-11] P2 8.78 282,516 32 449 287 Cy3Ga [2-4] P3 9.98 282,516 33 581 449,287 Cy3GX [10-11] P4 10.54 282,514 32 449 287 Cy3G [2-4] P5 25.64 281,315,516 33 611 449,287 Cy3GaECaf [2-4] P6 25.89 282,315,517 33 743 611,449,287 Cy3GaECafX [10-11] P7 26.76 284,311,515 34 727 595,449,287 Gy3GaZpCX [10-11] P8 27.19 285,310,516 34 595 449,287 Gy3GaZpC [10-11] P9 28.52 283,315,515 33 743 611,449,287 Cy3GECafX [10-11] P10 28.77 283,316,515 33 611 449,287 Cy3GECaf [2-4] P11 29.68 283,313,516 34 595 449,287 Cy3GaEpC [2-4] P12 30.07 282,312,516 33 727 595,449,287 Cy3GaEpCX [10-11] P13 31.89 284,314,514 34 727 595,449,287 Cy3GEpCX [10-11] P14 32.24 283,313,515 34 595 449,287 Cy3GEpC [2-4] 注:Cy3GaX: 矢车菊素-3-O-(2-O-β-木糖基)-β-半乳糖苷; Cy3Ga: 矢车菊素-3-O-β-半乳糖苷; Cy3GX: 矢车菊素-3-O-(2-O-β-木糖基)-β-葡萄糖苷; Cy3G: 矢车菊素-3-O-β-葡萄糖苷; Cy3GaECaf: 矢车菊素-3-O-[6-O-(E)–咖啡酰]-β-半乳糖苷; Cy3GaECafX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-咖啡酰]-β-半乳糖苷; Cy3GaZpCX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(Z)-p-香豆酰]-β-半乳糖苷; Cy3GaZpC: 矢车菊素-3-O-[6-O-(Z)-p-香豆酰]-β-半乳糖苷; Cy3GECafX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-咖啡酰]-β-葡萄糖苷; Cy3GECaf: 矢车菊素-3-O-[6-O-(E)-咖啡酰]-β-葡萄糖苷; Cy3GaEpC: 矢车菊素-3-O-[6-O-(E)-p-香豆酰]-β-半乳糖苷; Cy3GaEpCX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-p-香豆酰]-β-半乳糖苷; Cy3GEpCX: 矢车菊素-3-O-[2-O-β-木糖基-6-O-(E)-p-香豆酰]-β-葡萄糖苷; Cy3GEpC: 矢车菊素-3-O-[6-O-(E)-p-香豆酰]-β-葡萄糖苷。下同。
Notes: Cy3GaX: Cyanidin-3-O-(2-O-β-xylosyl)-β-galactoside; Cy3Ga: Cyanidin-3-O-β-galactoside; Cy3GX: Cyanidin- 3-O-(2-O-β-xylosyl)-β-glucoside; Cy3G: Cyanidin-3-O-β-glucoside; Cy3GaECaf: Cyanidin- 3-O-(6-O-(E)-caffeoyl)-β-galactoside; Cy3GaECafX: Cyanidin-3-O-(2-O-β-xylosyl-6-O-(E)-caffeoyl)-β-galactoside; Cy3GaZpCX: Cyanidin-3-O-(2-O-β-xylosyl-6-O-(Z)-p-coumaroyl)-β-galactoside; Cy3GaZpC: Cyanidin- 3-O-(6-O-(Z)-p-coumaroyl)-β-galactoside; Cy3GECafX: Cyanidin- 3-O-(2-O-β-xylosyl-6-O-(E)-caffeoyl)-β-glucoside; Cy3GECaf: Cyanidin-3-O-(6-O-(E)-caffeoyl)-β-glucoside; Cy3GaEpC: Cyanidin-3-O-(6-O-(E)-p-coumaroyl)-β-galactoside; Cy3GaEpCX: Cyanidin- 3-O-(2-O-β-xylosyl-6-O-(E)-p-coumaroyl)-β-galactoside; Cy3GEpCX: Cyanidin-3-O-(2-O-β-xylosyl-6-O-(E)-p-coumaroyl)-β-glucoside; Cy3GEpC: Cyanidin- 3-O-(6-O-(E)-p-coumaroyl)-β-glucoside. The same as below.表 2 杜鹃红山茶与山茶‘媚丽’杂交后代主要花青苷含量
Table 2. Content of main anthocyanins in hybrids of C. azalea and C. japonica 'Meili'
μg·g−1 样品 Samples Cy3GaX Cy3Ga Cy3GX Cy3G Cy3GaEpCX Cy3GaEpC Cy3GEpCX Cy3GEpC 合计 Total M — 28.23±2.15 — 215.43±15.70 — 43.75±2.55 — 239.21±12.68 526.61±30.67 D 141.54±0.47 5.75±0.00 466.23±1.57 44.88±0.16 23.38±0.07 2.07±0.01 40.05±0.12 11.90±0.03 735.81±2.43 MD2 8.38±0.43 1.70±0.02 120.51±0.78 25.76±0.20 24.35±0.74 21.04±0.52 125.89±4.31 34.18±1.42 361.80±7.28 MD4 5.56±0.46 6.81±0.33 85.48±4.70 163.12±9.15 57.52±2.64 10.50±0.47 108.55±5.92 293.89±17.35 731.42±40.29 MD6 17.82±0.10 29.37±0.96 189.85±4.36 561.41±16.24 78.56±2.88 6.62±0.34 136.90±4.33 385.62±11.67 1406.15±40.60 MD10 29.25±0.58 2.54±0.06 392.53±10.24 29.80±0.96 32.91±1.01 20.20±0.52 171.04±5.41 34.88±0.64 713.15±18.34 MD11 35.13±0.80 10.78±0.50 366.20±10.17 105.87±3.84 31.48±1.00 10.96±0.28 115.86±3.46 31.16±0.93 707.45±20.81 MD13 47.85±1.37 6.99±0.30 200.59±6.58 31.68±1.06 47.06±1.92 6.54±0.19 63.09±2.22 21.28±1.20 425.08±14.58 MD14 5.89±0.23 3.81±0.20 61.03±1.81 46.92±28.29 39.04±1.90 6.72±0.25 63.89±2.29 137.81±7.90 365.11±17.43 MD16 27.63±0.67 9.84±0.39 141.62±3.56 64.23±1.86 88.17±3.66 14.15±0.47 132.94±4.70 75.58±2.29 554.15±17.33 MD17 4.53±0.19 3.20±0.20 44.43±0.93 45.16±1.94 46.88±2.01 10.22±0.65 55.05±2.13 151.13±5.51 360.60±10.59 MD19 11.26±0.19 3.78±0.08 213.63±5.02 82.64±2.20 25.56±0.84 19.46±0.58 119.21±4.19 59.73±2.04 535.26±14.87 MD25 71.58±1.22 25.06±0.85 296.79±7.71 144.74±4.26 53.97±1.86 9.52±0.26 62.47±1.88 40.19±1.41 704.32±19.41 MD26 15.67±0.42 5.16±0.21 121.23±2.96 90.56±2.84 26.51±1.04 4.87±0.12 60.60±1.87 76.01±3.90 400.62±12.73 MD28 16.92±0.33 26.95±0.93 117.73±2.28 404.54±11.30 42.36±1.23 5.75±0.17 52.93±1.31 134.92±3.63 802.10±20.82 MD29 75.06±2.68 10.31±0.56 247.46±9.47 52.06±2.27 50.62±2.05 11.12±0.37 79.52±3.00 18.59±0.60 544.74±20.75 MD36 25.58±0.02 5.11±0.13 297.94±5.12 61.06±1.57 51.02±1.07 17.45±0.29 245.78±5.83 42.02±0.62 745.96±13.88 MD51 32.13±0.12 11.11±0.42 202.10±4.49 65.13±2.00 49.53±1.67 14.86±0.39 115.04±3.31 36.94±1.19 526.84±13.49 MD52 20.28±0.86 6.24±0.42 64.94±2.97 30.45±1.95 19.40±0.98 6.73±0.17 38.10±1.64 19.35±1.75 205.49±10.60 MD55 12.72±0.18 8.59±0.38 147.86±4.38 159.45±6.94 33.86±1.43 9.44±0.34 63.87±2.05 91.74±5.41 527.53±20.67 MD58 5.43±0.43 1.38±0.12 148.40±6.29 48.22±2.32 25.57±0.62 5.40±0.18 231.67±6.20 133.23±4.19 599.30±19.03 MD61 13.01±0.48 4.40±0.05 183.65±2.61 64.41±0.41 40.28±0.67 17.82±0.38 139.72±2.68 66.94±0.29 530.23±6.65 MD71 7.69±0.20 0.54±0.06 151.50±5.97 12.13±0.49 12.81±0.32 13.42±0.22 99.70±3.21 11.99±0.53 309.78±10.13 MD72 20.99±0.42 2.50±0.09 403.17±8.23 72.35±1.94 17.82±0.50 17.10±0.30 107.89±2.82 22.98±0.70 664.79±14.82 MD73 8.58±0.33 7.46±0.12 83.90±0.49 122.28±0.99 66.67±1.25 12.96±0.33 113.60±1.51 198.84±2.32 614.28±4.71 MD75 18.34±0.35 16.63±0.73 166.68±4.85 250.27±8.99 42.84±1.63 9.11±0.30 71.60±2.35 126.47±4.01 701.94±23.12 MD76 3.06±0.15 1.62±0.10 66.26±1.13 71.27±1.55 27.10±0.21 7.80±0.05 79.77±0.78 123.34±0.76 380.23±4.41 MD77 122.71±2.05 7.85±0.04 409.23±8.97 42.62±0.62 78.89±2.43 11.22±0.30 97.25±2.64 17.84±0.32 787.61±17.30 MD78 1.29±0.01 — 38.99±0.15 7.36±0.05 6.85±0.03 11.03±0.05 51.73±0.09 17.07±0.03 134.32±0.31 DM3 15.95±0.59 5.24±0.25 196.31±4.31 87.41±2.76 36.88±1.39 17.38±0.56 215.33±7.68 77.81±2.09 652.32±18.26 DM4 7.20±0.11 2.39±0.03 85.17±0.28 28.77±0.06 33.49±1.10 21.63±0.57 149.18±4.98 64.55±1.45 392.38±8.30 DM5 12.10±0.33 3.06±0.12 198.38±6.15 67.83±3.02 32.01±1.47 14.22±0.58 217.57±11.09 61.49±3.53 606.66±25.62 DM7 15.40±0.27 3.66±0.18 174.09±6.82 43.46±2.21 31.28±1.54 11.85±0.48 160.62±7.20 27.73±0.89 468.08±18.76 DM8 14.56±0.25 3.84±0.17 193.72±6.54 56.72±2.41 32.83±1.66 11.03±0.48 197.11±9.37 46.40±1.57 556.21±22.35 DM9 22.60±0.65 33.44±0.95 72.24±1.98 270.50±8.07 49.54±1.07 5.49±0.07 36.62±0.86 123.99±3.08 614.41±16.57 DM10 9.06±0.20 2.09±0.06 134.33±8.01 38.70±2.11 22.95±1.02 12.46±0.65 155.33±8.08 37.94±1.81 412.86±21.63 DM11 12.67±0.51 2.55±0.13 177.88±4.03 40.91±1.00 28.88±0.96 11.49±0.29 182.83±6.24 35.34±0.81 492.54±13.81 注: “-”表示未鉴定出。下同。
Notes: “—” : Not identified. The same below.表 3 杜鹃红山茶与山茶‘媚丽’杂交后代主要花青苷比例
Table 3. Proportion of main anthocyanins in hybrids of C. azalea and C. japonica 'Meili'
% 样品Samples Cy3GaX Cy3Ga Cy3GX Cy3G Cy3GaEpC Cy3GaEpCX Cy3GEpCX Cy3GEpC M — 5.27 — 40.26 8.18 — — 44.76 D 19.17 0.78 63.14 6.08 0.28 3.17 5.42 1.61 MD2 2.24 0.45 32.12 6.87 5.61 6.49 33.53 9.10 MD4 0.75 0.92 11.56 22.06 1.42 7.78 14.68 39.73 MD6 1.25 2.06 13.35 39.47 0.47 5.52 9.62 27.11 MD10 4.00 0.35 53.64 4.07 2.76 4.50 23.37 4.77 MD11 4.90 1.50 51.12 14.77 1.53 4.39 16.17 4.35 MD13 10.98 1.60 46.05 7.27 1.50 10.80 14.48 4.88 MD14 1.54 0.99 15.93 11.95 1.76 10.20 16.68 35.98 MD16 4.86 1.73 24.89 11.29 2.49 15.49 23.36 13.28 MD17 1.21 0.86 11.90 12.11 2.73 12.55 14.74 40.47 MD19 2.05 0.69 38.79 15.00 3.53 4.64 21.64 10.84 MD25 9.92 3.47 41.11 20.04 1.32 7.47 8.65 5.56 MD26 3.85 1.27 29.82 22.27 1.20 6.52 14.90 18.68 MD28 2.08 3.31 14.48 49.74 0.71 5.21 6.51 16.59 MD29 13.46 1.85 44.36 9.33 1.99 9.07 14.26 3.33 MD36 3.32 0.66 38.69 7.93 2.27 6.62 31.91 5.46 MD51 5.91 2.04 37.17 11.98 2.73 9.11 21.16 6.79 MD52 9.44 2.90 30.21 14.15 3.13 9.02 17.72 8.98 MD55 2.37 1.60 27.49 29.62 1.75 6.29 11.87 17.03 MD58 0.88 0.22 24.14 7.84 0.88 4.16 37.72 21.69 MD61 2.38 0.80 33.53 11.76 3.25 7.36 25.51 12.22 MD71 2.43 0.17 47.88 3.83 4.25 4.05 31.52 3.79 MD72 3.11 0.37 59.82 10.73 2.54 2.64 16.01 3.41 MD73 1.36 1.18 13.30 19.38 2.05 10.56 18.00 31.51 MD75 2.56 2.32 23.23 34.87 1.27 5.97 9.98 17.62 MD76 0.79 0.42 17.09 18.38 2.01 6.99 20.57 31.81 MD77 15.33 0.98 51.13 5.33 1.40 9.85 12.15 2.23 MD78 0.94 — 28.29 5.34 8.00 4.97 37.54 12.39 DM3 2.41 0.79 29.63 13.19 2.62 5.56 32.49 11.75 DM4 1.77 0.59 20.91 7.06 5.31 8.22 36.60 15.84 DM5 1.96 0.50 32.18 11.00 2.31 5.19 35.26 9.97 DM7 3.24 0.77 36.62 9.14 2.49 6.58 33.78 5.83 DM8 2.57 0.68 34.19 10.01 1.95 5.79 34.77 8.19 DM9 3.59 5.31 11.47 42.95 0.87 7.87 5.82 19.69 DM10 2.15 0.50 31.83 9.17 2.95 5.44 36.82 8.99 DM11 2.53 0.51 35.53 8.17 2.29 5.77 36.50 7.06 -
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