Analysis and Differences Research of Multiple Types of Chemical Components in Goat Horn from Different Producing Areas
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摘要:
目的 分析不同产地山羊角中多类型资源性化学成分差异, 为其资源利用提供依据。 方法 分别采用超高效液相色谱-串联质谱法(UHPLC-MS/MS)、电感耦合等离子体质谱法(ICP-MS)、分光光度法对32批不同产地山羊角中核苷类及氨基酸类成分、水溶性蛋白、含巯基类化合物、无机元素类成分含量进行分析评价; 利用主成分分析法(PCA)和偏最小二乘判别法(PLS-DA)对山羊角产地差异进行综合评价。 结果 山羊角中检测出9种核苷类成分、18种游离氨基酸类成分, 核苷类成分平均总量为20.88 μg · g-1, 氨基酸类成分平均总量为642.41 μg · g-1; 山羊角中水溶性蛋白、总巯基和游离巯基类成分的平均含量分别为51.07 mg · g-1，22.48 μmol · g-1，9.97 μmol · g-1；山羊角中检测出常量元素5种，必须微量元素9种，重金属元素4种, 平均总量分别为45.09 mg · g-1，295.32 μg · g-1，7.74 μg · g-1; PCA和PLS-DA结果表明, 基于48种化学成分分析结果, 不同产地山羊角存在显著差异, 西南地区产山羊角与华东、华北地区产山羊角差异较大, 华东地区产山羊角综合得分较高。 结论 不同产地山羊角所含资源性成分种类基本一致, 但各类型成分含量差异较大, 华东地区产山羊角资源性成分含量相比其他产区较高, 研究结果可为山羊角药用产地选择及综合利用提供一定参考。 Abstract:OBJECTIVE To investigate the multiple types of chemical constituents in goat horns from different areas, then to provide data support for its resource utilization. METHODS The contents of nucleosides, amino acids, inorganic elements, water-soluble proteins, total thiols and native thiols in 32 batches of goat horn from different producing areas were analyzed and evaluated by ultra performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), inductively coupled plasma mass spectrometry (ICP-MS) and spectrophotometry, respectively. Principal component analysis (PCA) and partial least squares discriminant (PLS-DA) of 48 chemical components were applied to comprehensively evaluate the differences in producing areas of goat horns. RESULTS 9 nucleosides and 18 free amino acids were detected in goat horns, and the average total nucleoside and amino acid contents were 20.88 μg · g-1 and 642.41 μg · g-1, respectively. The average contents of water-soluble protein, total sulfhydryl group and free sulfhydryl group in goat horn were 51.07 mg · g-1, 22.48 μmol · g-1 and 9.97 μmol · g-1, respectively. 5 major elements, 9 essential trace elements and 4 heavy metal elements were detected, with an average amounts of 45.09 mg · g-1, 295.32 μg · g-1 and 7.74 μg · g-1, respectively. The results of PCA and PLS-DA showed that based on the analysis results of 48 chemical components, there were significant differences among the goat horns from different regions of China. There was a significant difference between the goat horns from southwestern part of China and the ones from eastern and northern parts of China. The comprehensive score of goat horn in east China was higher. CONCLUSION The resource-based components contained in goat horn from different regions show little differences, but the contents of these components exhibit significant differences. The contents of resource-based components in east China were higher than those in other regions. The results of this study can provide some reference for the selection and comprehensive utilization of goat horns from different regions. -
Key words:
- goat horn /
- resource chemical composition /
- production area difference /
- nucleoside /
- amino acid /
- thiol
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图 1 27种对照品及IS的MRM图
注：1.胸腺嘧啶; 2.胸苷; 3.2′-脱氧腺苷; 4.次黄嘌呤; 5.2′-脱氧肌苷; 6.肌苷; 7.胞嘧啶; 8.胞苷; 9.L-亮氨酸; 10.L-苯丙氨酸; 11.L- 色氨酸; 12.L-异亮氨酸; 13.γ-氨基丁酸; 14.鸟苷; 15.甲硫氨酸; 16.L-缬氨酸; 17.L-酪氨酸; 18.L-苏氨酸; 19.L-谷氨酸; 20.谷氨酰胺; 21.L-丝氨酸; 22.天冬酰胺; 23.L-天冬氨酸; 24.L-瓜氨酸; 25.L-组氨酸; 26.L-精氨酸; 27.L-赖氨酸; IS.2-氯苯丙氨酸
Figure 1. Multi-reaction monitoring chromatograms of 27 reference substances and IS
图 2 不同产地山羊角中氨基酸类成分结果分析(n=3)
Figure 2. Analysis of amino acids in goat horn from different producing areas (n=3)
图 3 不同产地山羊角中核苷类成分结果分析(n=3)
Figure 3. Analysis of nucleoside compositions in goat horn from different producing areas (n=3)
图 4 不同产地山羊角中含巯基化合物含量测定结果分析(n=3)
Figure 4. Analysis of total sulfhydryl and free sulfhydryl in goat horn from different producing areas (n=3)
图 5 不同产地山羊角中常量元素含量结果分析(n=3)
Figure 5. Analysis of major elements in goat horn from different producing areas (n=3)
图 6 不同产地山羊角中必须微量元素含量结果分析(n=3)
Figure 6. Analysis of essential trace elements in goat horn from different producing areas (n=3)
图 7 不同产地山羊角中重金属元素含量结果分析(n=3)
Figure 7. Analysis of heavy metal contents in goat horn from different producing areas (n=3)
图 8 基于48种成分含量的不同产地山羊角PCA得分图(A)、PLS-DA得分图(B)、置换检验模型验证图(C)和VIP图(D)
Figure 8. Multivariate statistical analysis of goat horn samples from different producing areas based on 48 kinds of chemical components: (A)PCA score plot; (B)PLS-DA score plot; (C)200 times permutation test model validation plot and (D)VIP plot
表 1 不同产地山羊角样品信息
Table 1. Sample information of goat horn from different origins
编号 产地 地区 编号 产地 地区 S1 云南昆明 西南 S17 山东临沂 华东 S2 云南昆明禄劝 西南 S18 山东济宁 华东 S3 云南红河州 西南 S19 山东菏泽 华东 S4 云南罗平 西南 S20 山东沂源 华东 S5 四川凉山 西南 S21 河北唐山 华北 S6 广西南宁 西南 S22 河北武安 华北 S7 江苏宿迁 华东 S23 河北武安 华北 S8 江苏宿迁 华东 S24 内蒙古赤峰 华北 S9 江苏淮安 华东 S25 内蒙古赤峰 华北 S10 江苏淮安 华东 S26 内蒙古乌审旗 华北 S11 江苏南京 华东 S27 内蒙古苏尼特 华北 S12 江苏南京 华东 S28 内蒙古苏尼特 华北 S13 江苏南通 华东 S29 山西太原 华北 S14 江苏镇江 华东 S30 山西朔州 华北 S15 江苏盐城 华东 S31 山西高平 华北 S16 山东临沂 华东 S32 山西吕梁 华北 
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表 2 27种核苷类及氨基酸类成分和IS主要质谱参数
Table 2. Mass spectrometry information of 27 nucleoside, amino acid compositions and IS
化合物 tR/min 母离子m/z 子离子m/z 锥孔电压/V 碰撞电压/V 胸腺嘧啶 1.12 126.95 56.24*, 53.90 32 14 胸苷 1.17 243.07 126.95*, 148.94 10 10 2′-脱氧腺苷 1.57 252.08 135.98*, 118.92 18 14 次黄嘌呤 1.85 136.95 109.95*, 130.96 38 20 2′-脱氧肌苷 2.06 275.07 158.96*, 136.94 34 14 肌苷 2.82 269.13 136.94*, 158.96 12 8 胞嘧啶 2.82 112.00 51.97*, 68.34 20 22 胞苷 4.02 266.06 133.90*, 111.94 36 16 L-亮氨酸 4.38 132.08 86.01*, 43.74 14 6 L-苯丙氨酸 4.43 165.97 102.98*, 76.90 16 26 L-色氨酸 4.75 205.00 145.95*, 117.94 14 18 L-异亮氨酸 4.79 132.08 68.99*, 86.01 16 10 γ-氨基丁酸 4.80 103.90 42.88*, 68.69 14 12 鸟苷 4.82 284.08 151.92*, 173.94 14 12 甲硫氨酸 5.45 149.99 60.92*, 55.97 14 20 L-缬氨酸 5.87 118.13 72.00*, 55.10 13 8 L-酪氨酸 6.43 182.10 136.10*, 91.00 19 16 L-苏氨酸 8.44 119.96 73.90*, 55.97 16 10 L-谷氨酸 9.39 147.97 83.93*, 101.97 16 12 谷氨酰胺 9.84 147.05 83.99*, 55.97 14 14 L-丝氨酸 9.95 103.87 73.91* 24 10 天冬酰胺 10.26 133.02 73.94*, 86.98 16 14 L-天冬氨酸 11.21 134.00 73.90*, 87.91 14 12 L-瓜氨酸 11.25 176.03 69.93*, 113.00 16 20 L-组氨酸 16.07 155.99 110.00*, 92.93 18 24 L-精氨酸 16.15 175.11 69.96*, 115.98 22 18 L-赖氨酸 16.25 147.03 83.94*, 55.98 16 14 IS 3.82 200.10 154.10*, 118.10 10 16 注: *为定量离子。
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表 3 27种对照品线性关系考察
Table 3. Results of linear relationships of 27 reference substances
化合物 线性方程 r 线性范围/(μg·mL-1) LOD/(ng·mL-1) LOQ/(ng·mL-1) 胸苷 Y=1.304 7X-0.032 3 1.000 0 0.018 6~38.00 3.3 11.0 胸腺嘧啶 Y=0.163 3X-0.024 7 0.999 8 0.123 7~31.67 35.1 116.8 2′-脱氧腺苷 Y=31.618 0X+0.017 7 0.999 8 0.004 2~4.31 0.1 0.5 次黄嘌呤 Y=5.193 0X+0.298 7 0.999 7 0.033 1~8.47 1.5 4.9 2′-脱氧肌苷 Y=2.071 0X-0.013 2 0.999 7 0.015 8~8.08 3.0 10.0 肌苷 Y=1.872 2X-0.008 6 0.999 9 0.007 1~3.64 1.4 4.8 胞嘧啶 Y=1.011 9X-0.045 4 0.999 5 0.064 3~16.47 9.9 33.3 胞苷 Y=7.846 0X-0.111 0 0.999 3 0.008 4~17.10 2.5 8.2 L-亮氨酸 Y=0.958 2X-0.045 6 0.999 6 0.121 2~62.07 22.9 76.3 L-苯丙氨酸 Y=1.812 4X+0.434 3 0.999 7 0.062 2~63.65 3.3 10.9 L-色氨酸 Y=1.631 0X+0.165 0 0.999 9 0.015 6~31.98 3.1 10.3 L-异亮氨酸 Y=0.091 2X+0.071 3 0.999 2 0.569 0~36.42 155.7 519.1 γ-氨基丁酸 Y=2.369 3X+0.014 5 0.999 5 0.036 4~18.68 9.2 30.6 鸟苷 Y=2.040 8X+0.031 1 0.999 6 0.011 1~22.64 2.5 8.5 甲硫氨酸 Y=3.052 6X+0.801 1 0.998 9 0.036 1~73.17 8.3 27.6 L-缬氨酸 Y=3.059 4X+0.940 1 0.999 6 0.076 7~39.27 17.3 57.5 L-酪氨酸 Y=1.325 1X+0.335 4 0.999 8 0.056 6~57.95 8.6 28.8 L-苏氨酸 Y=1.591 0X+0.203 9 0.999 7 0.039 0~39.90 10.1 33.7 L-谷氨酸 Y=1.570 6X+0.465 6 0.999 5 0.062 4~31.98 11.0 36.7 谷氨酰胺 Y=12.022 0X-0.045 0 0.999 9 0.023 5~12.03 6.1 20.4 L-丝氨酸 Y=0.042 4X+0.122 8 0.999 6 0.524 5~67.13 126.6 422.0 天冬酰胺 Y=2.203 7X+0.077 6 0.999 7 0.008 8~1.13 2.1 6.8 L-天冬氨酸 Y=0.455 7X+0.063 2 0.999 8 0.006 6~33.88 13.3 44.9 L-瓜氨酸 Y=5.340 5X+0.199 3 0.999 5 0.004 2~35.15 1.2 3.9 L-组氨酸 Y=2.295 2X-1.042 8 0.998 4 0.040 8~41.80 5.5 18.2 L-精氨酸 Y=0.987 5X-0.699 7 0.999 5 0.098 3~50.35 23.9 79.6 L-赖氨酸 Y=0.249 3X+0.018 4 0.999 6 0.065 6~33.57 12.0 40.0
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表 4 不同产地山羊角水溶性蛋白含量(n=3)
Table 4. Content of water-soluble protein in goat horn from different producing areas (n=3)
编号 含量/(mg·g-1) 编号 含量/(mg·g-1) S1 35.92±0.47 S17 77.98±0.27 S2 28.95±0.99 S18 76.24±0.77 S3 19.45±0.87 S19 52.95±1.17 S4 18.47±1.13 S20 53.85±1.19 S5 31.29±1.16 S21 81.97±2.99 S6 35.11±1.13 S22 82.94±2.91 S7 71.42±1.20 S23 44.69±4.20 S8 70.22±0.62 S24 40.86±1.11 S9 67.19±0.78 S25 48.62±2.00 S10 66.81±1.16 S26 48.62±2.17 S11 34.95±0.36 S27 42.98±1.94 S12 35.62±0.66 S28 43.00±2.14 S13 41.10±0.50 S29 41.91±1.86 S14 39.71±0.93 S30 47.74±3.18 S15 68.63±2.31 S31 59.22±0.74 S16 66.32±0.95 S32 59.54±0.85
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表 5 含巯基类化合物含量测定方法考察结果(n=6)
Table 5. Methodological study of sulfhydryl compounds (n=6)
成分 精密度RSD/% 重复性RSD/% 稳定性RSD/% 加样回收率/% 平均值 RSD 游离巯基 3.36 4.38 1.96 100.43 3.97 总巯基 3.25 5.54 1.72 99.67 3.08
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表 6 18种元素线性关系和方法学考察
Table 6. Results of linear relationships and methodological study of 18 investigated elements
元素 回归方程 r 线性范围/(ng·mL-1) 精密度RSD/% 重复性RSD/% 稳定性RSD/% Mg Y=0.038 3X-0.117 7 0.9996 10~1 000 2.09 1.16 1.66 Ca Y=0.002 1X-0.182 6 0.999 6 10~1 000 0.92 1.53 0.97 P Y=0.001 8X+0.001 0 0.999 9 10~1 000 1.52 1.56 1.87 Na Y=0.105 3X-0.037 5 0.999 8 10~1 000 1.06 0.58 1.07 K Y=0.073 3X-0.222 1 0.999 9 10~1 000 0.59 1.22 6.37 Cr Y=0.056 2X-0.131 6 0.999 7 0~800 0.75 0.44 1.22 Mn Y=0.082 0X-0.130 8 0.999 6 0~800 1.07 0.86 1.00 Fe Y=0.001 3X+0.003 6 0.999 9 0~800 1.38 1.25 2.09 Ni Y=0.009 3X+0.007 5 0.999 9 0~800 0.39 1.48 2.77 Zn Y=0.005 1X-0.001 3 0.999 9 0~400 0.25 0.92 1.42 Sn Y=0.020 8X-0.151 4 0.998 3 0~400 0.64 0.76 2.93 Cu Y=0.024 3X-0.137 4 0.998 9 0~400 0.65 1.08 1.43 Cd Y=0.004 7X-0.003 2 0.999 8 0~200 0.76 0.61 0.68 Pb Y=0.017 4X-0.077 7 0.999 2 0~200 1.49 0.69 1.59 As Y=0.008 0X+0.004 7 0.999 9 0~200 0.88 0.83 3.36 Si Y=0.022 7X-0.089 3 0.999 5 0~200 0.38 1.18 1.89 Co Y=0.056 9X-0.158 6 0.999 7 0~200 0.39 0.96 0.43 Mo Y=0.015 2X-0.006 7 0.999 9 0~200 0.70 0.90 3.13
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表 7 不同产地山羊角各类成分含量综合评分结果
Table 7. Comprehensive score of various components of goat-horn from different producing areas
样品编号 地区 F1 F2 F3 F4 F5 F6 F S16 华东 6.871 3.419 -1.343 1.548 1.152 2.332 3.194 S15 华东 6.041 1.870 -1.730 1.603 0.910 1.365 2.606 S8 华东 4.988 3.671 0.648 -0.342 0.541 1.058 2.446 S17 华东 5.075 0.173 -1.684 1.150 -1.964 -2.206 1.667 S7 华东 3.462 1.850 0.611 -0.235 0.603 0.668 1.642 S18 华东 5.073 -0.800 -2.059 1.116 -1.618 -2.421 1.528 S11 华东 2.992 2.183 1.683 -4.235 -0.037 1.478 1.319 S12 华东 2.468 2.674 1.429 -3.345 0.546 1.003 1.222 S25 华北 0.495 -0.056 6.181 3.507 -2.317 0.796 0.903 S14 华东 3.227 -0.252 -2.977 0.524 -1.657 -2.132 0.771 S13 华东 3.162 -0.098 -2.489 -0.161 -2.148 -2.341 0.720 S23 华北 0.420 1.358 1.337 1.151 1.964 -2.423 0.499 样品编号 地区 F1 F2 F3 F4 F5 F6 F S26 华北 -0.107 -1.199 5.719 3.310 -1.879 0.287 0.485 S19 华东 1.999 -0.455 -1.597 -0.871 -3.805 2.049 0.402 S21 华北 0.586 0.827 0.146 -0.084 2.308 -1.774 0.361 S24 华北 -0.156 1.312 1.576 0.827 1.357 -2.488 0.235 S22 华北 0.304 0.410 0.443 -0.461 2.400 -2.039 0.198 S31 华北 0.951 -3.142 -0.749 0.600 1.748 1.280 0.150 S27 华北 -0.048 -2.013 -2.148 2.502 1.877 2.632 -0.018 S10 华东 0.418 -1.701 2.796 -3.331 -0.058 -0.570 -0.050 S9 华东 0.049 -1.514 1.811 -3.230 0.685 -0.961 -0.230 S28 华北 -0.617 -2.925 -1.332 1.459 2.001 2.293 -0.351 S32 华北 -0.196 -4.347 -0.164 -0.171 1.712 0.745 -0.458 S20 华东 -0.450 -1.207 -0.916 -2.220 -3.112 2.159 -0.610 S29 华北 -0.339 -4.544 -0.146 -0.699 0.344 -0.338 -0.705 S30 华北 -0.563 -5.141 0.215 -1.017 0.383 -0.724 -0.866 S1 西南 -6.959 2.781 -0.693 -0.548 0.125 0.312 -2.429 S3 西南 -7.642 2.336 -1.135 0.887 0.314 -0.172 -2.694 S2 西南 -7.645 2.504 -0.593 -0.525 0.161 -0.179 -2.736 S4 西南 -7.957 1.713 -0.772 2.014 -0.581 0.298 -2.797 S6 西南 -7.939 0.404 -0.994 -0.334 -1.105 -0.069 -3.174 S5 西南 -7.962 -0.092 -1.073 -0.392 -0.848 0.082 -3.228
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