南瓜多糖高通量提取测定体系优化及应用

doi:10.11923/j.issn.2095-4050.cjas2025-0019

摘要/Abstract

摘要：

为简化南瓜多糖育种过程中多糖含量测定方法，开展了优化高通量热水浸提南瓜多糖苯酚-硫酸法测定体系的试验。以23S18等南瓜果肉为试材，以单因素试验确定检测波长和浓硫酸用量，并明确测定结果与纯化后测定多糖结果的相关性。结果表明，苯酚-硫酸法测定南瓜多糖波长为490 nm，反应体系中样品用量:5%苯酚:浓硫酸为2:2:7。建立高通量测定多糖回归方程y=0.0061x+0.1476(R²=0.9965)，吸光值在0.084~3.684线性关系良好。微量提取高通量测定方法与纯化后测定多糖的结果显著相关。分析测定53份南瓜多糖含量，筛选到含量>100 mg/g的高多糖亲本（23S2和23S14）和组合（23FV5和23FV26）用以育种。南瓜多糖高通量提取测定体系操作简单、线性范围广，测定结果与纯化测定多糖结果显著相关，对提高南瓜多糖育种效率行之有效。

关键词: 南瓜, 多糖, 单因素试验, 苯酚-硫酸法, 育种

Abstract:

To simplify the determination methods of pumpkin polysaccharide content in breeding process, the optimized experiments of phenol sulfuric acid method after hot water extraction of pumpkin polysaccharide were carried out. The detection wavelength and H₂SO₄ amount were determined by single factor experiment using 23S18 and other pumpkin flesh. The correlation of determination results between unpurified and purified polysaccharide content was clarified. The results showed that the wavelength of pumpkin polysaccharide determined by phenol-sulfuric acid method was 490nm, and the sample in reaction system included: 5% phenol: H₂SO₄ was 2:2:7. The regression equation was established as y=0.0061x+0.1476 (R²=0.9965) and the linear relationship was good when the absorbance was in the range of 0.084-3.684. There was a significant correlation between the unpurified and purified polysaccharide content. The polysaccharide contents of 53 pumpkin parents and combinations were determined and analyzed. Two parents (23S2 and 23S14) and two combinations (23FV26 and 23FV5) with high polysaccharide content of more than 100mg/g were selected for breeding. The efficient detection system for polysaccharides was simple to operate, wide in linear range, and the determination results are significantly related to the results of purified polysaccharide, which would improve the efficiency of pumpkin polysaccharide breeding.

Key words: pumpkin, polysaccharides, single factor experiment, phenol-sulfuric acid method, breeding

宋慧, 安学君, 黄芸萍, 张香琴, 吕燕, 钱欣欣. 南瓜多糖高通量提取测定体系优化及应用[J]. 农学学报, 2025, 15(6): 1-9.

SONG Hui, AN Xuejun, HUANG Yunping, ZHANG Xiangqin, LV Yan, QIAN Xinxin. Optimization and Application of Efficient Detection System for Extraction of Polysaccharides in Pumpkin[J]. Journal of Agriculture, 2025, 15(6): 1-9.

图/表 10

参考文献 40

[1]	刘珈亦, 苗明三. 南瓜不同入药部位的本草考证及现代研究[J]. 中南药学, 2024, 22(9):2494-2500.
[2]	LI Q, FU C, RUI Y, et al. Effects of protein-bound polysaccharide isolated from pumpkin on insulin in diabetic rat[J]. Plant foods for human nutrition, 2005,60:13-16.
[3]	SONG H, SUN Z. Hypolipidaemic and hypoglycaemic properties of pumpkin polysaccharides[J]. Biotech, 2017, 7(3):159.
[4]	HUANG L, YANG Z, YUAN J, et al. Preparation and characteristics of pumpkin polysaccharides and their effects on abnormal glucose metabolism in diabetes mice[J]. Food bioscience, 2023,54:102792.
[5]	JI X, PENG B, DING H, et al. Purification, structure and biological activity of pumpkin polysaccharides, a review[J]. Food reviews international, 2023, 39(1):307-319.
[6]	YANG Z, CHEN S, SUN W, et al. Study on the mechanisms by which pumpkin polysaccharides regulate abnormal glucose and lipid metabolism in diabetic mice under oxidative stress[J]. International journal of biological macromolecules, 2024,270:132249.
[7]	ADAMS G G, IMRAN S, WANG S, et al. The hypoglycaemic effect of pumpkins as anti-diabetic and functional medicines[J]. Food research international, 2011,44:862-867.
[8]	GAO X F, YANG Z, HUANG L, et al. Protective effects of pumpkin polysaccharide hydrolysates on oxidative stress injury and its potential mechanism-Antioxidant mechanism of pumpkin polysaccharide hydrolysates[J]. International journal of biological macromolecules, 2023,241:124423.
[9]	WU H, MA Z, ZHANG D, et al. Sequential extraction, characterization, and analysis of pumpkin polysaccharides for their hypoglycemic activities and effects on gut microbiota in mice[J]. Frontiers in nutrition, 2021,8:769181.
[10]	樊龙江, 王卫娣, 王斌, 等. 作物育种相关数据及大数据技术育种利用[J]. 浙江大学学报(农业与生命科学版), 2016, 42(1):30-39.
[11]	张雯. 南瓜皮多糖铬的制备与降糖活性评价[D]. 泰安: 山东农业大学, 2022.
[12]	EL-GENDI H, ABUSERIE M M, KAMOUN E A, et al. Statistical optimization and characterization of fucose-rich polysaccharides extracted from pumpkin (Cucurbita maxima) along with antioxidant and antiviral activities[J]. International journal of biological macromolecules, 2023,232:123372.
[13]	XU Y, WANG M, ABBAS H M K, et al. Comparing the differences in quality profiles and antioxidant activity in seven pumpkin cultivars (Cucurbita moschata and Cucurbita maxima) at harvest and during postharvest storage[J]. Food chemistry,x, 2024,22:101383.
[14]	CHEN S, B M K, CHEONG K L, LIU Y. Pumpkin polysaccharides, purification, characterization and hypoglycemic potential[J]. International journal of biological macromolecules, 2019,139:842-849.
[15]	CHEN L, LONG R, HUANG G, et al. Extraction and antioxidant activities in vivo of pumpkin polysaccharide[J]. Industrial crops and products, 2020,146:112199.
[16]	伦麒景, 罗珮妍. 苯酚-硫酸法与硫酸蒽酮法测定灵芝孢子粉中灵芝多糖含量的比较[J]. 中国食品安全, 2024, 5,89-95.
[17]	金瑾, 刘延奇, 秦令祥. 蒸汽爆破辅助提取莼菜多糖及苯酚-硫酸法测定其多糖含量的研究[J]. 粮食与油脂, 2022, 35(5):116-120.
[18]	王彦平, 娄芳慧, 陈月英, 等. 苯酚-硫酸法测定紫山药多糖含量的条件优化[J]. 食品研究与开发, 2021, 42(4):170-174.
[19]	陈立江, 王菁, 刘宇, 等. 南瓜多糖含量测定方法比较[J]. 辽宁大学学报(自然科学版), 2014, 41(1):76-81.
[20]	池源, 王丽波. 苯酚-硫酸法测定南瓜籽多糖含量的条件优化[J]. 食品与机械, 2014, 30(1):89-92.
[21]	张彦军, 李梅. 热水提取及碱法提取对南瓜多糖提取率的影响[J]. 榆林学院学报, 2020, 30(2):32-35.
[22]	GUO Y, SHANG H, ZHAO J, et al. Enzyme-assisted extraction of a cup plant (Silphium perfoliatum L.) polysaccharide and its antioxidant and hypoglycemic activities[J]. Process biochemistry, 2020,92:17-28.
[23]	孔娜, 潘少斌, 李长庚, 等. 不同提取方式对炒麦芽多糖抗氧化及降血糖活性的影响[J]. 食品研究与开发, 2024, 45(16):122-129.
[24]	张力凡, 陈洁, 许飞, 等. 古田银耳粗多糖热水提取工艺正交优化及功能活性研究[J]. 河南工业大学学报(自然科学版), 2021, 42(6):48-55.
[25]	LI F, ZHAO J, WEI Y, et al. Holistic review of polysaccharides isolated from pumpkin, preparation methods, structures and bioactivities[J]. International journal of biological macromolecules, 2021,193:541-552.
[26]	JIAO X, LI F, ZHAO J, et al. Structural diversity and physicochemical properties of polysaccharides isolated from pumpkin (Cucurbita moschata) by different methods[J]. Food research international, 2023,163:112157.
[27]	孙国强, 董金颖, 董建生. 苯酚-硫酸法测定灵芝菌丝多糖含量的研究[J]. 山西化工, 2024,4:50-52.
[28]	张倩茹, 凌蕾, 何芋岐, 等. 苯酚-硫酸法测定苗药艾纳香中多糖含量[J]. 中国民族民间医药, 2019, 28(14):30-32.
[29]	罗春萍, 陆友利, 王星星. 苯酚-硫酸法快速测定多糖方法的优化[J]. 化工管理, 2021, 3,90-91,94.
[30]	JIN H, ZHANG Y, JIANG J, et al. Studies on the extraction of pumpkin components and their biological effects on blood glucose of diabetic mice[J]. Journal of food and drug analysis, 2013,21:184-189.
[31]	王文洁, 唐炜, 俞玲娜, 等. 蒽酮-硫酸法与苯酚-硫酸法测定凉粉草多糖的比较[J]. 食品科技, 2017, 42(9):274-279.
[32]	FU C, TIAN H, CAI T, et al. Some properties of an acidic protein-bound polysaccharide from the fruit of pumpkin[J]. Food chemistry, 2007,100:944-947.
[33]	ZHAO J, ZHANG F, LIU X, et al. Isolation of a lectin binding rhamnogalacturonan-I containing pectic polysaccharide from pumpkin[J]. Carbohydrate polymers, 2017,163:330-336.
[34]	CHEN X, CHEN L, LI J, et al. Pectins from food waste, characterization and functional properties of pectic polysaccharide extracted from pumpkin (Cucurbita moschata Duch.) peels[J]. European food research and technology, 2024, 250(6):1803-1814.
[35]	WANG S, LU A, ZHANG L, et al. Extraction and purification of pumpkin polysaccharides and their hypoglycemic effect[J]. International journal of biological macromolecules, 2017,98:182-187.
[36]	ZUO S, GE H, LI Z, et al. Effect of drying methods on the structural characteristics and antioxidant stress potential of pumpkin polysaccharides[J]. ACS Food Science & Technology, 2024, 4(2):404-417.
[37]	KESH H, YADAV S. Recent advances in genetics and breeding of pumpkin (Cucurbita moschata Duch.)[J]. The journal of horticultural science and biotechnology, 2023, 98(2):141-158.
[38]	宋玉浩, 史伟玲, 张娇, 等. 马铃薯块茎淀粉组分高效检测体系的建立及应用[J]. 园艺学报, 2021, 48(3):600-608. doi: 10.16420/j.issn.0513-353x.2020-0446
[39]	宋慧, 安学君, 黄芸萍, 等. 南瓜淀粉组分高效检测体系建立及应用[J]. 中国农学通报, 2024, 40(5):135-144. doi: 10.11924/j.issn.1000-6850.casb2023-0143
[40]	范文秀, 李新峥. 南瓜生长过程中多糖含量的测定[J]. 光谱试验室, 2005, 22(4):834-836.

编号	类型	多糖含量	编号	类型	多糖含量
23S1	板栗	87.22±0.76bcde	23S10	长条橄榄	60.30±4.18fg
23S2	板栗	108.87±2.19ab	23S11	长条橄榄	89.42±4.69bcd
23S3	板栗	51.55±2.67g	23S12	长条橄榄	80.37±6.66cdef
23S4	板栗	61.85±4.62efg	23S13	小甘龙	87.68±1.06bcde
23S5	板栗	88.54±15.50bcde	23S14	大青圆	115.75±1.39a
23S6	板栗	78.46±7.67cdef	23S15	红皮	55.87±2.79fg
23S7	贝贝	63.91±5.47defg	23S16	红皮	68.05±7.78cdefg
23S8	贝贝	49.61±7.76g	23S17	红皮	68.51±3.09cdefg
23S9	贝贝	92.06±3.24bc	23S18	中印红皮	79.64±7.38cdef

编号	类型	多糖含量	编号	类型	多糖含量
23S1	板栗	87.22±0.76bcde	23S10	长条橄榄	60.30±4.18fg
23S2	板栗	108.87±2.19ab	23S11	长条橄榄	89.42±4.69bcd
23S3	板栗	51.55±2.67g	23S12	长条橄榄	80.37±6.66cdef
23S4	板栗	61.85±4.62efg	23S13	小甘龙	87.68±1.06bcde
23S5	板栗	88.54±15.50bcde	23S14	大青圆	115.75±1.39a
23S6	板栗	78.46±7.67cdef	23S15	红皮	55.87±2.79fg
23S7	贝贝	63.91±5.47defg	23S16	红皮	68.05±7.78cdefg
23S8	贝贝	49.61±7.76g	23S17	红皮	68.51±3.09cdefg
23S9	贝贝	92.06±3.24bc	23S18	中印红皮	79.64±7.38cdef

编号	类型	多糖含量	编号	类型	多糖含量
23FV1	板栗	59.52±0.41defg	23FV19	红皮	86.18±5.90abc
23FV2	板栗	81.50±7.50bcde	23FV20	红皮	62.22±3.15cdefg
23FV3	板栗	45.43±0.78ghi	23FV21	红皮	64.36±5.56bcdefg
23FV4	板栗	76.29±0.73bcdef	23FV22	红皮	63.18±1.94cdefg
23FV5	板栗	102.94±5.01a	23FV23	红皮	52.98±1.05fghi
23FV6	板栗	64.54±1.70bcdefg	23FV24	红皮	62.94±1.22cdefg
23FV7	板栗	83.66±1.33abcd	23FV25	红皮	63.10±0.83cdefg
23FV8	板栗	79.88±1.51bcde	23FV26	红皮	102.61±5.91a
23FV9	板栗	75.89±2.27bcdef	23FV27	其他	47.62±4.29ghi
23FV10	贝贝	68.74±2.33bcdefg	23FV28	红皮	57.04±3.70efgh
23FV11	贝贝	68.78±7.00bcdefg	23FV29	其他	35.09±3.51i
23FV12	贝贝	88.40±1.77ab	23FV30	其他	39.95±2.24hj
23FV13	贝贝	80.58±2.16bcde	23FV31	其他	68.61±1.34bcdefg
23FV14	贝贝	83.64±2.10abcd	23FV32	其他	63.05±4.58cdefg
23FV15	贝贝	80.14±0.70bcde	23FV33	其他	66.23±1.63bcdefg
23FV16	橄榄	76.90±7.38bcde	23FV34	锦栗南瓜	73.18±2.43bcdef
23FV17	橄榄	79.01±3.31bcde	23FV35	贝栗4号	78.69±0.29bcde
23FV18	橄榄	77.29±3.50bcde

编号	类型	多糖含量	编号	类型	多糖含量
23FV1	板栗	59.52±0.41defg	23FV19	红皮	86.18±5.90abc
23FV2	板栗	81.50±7.50bcde	23FV20	红皮	62.22±3.15cdefg
23FV3	板栗	45.43±0.78ghi	23FV21	红皮	64.36±5.56bcdefg
23FV4	板栗	76.29±0.73bcdef	23FV22	红皮	63.18±1.94cdefg
23FV5	板栗	102.94±5.01a	23FV23	红皮	52.98±1.05fghi
23FV6	板栗	64.54±1.70bcdefg	23FV24	红皮	62.94±1.22cdefg
23FV7	板栗	83.66±1.33abcd	23FV25	红皮	63.10±0.83cdefg
23FV8	板栗	79.88±1.51bcde	23FV26	红皮	102.61±5.91a
23FV9	板栗	75.89±2.27bcdef	23FV27	其他	47.62±4.29ghi
23FV10	贝贝	68.74±2.33bcdefg	23FV28	红皮	57.04±3.70efgh
23FV11	贝贝	68.78±7.00bcdefg	23FV29	其他	35.09±3.51i
23FV12	贝贝	88.40±1.77ab	23FV30	其他	39.95±2.24hj
23FV13	贝贝	80.58±2.16bcde	23FV31	其他	68.61±1.34bcdefg
23FV14	贝贝	83.64±2.10abcd	23FV32	其他	63.05±4.58cdefg
23FV15	贝贝	80.14±0.70bcde	23FV33	其他	66.23±1.63bcdefg
23FV16	橄榄	76.90±7.38bcde	23FV34	锦栗南瓜	73.18±2.43bcdef
23FV17	橄榄	79.01±3.31bcde	23FV35	贝栗4号	78.69±0.29bcde
23FV18	橄榄	77.29±3.50bcde

仪器	用量	23FV5	23FV12	23FV19	24S8	24S17
分光光度计	1 mL	0.078±0.000e	0.060±0.001e	0.046±0.002e	0.036±0.001e	0.038±0.001e
	3 mL	1.218±0.000d	0.991±0.004d	1.142±0.005d	0.165±0.001d	0.305±0.003d
	5 mL	2.578±0.035c	2.241±0.011c	2.459±0.013c	0.852±0.001c	1.486±0.012c
	7 mL	2.699±0.043b	2.701±0.010a	2.7±0.003a	1.192±0.001a	2.017±0.006a
	9 mL	2.673±0.019b	2.371±0.006b	2.546±0.017b	0.98±0.002b	1.637±0.005b
多功能微孔板检测仪	100 μL	0.063±0.001e	0.06±0.001e	0.06±0.002e	0.05±0.001e	0.08±0.001e
	300 μL	0.478±0.001d	0.331±0.00d	0.378±0.002d	0.123±0.001d	0.196±0.001d
	500 μL	1.651±0.009c	1.291±0.01c	1.528±0.006c	0.492±0.001c	0.845±0.010c
	700 μL	2.847±0.018a	2.203±0.004a	2.373±0.01a	0.737±0.002a	1.22±0.008a
	900 μL	2.349±0.008b	1.534±0.012b	1.802±0.003b	0.602±0.006b	0.99±0.002b