Research Progress on Structure Characteristics, Biological Activity, Structure-Activity Relationship and Product Development of Dendrobium Polysaccharides
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摘要: 石斛是我国传统名贵的滋补药材,具有益胃生津、滋阴清热的功效,多糖是其主要的活性成分之一。通过查阅国内外近10年相关文献,系统总结常见石斛多糖的结构及生物活性。结果发现石斛多糖具有抗氧化、抗肿瘤、抗炎、免疫调节、调节肠道菌群、降血糖、抗白内障等多种功效,且石斛多糖的生物活性与结构密切相关。研究认为富含甘露糖和葡萄糖的石斛多糖显示出更强的活性,乙酰基含量越高,其免疫活性越强;糖醛酸含量越高,其抗氧化能力越强。主链上的(1→4)-α-D-Glcp和(1→4)-α-D-Manp可能是与石斛多糖活性相关的关键结构,化学修饰如硫酸化、硒化和乙酰化能够增强石斛多糖的活性,而羧甲基化则降低了其活性。本文对近年来国内外关于石斛多糖结构特征、生物活性、产品开发的研究进展进行总结,并阐明其构效关系,旨在为促进石斛多糖的深度开发和应用提供重要的参考依据。Abstract: Dendrobium is a traditional, valuable, and nourishing medicinal herb in China. It benefits the stomach, promotes fluid production, nourishes Yin, and clears heat. Polysaccharides are among the main active ingredients of Dendrobium. By reviewing literature over the past decade, the common structures and biological activities of Dendrobium polysaccharides are systematically summarized. The results illustrate that Dendrobium polysaccharides possess various effects, including antioxidant, antitumor, anti-inflammatory, and immunoregulatory effects, gut microbiota modulation, blood sugar reduction, and anticataract properties. The chemical structures of these polysaccharides are closely related to their biological activities. Previous studies suggest that polysaccharides rich in mannose and glucose exhibit stronger activity, whereas higher acetylation levels enhance immune activity. Higher uronic acid content corresponds to greater antioxidant ability, which may be due to the presence of (1→4)-α-D-Glcp and (1→4)-α-D-Manp on the main chain. Additionally, chemical modifications such as sulfation, selenization, and acetylation can enhance activity, whereas carboxymethylation decreases it. This article summarizes the research progress in recent years on the structure, biological activity, and product development of Dendrobium polysaccharides both home and abroad, elucidating the structure-activity relationship, aiming to provide important references for their in-depth development and application.
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石斛,又名林兰、禁生,是兰科(Orchidaceae)石斛属(Dendrobium)多种植物的茎,主要分布在秦岭、淮河以南等地区[1]。据《中国植物志》记载,我国有74种和2变种石斛属植物,其中30余种有可食用[1]记录[2]。2020年版《中国药典》收录的石斛包括金钗石斛、霍山石斛、鼓槌石斛、流苏石斛以及铁皮石斛的栽培品种及其同属植物中与之相似的新鲜或干燥茎[3]。石斛被誉为“植物黄金”,拥有极高的食用和药用价值。其功效最早记载于《神农本草经》,称其“能补五脏虚劳,羸瘦,强阴,久服厚肠胃,轻身延年”[4]。《本草纲目》更将其推崇为“补五脏虚劳”的上医良药[5]。石斛的化学成分十分丰富,包括多糖、香豆素、生物碱、倍半萜类化合物等[6]。其中,多糖作为石斛的主要活性成分(约占鲜重的9.90%~48.60%),具有抗肿瘤、抗氧化、抗炎、免疫调节、肠道菌群调节、降血糖等多种作用,在医药、保健食品及化妆品等领域应用广泛[7−8]。石斛多糖的生物活性与其结构特征密切相关,研究石斛多糖的结构特性是探索生物活性的基础。本文对国内外近年来有关石斛多糖的研究报道进行了综合整理,总结了常见石斛多糖的结构特征和生物活性,旨在揭示石斛多糖与生物活性之间的关联,以进一步推动石斛多糖的开发与应用。
1. 石斛多糖的结构特征
石斛多糖属植物多糖,由至少10个单糖通过糖苷键连接而成,相对分子质量较大,可高达数万甚至几百万。石斛多糖的结构复杂,易受产地、品种、提取方法及纯化工艺等多种因素影响[9]。目前,对石斛多糖结构的研究主要集中在一级结构方面,包括分子量测定、单糖组成及比例、单糖的连接方式以及糖苷键类型等。
1.1 石斛多糖的单糖组成
明确石斛多糖的单糖组成,是其质量控制过程中必不可少的环节。一般采用酸水解-衍生化,高效液相色谱法(HPLC)或气相色谱法测定石斛多糖的单糖组成。由表1和表2可知,石斛多糖是一种杂多糖,主要由六种单糖(甘露糖、葡萄糖、半乳糖、鼠李糖、阿拉伯糖和木糖)和两种糖醛酸(半乳糖醛酸和葡萄糖醛酸)构成,其中葡萄糖和甘露糖的含量相对较高,糖醛酸只存在于极少数的石斛多糖中。需要注意的是,石斛多糖的单糖组成和含量受品种、产地、生长年限、采收期、提取和纯化方法等多种因素影响。
表 1 提取和纯化方法对石斛多糖单糖组成及分子量的影响Table 1. Effects of extraction and purification methods on the monosaccharide composition and molecular weight of Dendrobium polysaccharides多糖来源 提取纯化方法/条件 单糖组成和摩尔比 分子量(Da) 文献 铁皮石斛多糖 热水浸提 Man:Glc=1.06:1.0 5.4×107 [17] 超高压提取 Man:Glc=1.71:1.0 3.2×105 铁皮石斛多糖 热水浸提 NA 2.01×105 [18] 酶法提取 NA 1.67×105 闪式提取 NA 1.79×105 超声波提取 NA 1.96×105 冻融提取 NA 2.28×105 霍山石斛总多糖ST 40%终浓度的乙醇沉淀 NA 4.79×105 [19] 50%终浓度的乙醇沉淀 NA 3.16×105 60%终浓度的乙醇沉淀 NA 3.07×105 80%终浓度的乙醇沉淀 NA 8.59×104
霍山石斛多糖HPS40 ℃水提取 Man:Glc:Gal:Ara:Rha:Xyl=2.24:50.22:35.88:8.79:1.66:1.21 9.42×106 [20] 60 ℃水提取 Man:Glc:Gal:Ara:Rha:Xyl=2.11:58.44:29.43:8.05:1.11:0.86 9.27×106 80 ℃水提取 Man:Glc:Gal:Ara:Rha:Xyl=1.78:65.36:24.37:6.87:0.85:0.75 9.20×106 100 ℃水提取 Man:Glc:Gal:Ara:Rha:Xyl=1.19:75.34:16.44:5.26:1.24:0.54 9.12×106 铁皮石斛多糖DOP 超滤膜分级分离 Man:Glc=1.38:1.0 4.92×105 [21] 3500 Da透析袋透析 Man:Glc=4.06:1.0 3.33×104 铁皮石斛多糖 超声提取/14 ℃,2 h Man:Glc:Gal:Ara:Rha:Xyl=79.17:16.33:0.90:0.86:0.45:1.21 2.95×105 [22] 超声提取/20 ℃,100 W,3 min Man:Glc:Gal:Ara:Rha:Xyl=75.58:21.02:0.35:0.35:0.20:1.14 3.28×105 超声提取/20 ℃,300 W,3 min Man:Glc:Gal:Ara:Rha:Xyl=76.63:18.81:0.69:0.42:0.15:1.05 3.31×105 超声提取/20 ℃,500 W,3 min Man:Glc:Gal:Ara:Rha:Xyl=77.69:17.82:0.95:0.48:0.27:0.96 3.68×105 超声提取/100 ℃,2 h Man:Glc:Gal:Ara:Rha:Xyl=77.39:17.82:0.85:0.34:0.34:0.64 3.13×105 注:Man-甘露糖;Glc-葡萄糖;Gal-半乳糖;Rha-鼠李糖;Ara-阿拉伯糖;Xyl-木糖;NA-文献中未提及。 表 2 石斛多糖的结构特征信息Table 2. Structure information of Dendrobium polysaccharides来源 多糖名称 分子量(Da) 单糖组成和摩尔比 结构特征 生物活性 文献 霍山石斛 DHP1A 6.7×103 Man:Glc:Gal=2.5:16.0:1.0 主链(1→4)-α-D-Glcp、(1→6)-β-D-Glcp、(1→4)-β-D-Manp,末端β-D-Glcp(1→ 抗氧化 [31] DHP-4A 2.32×105 Glc:Ara:Man:Rha=13.8:3.0:6.1:2.1 主链(1→6)-β-D-Glcp、(1→6)-β-Manp,侧链(1→2)-α-L-Rhap、(1→4)-β-D-Manp、(1→3)-α-L-Araf,末端α-L-Rhap(1→、α-L-Araf(1→ 免疫调节 [32] DHPs-1 5.0×104 Glc:Man:Gal:GalA:Rha =
65.04:14.23:8.17:6.41:2.34主链(1→4)-Manp、(1→4)-Galp、(1→4)-Glcp、(1→3,4)-Galp、(1→4,6)-Galp、(1→3,4,6)-Galp,侧链(1→4)-Manp、(1→4)-Galp、(1→3,4)-Galp、(1→3,4,6)-Galp,末端Galp-(1→ 免疫调节 [33] TCDHPA4 8.0×105 Rha:Ara:Man:Glc:Gal=
1.28:1.67:4.71:10.43:1.42(1→6)-β-Galp、(1→4/6)-β-Glcp,侧链(1→2/4)-α-Rhap、(1→3)-β-Galp、(1→2/6)-β-D-Manp、(1→2)-α-Araf,末端β-Glcp(1→、α-Araf(1→ NA [34] DHPW1 2.3×103 Man:Glc:Gal=37.8:21.9:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp 促进成骨 [35] DHPD2 8.09×106 Gal:Glc:Ara=0.9:0.7:0.2 主链(1→4/6)-β-D-Galp、(1→5)-α-L-Araf、(1→6)-α-D-Glcp、(1→3,6)-β-D-Galp、(1→6)-α-D-Galp,侧链(1→3)-β-D-Manp,末端α-D-Xylp(1→、β-D-Manp(1→ 免疫调节 [36] DHPD1 3.2×103 Gal:Glc:Ara=0.021:1.023:0.023 由(1→4/6/4,6)-Glcp、(1→6)-Galp、(1→5)-Araf糖残基组成,末端Glcp(1→ 抗糖基化 [37] DHP-W2 7.3×104 Glc:Xyl:Gal=1.0:1.0:0.5 主链(1→4/6)-β-D-Glcp,侧链(1→6)-α-D-Xylp,末端α-D-Galp(1→、α-D-Xylp(1→ 抗糖基化 [38] GXG 1.78×106 Xyl :Gal :Glc=2.13:1.0:2.85 由(1→4/2,4)-Xylp、(1→4)-Galp、(1→4/6/3,6/4,6)-Glcp糖残基组成 促进胃肠消化 [39] cDHPS 2.59×105 Man:Glc=3.04:1.0 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-3-O-acetyl-β-D-Manp 抗肿瘤 [40] cDHPR 1.41×104 Man:Glc:Gal=2.38:1.00:8.49 主链(1→3,5)-α-L-Araf、(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4,6)-β-D-Manp、(1→6)-α-D-Galp,末端 β-L-Araf(1→ 抗肿瘤 [40] cDHPL 2.09×105 Man:Glc:Gal=19.15:1.32:1.0 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-O-3-acetyl-β-D-Manp、(1→3,6)-β-D-Manp,末端 α-D-Galp(1→ 抗肿瘤 [40] cDHPF 4.78×105 Man:Glc:Gal=9.68:3.26:1.0 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→3,6)-β-D-Manp,末端 α-D-Galp(1→ 抗肿瘤 [40] HPS-1B23 2.2×104 Glc: Man :Gal=31:10:8.0 主链(1→4/6)-α-D-Glcp、(1→6)-α-D-Manp、(1→6)-O-2-acetyl-β-D-Glcp,侧链(1→6)-α-D-Manp 免疫调节 [41] HPS1-A 6.74×106 Man:Glc:Gal=1.0:6.78:2.14 主链都由(1→4/6/4,6)-D-Glcp、1-D-Galp、(1→3,6)-D-Manp糖苷键组成,但摩尔比不同 免疫调节 [42] HPS1-B 3.39×105 Man:Glc:Gal=1.0:41.88:2.45 HPS1-C 8.90×103 Man:Glc:Gal=1.0:71.35:3.13 HPS1-D 6.81×103 Man:Glc:Gal=1.0:93.71:3.33 HPS1-E 4.97×103 Man:Glc:Gal=1.0:68.55:2.58 HPS1-F 3.72×103 Man:Glc:Gal=1.0:85.05:2.45 HPS1-G 2.59×103 Man:Glc:Gal=1.0:84.99:1.38 铁皮石斛 DOPS-1 1.53×103 Man:Glc:Gal=3.2:1.3:1.0 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、2-O-acetyl-(1→4)-β-D-Manp 抗氧化 [43] DOP1-DES 2.98×105 Man:Glc=2.2:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp 抗氧化 [44] DOP2-DES 3.04×104 Glc:Man=3.7:1.0 主链(1→4)-β-D-Manp、(1→3)-α-D-Glcp 抗氧化 [44] DOP-1 4.47×105 Gal:Glc:Man=1.0:1.79:6.71 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-α-D-Glcp、(1→4)-O-2-acetyl-β-D-Manp,末端α-D-Glap(1→ 抗炎 [45] LDOP-1 9.18×103 Man:Gla:Glc:GlcA:Ara=
2.0:1.3:1.6:1.7:0.7主链(1→6)-α-D-Glcp、(1→4)-α-D-Manp 抗炎 [46] DOP-1-A1 1.3×105 Man:Glc:Ara=40.2:8.4:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp、(1→4)-2-O-acetyl-β-D-Manp、(1→4)-O-2-acetyl-β-D-Glcp,侧链(1→3)-β-D-Manp、(1→3)-β-D-Glcp,末端 β-D-Manp(1→、D-Araf(1→ NA [47] DOP-1 6.8×103
Man:Glc=5.18:1.0主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-O-2-acetyl-β-D-Manp、(1→4)-O-3-acetyl-D-Manp 降血糖 [48] DOP-W3-b 1.54×104 Man:Glc=4.5:1.0 主链(1→4)-D-Manp、β-(1→4)-D-Glcp、β-(1→3,6)-D-Manp、(1→4)-O-2-acetyl-β-D-Manp,侧链(1→4)-D-Manp、β-(1→4)-D-Glcp,末端β-D-Glcp(1→
免疫调节[49] DOPW-1 3.90×105 Man:Glc=10.75:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp、(1→4)-O-2-acetyl-β-D-Manp,末端β-D-Manp(1→ 抗糖基化 [50] DOPA-1 3.90×105 Man:Glc=5.8:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp、(1→4)-O-2-acetyl-β-D-Manp 免疫调节 [51] WDOP-1 1.58×106 Man:Glc:Gal:Xyl:Ara:Rha=
35.65:44.89:13.99:12.25:9.13:1.76主链(1→4)-Manp、(1→4)-Glcp,侧链(1→3,4)-Glcp,末端Galp-(1→、Araf(1→ 降血糖 [52] WDOP-2 2.68×106 Man:Glc:Gal:Xyl:Ara:Rha=
72.34:25.87:11.02:10.09:0.5:0.09主链(1→4)-Manp、(1→4)-Glcp,侧链(1→3,4/2,4)-Manp,末端Galp(1→、Manp(1→ 降血糖 [52] 金钗石斛 DNPE6(4) 9.92×104 Ara:Glc:Gal:Man=2.5:0.9:0.3:0.8 主链(1→3,6)-L-Araf、(1→3)-D-Glcp、(1→3/4)-D-Galp、(1→6)-D-Manp,末端β-D-Manp(1→ 抗病毒 [53] DNP1 6.80×103 Man:Glc=3.14:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp、(1→4)-O-2/3-acetyl-β-D-Manp 抗炎 [54] JCS1 2.3×104 Glc:Man:Xyl:Ara=40.2:2.3:1.7:1.0 主链(1→4)-β-Manp、(1→4)-α-Glcp,侧链(1→6)-α-Xylp、(1→4)-α-Glcp,末端 α-Araf(1→ 调节神经系统 [55] DNPE6(11) 3.01×103 Man:Glc:Gal=3.0:11.0:3.0 主链(1→4)-D-Glcp、(1→6)-D-Manp、(1→4)-O-2/3-acetyl-β-D-Manp,末端D-Galp(1→ 抗病毒 [56] DNP-W2 1.80×104 Glc:Man:Gal=6.1:2.9:2.0 主链(1→4/6)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-O-2-acetyl-β-D-Manp,侧链(1→4)-β-D-Glcp、(1→4)-β-D-Manp,末端 α-D-Galp(1→ 免疫调节 [57] DNP-W3 7.1×105 Gal:Rha:Ara=3.1:1.1:1.0 主链(1→3)-β-D-Galp,侧链(1→4)-α-L-Rhap,末端β-L-Arap(1→ 免疫调节 [58] DNP-W4 5.0×105 Man:Glc:Gal:Xyl:Rha:GalA=
1.0:4.9:2.5:0.5:1.0:0.9主链(1→4/6)-β-D-Glcp、(1→6)-β-D-Galp、(1→4)-O-4/6-acetyl-β-D-Glcp、(1→6)-O-3-acetyl-β-D-Galp,侧链(1→6)-β-D-Manp、(1→3)-β-D-Glcp、(1→4)-α-D-GalAp、(1→2)-α-L-Rhap/Xylp,末端 β-D-Manp(1→ 免疫调节 [59] DNP 8.76×104 Rha:Ara:Xyl:Man:Glc:Gal=
1.0:2.8:2.2:30.76:117.96:31.76主链(1→6)-α-D-Glcp、(1→6)-α-D-Galp,侧链(1→4)-α-D-Glcp、(1→4)-α-D-Manp 抗氧化 [60] 鼓槌石斛 DCPP-I-a 6.7×104 Xyl:Glc:Gal=1.44:6.93:12.79 NA 抗增殖 [61] 密花石斛 DDP-1-D 9.44×103 Glc:Man=1.0:3.01 主链(1→4/6)-α-D-Glcp、(1→2)-α-D-Manp、(1→4)-β-D-Manp NA [62] 细茎石斛 DMP4a-1 3.04×103 Glc:Man:Rha:Ara:Gal=
2.87:2.85:1.76:1.27:1.0由1→4、1→3及1→6糖苷键组成 免疫调节 [63] DMP2-A 1.07×104 Ara:Xyl:Man:Glc:Gal=1.0:1.5:0.8:4.5:1.5 由(1→3/4/3,6)-D-Glcp、(1→3/4/3,6)-D-Galp、(1→3,5)-L-Araf、(1→3,6)-D-Manp糖残基构成,末端Xylp(1→ NA [64] 注:Man-甘露糖;Glc-葡萄糖;Gal-半乳糖;Rha-鼠李糖;Ara-阿拉伯糖;Xyl-木糖;GlcA-葡萄糖醛酸;GalA-半乳糖醛酸;acetyl-乙酰基;NA-文献中未提及。 不同产地和品种的石斛多糖,其单糖组成和含量存在差异。云南和浙江产地的铁皮石斛多糖中的甘露糖含量比广西和安徽产地高[10]。与广东丹霞和浙江相比,云南产地的铁皮石斛多糖中甘露糖与葡萄糖的比例更高,而广东丹霞和浙江产地的铁皮石斛多糖在单糖组成比例方面相似[11]。铁皮石斛、钩状石斛、鼓槌石斛中葡萄糖含量比甘露糖高,而在重唇石斛、细茎石斛、金钗石斛中甘露糖含量则高于葡萄糖[12]。同一产地的不同品种石斛,其单糖含量也有差异。如来自红河群鑫种植公司不同品种的铁皮石斛(红鑫1号青杆、红鑫1号红杆和红鑫5号)多糖中,红鑫1号红杆铁皮石斛多糖中的甘露糖含量最高,达46.71%,而红鑫1号青杆含量最低,为35.92%[13]。
不同生长年限、采收期的石斛多糖中单糖组成和含量存在差异。一年生金钗石斛多糖由甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖7种单糖组成,摩尔比为68.33:1.11:1.0:18.96:3.44:5.63:7.26;二年生金钗石斛多糖由甘露糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖6种单糖组成,摩尔比为5.54:1.07:26.26:1.0:3.15:5.75[14]。铁皮石斛开花后会显著消耗多糖中甘露糖、半乳糖醛酸、葡萄糖的含量,而开花过程有利于木糖、阿拉伯糖积累,在铁皮石斛开花前采收或者通过选育不开花品种、人工摘除花蕾控制开花,可以显著提高铁皮石斛多糖中葡萄糖和甘露糖含量[15]。
不同部位石斛多糖的单糖组成和含量也有明显差异。铁皮石斛叶多糖多为酸性杂多糖,由葡萄糖、甘露糖、半乳糖、半乳糖醛酸、阿拉伯糖组成,而茎多糖由葡萄糖和甘露糖组成,茎中甘露糖和葡萄糖含量均比叶中高[16]。
石斛多糖中单糖组成还会受到提取和纯化方法影响。不同的提取方法和条件,如热水浸提、酶解、超声提取等,可能会导致石斛多糖部分或完全分解,影响多糖中各单糖的相对含量和组成。此外,不同的纯化方法可能选择性地富集特定类型的多糖,最终也会影响石斛多糖的单糖组成[17−22],见表1。由表1可知,超高压提取的石斛多糖的甘露糖含量明显高于传统热水浸提,这可能是其具有更高抗氧化能力的因素之一[17];随提取温度的升高,石斛多糖中的葡萄糖含量逐渐上升,而甘露糖、半乳糖、阿拉伯糖和木糖组分均降低[20];随超声功率的增强,提取得到的石斛多糖中的甘露糖、阿拉伯糖和半乳糖含量升高,而葡萄糖和木糖降低[22]。
1.2 石斛多糖的分子量
通过分子量的测定,可以获取关于石斛多糖结构的重要信息,包括分子大小、聚合程度和分子链的结构等。石斛多糖分子量多采用高效尺寸排阻色谱(HPSEC)、HPLC结合示差折光检测器、蒸发光散射检测器及多角度激光散射(MALLS)测定。其中,HPSEC可根据样品中化合物分子量的大小进行分离,而MALLS可以直接测定分子量,经常通过联用这两种方法,不仅可以测定化合物分子量,还可以测定多分散系数和旋转半径等信息[23]。胡卫珍等[24]采用HPSEC-MALLS对不同种源地的铁皮石斛多糖分子量及其分布进行测定,结果表明它们的重均分子量、多分散系数和旋转半径分别为2.975×105~3.741×105 Da、1.289~1.502和60.3~61.1 nm。HPLC是一种分离和定量分析的技术,适用于分子质量大、分子质量范围广的化合物测定[25]。如表2所示,石斛多糖的分子质量范围比较宽广,主要分布在103~107 Da范围内。目前通过分离纯化得到的石斛多糖的分子量差异较大,这可能与石斛的品种、提取纯化方法及分子量测定方法相关。如酶解提取法所得的铁皮石斛多糖组分较少,分子量在7×104~105 Da;超声辅助提取的多糖组分比较多,分子量分布广泛,在103~205 Da[26]。从表1也可以看出,超高压提取的石斛多糖分子量远小于热水浸提,这可能是因为超高压产生的强烈机械剪切作用破坏了石斛多糖的糖链[17];超声提取和酶法提取所得石斛多糖分子量相比于热水浸提、闪式提取、冻融提取的小,可能是由于超声波的空化作用、机械振动以及酶对植物细胞壁结构的酶解作用使石斛多糖分子量减小[18];采用醇沉法分离石斛多糖,分子量随乙醇浓度升高而降低[19]。
1.3 石斛多糖的化学结构
深入了解石斛多糖的生物活性,关键在于研究其化学结构,揭示其结构有助于寻找具有相似生物活性石斛多糖的结构共性,确立构效关系。研究石斛多糖的一级结构方法主要分为两大类:化学分析和物理分析。常用的化学分析方法包括甲基化反应、Smith降解法、酸水解法和高碘酸氧化法等,可确定石斛多糖中单糖组成、糖苷键类型和比例、糖苷键位置以及支链多糖的分支数目;物理分析方法涵盖红外光谱、核磁共振光谱、HPLC、气相色谱和质谱等,不仅适用于确定石斛多糖糖苷键类型和构型,还能确认多糖链上的取代基类型、连接顺序和重复单元等[27]。其中,核磁共振技术特别是二维核磁共振(2D-NMR)对推断多糖的精细结构起至关重要的作用,2D-NMR可以提供石斛多糖结构中单糖残基的类型、各糖残基中 C、H 化学位移归属,各糖残基间的连接位置和连接顺序等诸多信息[28]。肖婧婧[29]结合全甲基化对霍山石斛多糖(DHP-D1)的异核单量子相关(HSQC)谱图进行分析,推测谱图的异头区中交叉峰的对应关系为:5.32/99.7 对应→4-α-D-Glcp(1→、5.31/99.9 对应α-Glcp(1→、5.29/99.6 对应→4,6-α-Glcp(1→、4.91/98.6 对应→6-α-Glap(1→、4.90/99.4 对应→6-α-Glcp(1→。高云霄等[30]利用同核化学位移相关谱(COSY)对铁皮石斛多糖(SDOP)糖残基中所有 H 的信号进行归属,并根据H 的化学位移在 HSQC中确认所有糖残基中 C 的信号,最终将难以确认的信号结合全相关谱( TOCSY)和核欧佛豪瑟效应频谱(NOESY) 进行验证和补充,并结合全甲基化的分析结果认为SDOP中仅有少量分支结构,主要为1,2,4-, 1,3,4-和1,4,6-甘露糖残基,是 1 个分支结构少的长链大分子。由于石斛多糖结构的复杂性,任何一种单一的方法都难以确定其结构,需要结合多种方法。石斛多糖的主链主要由α或β构型的葡萄糖、甘露糖及少量的半乳糖残基通过1→3、1→4、1→6、1→3,6及1→4,6糖苷键交替连接而成,支链多由木糖、阿拉伯糖和鼠李糖单元通过O-2、O-3、O-6与主链相连,支链末端多为甘露糖基1→、阿拉伯糖基1→和鼠李糖基1→等。此外,大多数铁皮石斛多糖还含有乙酰基基团,通常连接在主链葡萄糖或甘露糖残基的O-2或O-3位,表2总结了近年来常见石斛多糖的结构特征。
2. 石斛多糖的生物活性
2.1 抗肿瘤
癌症是全球死亡率第二高的疾病,仅次于心血管疾病。传统治疗方式如化疗、放疗、手术在一定程度上可以缓解病情,但其副作用较大。为了寻找副作用较小的治疗方式,人们开始关注天然植物的价值,尤其是天然植物多糖备受关注。石斛多糖可以通过多种途径发挥抗肿瘤作用。研究发现,霍山石斛多糖DOPA-1能够减少肝癌HepG-2细胞中抗凋亡蛋白Bcl-2表达,增加促凋亡B细胞淋巴相关X蛋白(Bax)的表达,过量的Bax会形成同源二聚体,导致HepG-2细胞凋亡[65]。不同部位提取的石斛多糖抗癌效果存在差异,霍山石斛茎多糖cDHPS对胃癌细胞的增殖抑制作用最为显著,叶多糖cDHPL次之,而根多糖cDHPR和花多糖cDHPF仅显示微弱的作用,其凋亡机制可能是上调抑癌基因p53的表达,下调原癌基因c-myc的表达[40]。铁皮石斛茎多糖FWDOP1通过刺激c-Jun氨基末端激酶(JNK)、胞外信号调节蛋白激酶(ERK)及p38丝裂原活化蛋白激酶磷酸化,激活与肿瘤细胞凋亡相关信号通路[66]。另外,铁皮石斛多糖可以增强CD8+细胞毒性T淋巴细胞(CTL)的代谢能力,减少CTL上程序性死亡受体(PD-1)的表达,增强T细胞在肿瘤微环境(TME)中抗肿瘤免疫反应,提升机体清除肿瘤细胞的能力[67]。石斛多糖还能够调节氧化应激产生活性氧(ROS),使线粒体功能障碍,进而抑制三磷酸腺苷(ATP)生成;低比例的三磷酸腺苷/单磷酸腺苷(ATP/AMP)激活了腺苷酸活化蛋白激酶(AMPK)-雷帕霉素靶蛋白(mTOR)信号通路,促使结肠癌CT26细胞过度自噬凋亡[68]。综上,石斛多糖能够调节凋亡蛋白和癌基因的表达、增强免疫代谢、增强氧化应激等抑制肿瘤细胞的生长,为今后石斛多糖在癌症治疗、预防等方面中的应用提供了理论依据。
2.2 免疫调节
免疫调节是指通过调节机体的免疫系统来维持免疫平衡和增强免疫功能。石斛多糖对免疫系统具有调节作用。研究发现,霍山石斛多糖DHP能刺激肠道细胞分泌γ-干扰素(IFN-γ)和白细胞介素-4(IL-4),恢复甲氨蝶呤损伤的小肠免疫功能[69]。铁皮石斛多糖DOP-1-1通过刺激吞噬细胞释放肿瘤坏死因子(TNF-α)、白细胞介素-1β(IL-1β)等细胞因子,并上调核因子-κB(NF-κB)和细胞外调节蛋白激酶(ERK1/2)蛋白磷酸化水平,因而表现出显著的免疫增强特性[70]。铁皮石斛茎多糖DOP-W3-b能促进派尔集合淋巴结(PPs)和肠系膜淋巴结细胞因子(MLN)的分泌,增加固有层分泌型免疫球蛋白(AsIgA)的产生,调节肠黏膜免疫活性[32]。此外,石斛多糖cDFP-W1可以增加脾脏中CD4+T细胞亚群、B细胞、自然杀伤细胞(NK)和树突状细胞的比例,降低CD8+T细胞亚群数量,上调活化巨噬细胞的比例来缓解免疫抑制[71]。综上分析发现石斛多糖主要通过促进免疫细胞的增殖与分化、调节细胞因子的释放、促进抗体的分泌等发挥其免疫调节活性。
2.3 抗炎
炎症是机体对刺激的一种防御反应,但长期或过度的炎症反应可能导致疾病的发展和组织损伤。石斛多糖具有抗炎活性,有助于减轻炎症反应。研究发现,石斛多糖能通过抑制TNF-α、IL-1β和白细胞介素-6(IL-6)等炎症因子的产生和释放,使炎症平衡恢复到正常水平[72]。石斛多糖还能够上调核因子E2相关因子2(Nrf-2)、血红素氧合酶1(HO-1)和醌氧化还原酶-1(NQO-1)的mRNA或蛋白表达水平,激活Nrf-2信号通路,减轻葡聚糖硫酸钠(DSS)诱导的急性结肠炎继发性肝损伤[73]。基质金属蛋白酶(MMPs)在炎症过程中发挥重要的作用,可以增强炎症反应强度。研究发现,金钗石斛多糖JCP可以通过抑制MMPs 信号通路的激活逆转乙醇诱导的胃溃疡,在炎症细胞浸润的状态下,乙醇诱导的大鼠中MMP-2和MMP-9的水平均会升高,导致胃黏膜结构受损;经过JCP干预后,MMP-2和MMP-9蛋白的表达明显降至正常水平[74]。石斛多糖还可以抑制炎症相关信号通路激活,减轻炎症反应。霍山石斛茎多糖CDHPS能有效降低Ⅱ型胶原诱导关节炎小鼠中JNK、p38、ERK1/2、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(PKB)、Janus激酶1(JAK1)信号转导及转录激活蛋白3(STAT3)的磷酸化水平,抑制NF-κB、丝裂原活化蛋白激酶(MAPKs)、PI3K/蛋白激酶B(Akt)和JAK1/STAT3炎症信号通路的激活[75]。CDHPS还可以降低MAPKs信号转导中的血清核转录因子激活蛋白-1(AP-1)的活性,抑制许多与炎症相关的基因的表达[76]。综上可知,石斛多糖减少炎症反应的作用机制在于通过调控炎症因子的释放、减轻氧化应激、抑制炎症信号通路激活等来减少炎症反应。
表 3 石斛多糖的生物活性及作用机制Table 3. Biological activity and mechanism of action of Dendrobium polysaccharides生物活性 动物模型/体外实验 剂量 药效/机制 文献 抗肿瘤 肝癌HepG-2细胞 50、100、200、400 μg/mL 减少抗凋亡蛋白Bcl-2表达,增加促凋亡蛋白Bax表达 [65] 胃癌MFC细胞 0.025、2.5 mg/mL 上调抑癌基因p53的表达,下调原癌基因c-myc的表达 [40] 子宫颈癌Hela细胞 25、400 μg/mL 上调JNK、ERK和p38丝裂原活化蛋白激酶表达 [66] AOM/DSS诱导的小鼠结直肠癌模型 50、100、200 mg/kg 减少CTL上PD-1表达,增强T细胞在TME中抗肿瘤免疫反应 [67] AOM/DSS诱导的小鼠结直肠癌模型 400、800 μg/mL 激活AMPK-mTOR信号通路,促进CT26细胞过度自噬凋亡 [68] 免疫调节 甲氨蝶呤诱导低免疫小鼠模型 50、100、200 mg/kg 激活免疫因子IFN-γ和IL-4分泌 [69] 急性单核细胞白血病细胞系 25、50、100 μg/mL 提升NF-κB和 ERK1/2蛋白磷酸水平 [70] 雌性ICR小鼠骨髓细胞 0.05、0.1、0.2 mg/mL 促进PPs和MLN的分泌,增加AsIgA的产生 [32] 雄性C57BL/6小鼠脾脏 10~200 μg/mL 增加CD4+T亚群、B淋巴细胞亚群和NK细胞比例,降低CD8+T 亚群比例 [71] 抗炎 DSS 诱导的小鼠急性结肠炎模型 50、100、200 mg/kg 抑制TNF-α、IL-1β和IL-6等炎症因子的产生和释放 [72] DSS诱导小鼠急性结肠炎继发性肝
损伤模型50、100、200 mg/kg 上调Nrf-2、HO-1和NQO-1的mRNA或蛋白表达水平,激活Nrf-2信号通路 [73] 乙醇诱导的大鼠胃溃疡模型 100、300 mg/kg 抑制MMP信号通路的激活 [74] Ⅱ型胶原诱导关节炎CIA小鼠模型 0.1095、0.438 g/kg 抑制NF-κB、MAPKs、PI3K/Akt和JAK1/STAT3信号通路的激活 [75] 香烟诱导的小鼠肺炎模型 100、200、400 mg/kg 降低MAPKs信号转导中的血清AP-1的活性 [76] 抗氧化 体外抗氧化实验 0~3.0 mg/mL 对·OH和ABTS+·均具有一定的清除作用 [78] 体外抗氧化实验 1.0~10.0 mg/mL 对DPPH·具有一定的清除作用 [79] 衰老小鼠、6周龄的年轻雌性小鼠 70 mg/kg 降低MDA的含量,提高SOD、CAT、GSH-Px的活性 [80] 调节肠
道菌群健康小鼠 50、200 mg/kg 调节肠道微生物菌群组成以及代谢,增强肠道物理、生化和免疫屏障功能 [81] 健康小鼠 0.4 g/kg 增加SCFA含量,提高有益微生物群属的多样性 [83] 健康小鼠 不明 代谢产生更多丁酸盐,介导肠道的免疫反应 [84] 降血糖 高脂饮食联合STZ建立的糖尿病小鼠模型 100、200、400 mg/kg 影响cAMP-PKA和Akt/FoxO1信号通路,促进肝糖原合成,抑制肝糖原降解和异生 [85] STZ诱导的糖尿病大鼠 15、25、100 mg/kg 增加血清胰岛素和GLP-1的分泌水平 [48] 棕榈酸诱导的高糖细胞胰岛素抵抗 100、200、400 μg/mL 增加PPAR-γ表达水平,调节细胞胰岛素敏感性 [86] 抗白内障 H2O2 诱导小鼠晶状体损伤 2、4 mg/mL 提高晶状体水溶性蛋白含量及GSH-Px和SOD活性 [87] 腹腔注射链脲菌诱导的糖尿病大鼠模型 25、50、100 mg/kg 抑制介导氧化应激的ERK/Raf信号通路激活 [88] 抗病毒 含有CMV的外壳蛋
白基因质粒的大肠杆菌125、500 μg/mL 对农作物PVY和CMV病毒有较好的杀灭效果 [56] 抗疲劳 负重游泳小鼠疲劳模型 50 mg/kg 减弱疲劳相关代谢物LDH、CK、TG和MDA的水平 [89] 保肝 酒精诱导肝损伤小鼠模型 100、200、400 mg/mL 增加肝脏中GSH-Px水平,平衡酒精诱导下氧化还原 [90] 2.4 抗氧化
氧化过程在人体内产生自由基,对细胞和组织造成损害,并诱发多种疾病,包括心脏病、癌症和神经退行性疾病等。抗氧化剂可以帮助中和自由基,减少氧化损伤。研究发现,石斛多糖具有体外清除多种自由基的能力,并呈剂量依赖性[77]。经体外抗氧化实验发现,铁皮石斛茎多糖DFHP在浓度为1.0 mg/mL时表现出良好的OH自由基清除活性,在浓度为3 mg/mL时,对ABTS+自由基的清除效果高达90.05%[78]。不同的提取方法对石斛多糖抗氧化能力有较大影响,采用水提和生物发酵法从铁皮石斛提取到了两种多糖组分DOP-S和DOP-F,均具有DPPH自由基清除活性,半抑制浓度值分别为4.9和1.0 mg/mL,发酵法提取的多糖表现出更加明显的抗氧化效果[79]。石斛多糖还能够降低丙二醛(MDA)的含量,提高超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)的活性,减少氧化损伤,以改善自然衰老小鼠卵巢功能早衰[80]。通过分析上述文献发现,石斛多糖具有显著的抗氧化能力,能通过清除自由基、提高抗氧化酶活性、抑制氧化应激等方式,帮助保护细胞免受氧化损伤,减缓衰老过程。
2.5 调节肠道菌群
肠道菌群是人体肠道内存在的各种微生物的总称。肠道菌群的稳定和平衡对于人体的健康至关重要,它们参与调节免疫系统、营养代谢和肠道屏障功能等多种生理过程。石斛多糖能够调节肠道微生物组成,维护肠道菌群平衡。最新研究表明,霍山石斛多糖GXG能影响小鼠结肠和粪便微生物群的香农指数、均匀度和丰富度指数;显著提高了微生物菌群中优势门(厚壁菌门F和拟杆菌门B)的相对丰度,下调了具有内毒素活性的革兰氏阴性细菌中变形杆菌门的丰度[81]。短链脂肪酸(SCFAs)是肠道微生物群代谢产物之一,包括丙酸、丁酸和乙酸等,对肠道健康起着重要的作用。研究表明,石斛多糖、SCFAs和结肠微生物三者之间密切关联,石斛多糖能使短链脂肪酸GPR41和GPR43蛋白偶联受体的mRNA表达恢复到正常水平,使短链脂肪酸发挥作用[82];同时结肠微生物能够利用体内未被吸收的石斛多糖产生糖酵解,促使SCFAs含量的增加[83]。石斛多糖还可以增加肠道中类副杆菌sp-HGS0025的数量,进而代谢产生更多的丁酸盐;丁酸盐可以介导肠道免疫反应,增强肠道的保护作用[84]。综上分析发现石斛多糖能够通过维持肠道菌群的多样性,调节菌群丰度,进而促进肠道微生物平衡;而肠道菌群反过来也可以吸收机体无法直接代谢的石斛多糖,促进机体健康,形成一种相互促进的关系。
2.6 降血糖
糖尿病是一种代谢紊乱的慢性疾病,其特征是血液中的血糖水平持续升高,控制血糖水平对于糖尿病患者的健康至关重要。石斛多糖在维持血糖平衡中有较好的效果。研究发现,铁皮石斛多糖可以减轻高脂饮食联合链脲佐菌素(STZ)诱导的2型糖尿病小鼠的高血糖,其作用机制是通过影响胰高血糖素介导的环磷酸腺苷-蛋白激酶A(cAMP-PKA)和蛋白激酶B-叉头转录因子1(Akt-FoxO1)信号通路,以促进肝糖原合成,抑制肝糖原降解和异生;并形成更稳定的α颗粒结构的糖原,能在一定程度上减缓糖原降解速度[85]。另有研究发现,STZ诱导的糖尿病大鼠连续口服铁皮石斛多糖28 d后,能够增加血清胰岛素和胰高血糖素样肽-1(GLP-1)的分泌水平,降低糖尿病大鼠的血糖水平[48]。另外,石斛多糖可以激活过氧化物酶体增殖物激活受体γ(PPAR-γ),调节细胞对胰岛素敏感性,改善棕榈酸诱导的高糖细胞的胰岛素的抵抗[86]。因此,在未来的研究中,可以深入探索石斛多糖降血糖的机制,明确其信号传导途径和相关作用靶点,以便更好利用石斛多糖的降血糖活性,并推动相关产品的开发。
2.7 抗白内障
石斛多糖通过抑制氧化应激,减轻晶状体细胞受到的损伤和减缓晶状体老化的进程,对于预防和治疗白内障具有一定的作用。体外实验研究表明,金钗石斛多糖在小鼠过氧化氢损伤的晶状体模型中显著提升了晶状体水溶性蛋白的含量,并增强了GSH-Px和SOD的活性,有效防止或逆转了晶状体浑浊现象,从而抑制和延缓白内障的发生与发展[87]。另一项研究表明,铁皮石斛多糖通过降低STZ诱导的糖尿病性白内障中ERK1、ERK2、丝裂原活化蛋白激酶、丝氨酸/苏氨酸蛋白激酶(Raf)、大鼠肉瘤蛋白(Ras)mRNA的基因表达水平,抑制介导氧化应激的ERK/Raf信号通路激活,延缓糖尿病性白内障发展进程[88]。以上研究证实了石斛多糖在治疗白内障方面的潜力,但目前仍需要更多的临床和科学研究来证实其具体的作用机制以及疗效。
2.8 其他活性
石斛多糖还具有保肝、抗疲劳及抗病毒等作用。据报道,一种从金钗石斛中提取出的新型同质杂多糖DNPE6(11)对农业病毒有显著的灭活作用,可以作为一种低毒、有效、环保的杀虫剂。实验结果显示,当DNPE6(11)浓度达到500 μg/mL时,对马铃薯Y病毒(PVY)的杀灭效果达到51.5%,与宁南霉素(50.8%)相似;对黄瓜花叶病毒(CMV)的杀灭效果为55.1%,比农药壳聚糖寡糖(37.3%)和香菇多糖(37.8%)的效果更优越[56]。研究还发现,在小鼠负重游泳试验中,铁皮石斛多糖表现出良好的抗疲劳效果,能减弱疲劳相关代谢物如乳酸脱氢酶(LDH)、肌酸激酶(CK)、甘油三酯(TG)和MDA的水平[89]。另外,铁皮石斛多糖可以增加肝脏中的GSH-Px水平,平衡酒精诱导下的氧化还原,以保护肝脏免受损伤[90]。现有的石斛多糖的生物活性及主要作用机制见图1和表3。
3. 石斛多糖的构效关系
3.1 石斛多糖分子量与其生物活性的关系
分子量的高低与石斛多糖的活性密切相关。通常,多糖的分子量越大,活性位点越多,活性可能会更强,但过高的分子质量会影响多糖穿透细胞膜进入细胞内部与作用靶点,限制了其生物活性的发挥。相比之下,若相对分子质量太低,则无法形成产生生物活性的聚合结构,只有分子量分布在一个比较合理的区间才有利于多糖发挥其生物活性[91]。研究表明,铁皮石斛的多糖只有在降解到较小的分子量片段后才表现出显著的抗肿瘤作用[66]。采用分级醇沉从铁皮石斛粗多糖中得到3个组分,其中分子量最低的总抗氧化能力最强,且认为分子量小于3.53×104 Da或大于 7.44×104 Da的铁皮石斛多糖抗氧化活性较高[92]。通过DEAE-52纤维素层析,分别用不同浓度的NaCl从密花石斛粗多糖中洗脱出4个酸性石斛多糖组分,结果发现相对分子量集中在Ⅲ区(10000~5000)的石斛多糖药效最强,而集中于Ⅱ区(50000~28000) 的相对较弱[93]。
尽管如此,也有研究表明高分子量的石斛多糖活性优于低分子量多糖。例如,霍山石斛多糖DHPD1对非酶糖化的蛋白质抑制的效果随着分子量的降低而减弱[94]。铁皮石斛粗多糖经过超声波联合过氧化氢降解可以得到4种不同分子量组分的多糖,分子量最小的多糖的免疫调节能力最弱,可能是因为分子量小、链长短、免疫调节活性点位较少[95]。综上,不同分子量大小的石斛多糖对活性的影响是不同的,单纯根据分子量来预测石斛多糖的活性可能是不准确的,还需要综合考虑糖链结构、糖组分以及空间排列等因素的影响。
3.2 石斛多糖单糖组成与其生物活性的关系
从表2可以看出,大部分石斛多糖含有甘露糖和葡萄糖,并且占据了较高的比例;这提示富含活性的石斛多糖中,甘露糖和葡萄糖可能在结构上具有明显的优势。已有研究表明,富含这两种单糖的多糖能发挥更强的抗癌活性,可能由于巨噬细胞中具有对甘露糖和葡萄糖特异性结合的多糖受体[96]。铁皮石斛多糖中的甘露糖在介导细胞活化中扮演着重要的角色,可以与免疫细胞表面上的甘露糖受体结合,并与发挥免疫调节的作用有关[36]。霍山石斛多糖对食管癌细胞生长的抑制作用会随甘露糖和葡萄糖的摩尔比的上升而增强[97]。值得注意的是,WDOP-1和WDOP-2是从同一铁皮石斛中提取分离出来的多糖组分,但WDOP-2在治疗二型糖尿病方面的效果优于WDOP-1,可能与WDOP-2中甘露糖含量更高有关[52]。另外,石斛多糖中的糖醛酸含量与抗氧化活性之间存在一定的正相关性。糖醛酸含量越高,石斛多糖的抗氧化能力越强,越容易激活异头上氢原子与氧自由基发生氧化还原反应。例如,碱溶性石斛多糖中糖醛酸的质量分数比水溶性石斛多糖中的高,因而表现出更强的抗氧化性[98];酸性石斛多糖的抗氧化活性与糖醛酸含量呈正相关,而与硫酸糖含量呈负相关[99]。石斛多糖中乙酰的含量也对活性有较大影响。霍山石斛多糖中乙酰基含量越高,对肠黏膜免疫调节活性越强[97]。存在乙酰基的石斛多糖在浓度为25 μg/mL时已具有免疫活性,而去乙酰基后的石斛多糖仅在100 μg/mL浓度具有免疫增强活性[100]。
3.3 石斛多糖糖苷键与其生物活性的关系
糖苷键是石斛多糖中的重要结构单元,对活性具有重要影响。如表2所示,石斛多糖中的糖苷键为α和β构型。已有研究表明,具有β构型吡喃糖形式的石斛多糖比α构型表现出更强的活性[7]。构成石斛多糖重复单元的糖苷键连接类型主要包括(1→4)、(1→6)、(1→3)、(1→3,6)及(1→4,6),其中(1→4)-β-D-Glcp和(1→4)-β-D-Manp被认为是构成石斛多糖结构中的核心单元,可能是石斛多糖活性相关的关键片段。例如,霍山石斛多糖中的(1→4)-β-D-Glcp与其促进成骨作用密切相关[35]。铁皮石斛多糖的免疫调节活性可能是主链中的(1→4)-β-D-Manp起主导作用[101]。β-(1→4)-Manp可能是铁皮石斛多糖抗结直肠癌的主要原因[51]。石斛多糖中的(1→4)-β-D-Manp和O-乙酰基被认为是具有抗氧化活性的主要结构特征[102]。霍山石斛多糖的免疫调节活性与(1→4)-D-Glcp的含量呈正相关趋势[42]。此外,还发现铁皮石斛多糖免疫调节活性与α-(1→3)、α-(1→4)、β-(1→3)、β-(1→4)糖苷键相关。细茎石斛多糖中的(1→3)糖苷键与免疫增强作用直接相关[63]。具有(1→3)-Manp糖苷键的铁皮石斛多糖的抑制肿瘤效果更为显著[103]。霍山石斛多糖中→6)-β-Galp-(1→和→3,6)-β-Manp-(1→结构是发挥抗胃癌活性的重要因素[104]。
3.4 石斛多糖改性与其生物活性的关系
多糖的改性方法主要有化学修饰、生物修饰和物理修饰,通过改性可改变多糖的分子量、稳定性和溶解度等特性,从而影响生物活性[105−106]。化学修饰是石斛多糖常用的改性方法,硫酸化、乙酰化、硒化、脱乙酰化及氧化降解等有效修饰能增强石斛多糖的抗糖基化、抗氧化、免疫调节、抗肿瘤、抗菌等作用,而羧甲基化则降低了石斛多糖的免疫调节作用,见表4。通过基因工程技术和微生物发酵等生物改性也能够改变石斛多糖的活性。如石斛粗多糖水提取液经面包酵母发酵后小分子多糖和酸性多糖增多,提高了其抗氧化和降血糖活性[110];利用基因工程技术获得黑曲霉β-甘露聚糖酶将石斛多糖水解成甘露低聚糖不但保留了石斛多糖原有的功能活性,还能使其功能得到延伸,很大程度上提高了石斛多糖生物利用度[111]。此外,还可以利用物理方法,如超声波降解,通过切断石斛多糖中的某些化学键,以降低多糖分子量,增强其水溶性,从而提高多糖的生物活性[95]。因此,选用特定的修饰方式对石斛多糖进行定向修饰,可以获得具有更强生物活性的石斛多糖,具备广泛的应用前景。
表 4 石斛多糖的化学修饰Table 4. Chemical modification of Dendrobium polysaccharides4. 石斛保键食品开发利用现状
通过检索药智网数据库发现,具有批准文号的石斛(铁皮石斛、霍山石斛、金钗石斛、细茎石斛、铜皮石斛和叠鞘石斛)保健食品有近200种,多数用于增强免疫力、抗疲劳、改善睡眠和延缓衰老。其中,铁皮石斛的种类最多,达185种;金钗石斛次之,有7种;霍山石斛有4种;而其他品种都只有1种产品。这些石斛产品主要以胶囊剂型居多,其次是颗粒剂,还包括片剂、剂、膏剂、茶剂、丸剂、粉剂、口服液和液体饮料等,见图2。从数据可以看出,其他品种的石斛为原料开发的保健食品的剂型种类方面远远少于铁皮石斛,因此,需要进一步加强对其他石斛类产品的深度开发和综合利用。
5. 小结与展望
近几十年来,石斛多糖的化学结构和生物活性的研究取得较大的进展,但仍然存在诸多亟待解决的问题。a.在结构解析中,由于石斛多糖的结构复杂,易受到多种因素的影响,目前的结构分析多限于分子量和单糖组成,对于糖苷键的精准连接方式及高级结构解析较少,无法建立较完善的构效关系,妨碍了石斛多糖的进一步开发利用。b.在生物活性研究方面,现阶段对石斛多糖的生物活性研究多停留在药效层面,相关研究缺乏深度,许多药效的具体作用机制也尚不清楚。c.在开发应用方面,当前市面上与石斛多糖相关的功能性保健食品较为单一,多以增强免疫力及抗疲劳为主,且大多数产品是基于铁皮石斛多糖开发的,其他品种的石斛多糖产品较少,仍具有很大的开发潜力。基于以上几点总结,未来可以从以下几个方面展望石斛多糖的研究:a.利用更先进的设备和技术,深入研究石斛多糖的一级结构和高级结构,为进一步了解其生物活性奠定坚实基础;b.通过动物试验及体外试验等方法,探索石斛多糖在机体内的作用途径和信号转导机制等,为其应用开发提供更可靠的理论支持;c.探索不同结构特征的石斛多糖与生物活性之间的关联,确立构效关系,筛选出具有特定且活性更优的石斛多糖;d.基于石斛多糖的特殊功能和潜在应用,加大开发力度,研制出更多种类的功能性食品或药品,以满足不同人群的需求。
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表 1 提取和纯化方法对石斛多糖单糖组成及分子量的影响
Table 1 Effects of extraction and purification methods on the monosaccharide composition and molecular weight of Dendrobium polysaccharides
多糖来源 提取纯化方法/条件 单糖组成和摩尔比 分子量(Da) 文献 铁皮石斛多糖 热水浸提 Man:Glc=1.06:1.0 5.4×107 [17] 超高压提取 Man:Glc=1.71:1.0 3.2×105 铁皮石斛多糖 热水浸提 NA 2.01×105 [18] 酶法提取 NA 1.67×105 闪式提取 NA 1.79×105 超声波提取 NA 1.96×105 冻融提取 NA 2.28×105 霍山石斛总多糖ST 40%终浓度的乙醇沉淀 NA 4.79×105 [19] 50%终浓度的乙醇沉淀 NA 3.16×105 60%终浓度的乙醇沉淀 NA 3.07×105 80%终浓度的乙醇沉淀 NA 8.59×104
霍山石斛多糖HPS40 ℃水提取 Man:Glc:Gal:Ara:Rha:Xyl=2.24:50.22:35.88:8.79:1.66:1.21 9.42×106 [20] 60 ℃水提取 Man:Glc:Gal:Ara:Rha:Xyl=2.11:58.44:29.43:8.05:1.11:0.86 9.27×106 80 ℃水提取 Man:Glc:Gal:Ara:Rha:Xyl=1.78:65.36:24.37:6.87:0.85:0.75 9.20×106 100 ℃水提取 Man:Glc:Gal:Ara:Rha:Xyl=1.19:75.34:16.44:5.26:1.24:0.54 9.12×106 铁皮石斛多糖DOP 超滤膜分级分离 Man:Glc=1.38:1.0 4.92×105 [21] 3500 Da透析袋透析 Man:Glc=4.06:1.0 3.33×104 铁皮石斛多糖 超声提取/14 ℃,2 h Man:Glc:Gal:Ara:Rha:Xyl=79.17:16.33:0.90:0.86:0.45:1.21 2.95×105 [22] 超声提取/20 ℃,100 W,3 min Man:Glc:Gal:Ara:Rha:Xyl=75.58:21.02:0.35:0.35:0.20:1.14 3.28×105 超声提取/20 ℃,300 W,3 min Man:Glc:Gal:Ara:Rha:Xyl=76.63:18.81:0.69:0.42:0.15:1.05 3.31×105 超声提取/20 ℃,500 W,3 min Man:Glc:Gal:Ara:Rha:Xyl=77.69:17.82:0.95:0.48:0.27:0.96 3.68×105 超声提取/100 ℃,2 h Man:Glc:Gal:Ara:Rha:Xyl=77.39:17.82:0.85:0.34:0.34:0.64 3.13×105 注:Man-甘露糖;Glc-葡萄糖;Gal-半乳糖;Rha-鼠李糖;Ara-阿拉伯糖;Xyl-木糖;NA-文献中未提及。 表 2 石斛多糖的结构特征信息
Table 2 Structure information of Dendrobium polysaccharides
来源 多糖名称 分子量(Da) 单糖组成和摩尔比 结构特征 生物活性 文献 霍山石斛 DHP1A 6.7×103 Man:Glc:Gal=2.5:16.0:1.0 主链(1→4)-α-D-Glcp、(1→6)-β-D-Glcp、(1→4)-β-D-Manp,末端β-D-Glcp(1→ 抗氧化 [31] DHP-4A 2.32×105 Glc:Ara:Man:Rha=13.8:3.0:6.1:2.1 主链(1→6)-β-D-Glcp、(1→6)-β-Manp,侧链(1→2)-α-L-Rhap、(1→4)-β-D-Manp、(1→3)-α-L-Araf,末端α-L-Rhap(1→、α-L-Araf(1→ 免疫调节 [32] DHPs-1 5.0×104 Glc:Man:Gal:GalA:Rha =
65.04:14.23:8.17:6.41:2.34主链(1→4)-Manp、(1→4)-Galp、(1→4)-Glcp、(1→3,4)-Galp、(1→4,6)-Galp、(1→3,4,6)-Galp,侧链(1→4)-Manp、(1→4)-Galp、(1→3,4)-Galp、(1→3,4,6)-Galp,末端Galp-(1→ 免疫调节 [33] TCDHPA4 8.0×105 Rha:Ara:Man:Glc:Gal=
1.28:1.67:4.71:10.43:1.42(1→6)-β-Galp、(1→4/6)-β-Glcp,侧链(1→2/4)-α-Rhap、(1→3)-β-Galp、(1→2/6)-β-D-Manp、(1→2)-α-Araf,末端β-Glcp(1→、α-Araf(1→ NA [34] DHPW1 2.3×103 Man:Glc:Gal=37.8:21.9:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp 促进成骨 [35] DHPD2 8.09×106 Gal:Glc:Ara=0.9:0.7:0.2 主链(1→4/6)-β-D-Galp、(1→5)-α-L-Araf、(1→6)-α-D-Glcp、(1→3,6)-β-D-Galp、(1→6)-α-D-Galp,侧链(1→3)-β-D-Manp,末端α-D-Xylp(1→、β-D-Manp(1→ 免疫调节 [36] DHPD1 3.2×103 Gal:Glc:Ara=0.021:1.023:0.023 由(1→4/6/4,6)-Glcp、(1→6)-Galp、(1→5)-Araf糖残基组成,末端Glcp(1→ 抗糖基化 [37] DHP-W2 7.3×104 Glc:Xyl:Gal=1.0:1.0:0.5 主链(1→4/6)-β-D-Glcp,侧链(1→6)-α-D-Xylp,末端α-D-Galp(1→、α-D-Xylp(1→ 抗糖基化 [38] GXG 1.78×106 Xyl :Gal :Glc=2.13:1.0:2.85 由(1→4/2,4)-Xylp、(1→4)-Galp、(1→4/6/3,6/4,6)-Glcp糖残基组成 促进胃肠消化 [39] cDHPS 2.59×105 Man:Glc=3.04:1.0 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-3-O-acetyl-β-D-Manp 抗肿瘤 [40] cDHPR 1.41×104 Man:Glc:Gal=2.38:1.00:8.49 主链(1→3,5)-α-L-Araf、(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4,6)-β-D-Manp、(1→6)-α-D-Galp,末端 β-L-Araf(1→ 抗肿瘤 [40] cDHPL 2.09×105 Man:Glc:Gal=19.15:1.32:1.0 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-O-3-acetyl-β-D-Manp、(1→3,6)-β-D-Manp,末端 α-D-Galp(1→ 抗肿瘤 [40] cDHPF 4.78×105 Man:Glc:Gal=9.68:3.26:1.0 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→3,6)-β-D-Manp,末端 α-D-Galp(1→ 抗肿瘤 [40] HPS-1B23 2.2×104 Glc: Man :Gal=31:10:8.0 主链(1→4/6)-α-D-Glcp、(1→6)-α-D-Manp、(1→6)-O-2-acetyl-β-D-Glcp,侧链(1→6)-α-D-Manp 免疫调节 [41] HPS1-A 6.74×106 Man:Glc:Gal=1.0:6.78:2.14 主链都由(1→4/6/4,6)-D-Glcp、1-D-Galp、(1→3,6)-D-Manp糖苷键组成,但摩尔比不同 免疫调节 [42] HPS1-B 3.39×105 Man:Glc:Gal=1.0:41.88:2.45 HPS1-C 8.90×103 Man:Glc:Gal=1.0:71.35:3.13 HPS1-D 6.81×103 Man:Glc:Gal=1.0:93.71:3.33 HPS1-E 4.97×103 Man:Glc:Gal=1.0:68.55:2.58 HPS1-F 3.72×103 Man:Glc:Gal=1.0:85.05:2.45 HPS1-G 2.59×103 Man:Glc:Gal=1.0:84.99:1.38 铁皮石斛 DOPS-1 1.53×103 Man:Glc:Gal=3.2:1.3:1.0 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、2-O-acetyl-(1→4)-β-D-Manp 抗氧化 [43] DOP1-DES 2.98×105 Man:Glc=2.2:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp 抗氧化 [44] DOP2-DES 3.04×104 Glc:Man=3.7:1.0 主链(1→4)-β-D-Manp、(1→3)-α-D-Glcp 抗氧化 [44] DOP-1 4.47×105 Gal:Glc:Man=1.0:1.79:6.71 主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-α-D-Glcp、(1→4)-O-2-acetyl-β-D-Manp,末端α-D-Glap(1→ 抗炎 [45] LDOP-1 9.18×103 Man:Gla:Glc:GlcA:Ara=
2.0:1.3:1.6:1.7:0.7主链(1→6)-α-D-Glcp、(1→4)-α-D-Manp 抗炎 [46] DOP-1-A1 1.3×105 Man:Glc:Ara=40.2:8.4:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp、(1→4)-2-O-acetyl-β-D-Manp、(1→4)-O-2-acetyl-β-D-Glcp,侧链(1→3)-β-D-Manp、(1→3)-β-D-Glcp,末端 β-D-Manp(1→、D-Araf(1→ NA [47] DOP-1 6.8×103
Man:Glc=5.18:1.0主链(1→4)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-O-2-acetyl-β-D-Manp、(1→4)-O-3-acetyl-D-Manp 降血糖 [48] DOP-W3-b 1.54×104 Man:Glc=4.5:1.0 主链(1→4)-D-Manp、β-(1→4)-D-Glcp、β-(1→3,6)-D-Manp、(1→4)-O-2-acetyl-β-D-Manp,侧链(1→4)-D-Manp、β-(1→4)-D-Glcp,末端β-D-Glcp(1→
免疫调节[49] DOPW-1 3.90×105 Man:Glc=10.75:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp、(1→4)-O-2-acetyl-β-D-Manp,末端β-D-Manp(1→ 抗糖基化 [50] DOPA-1 3.90×105 Man:Glc=5.8:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp、(1→4)-O-2-acetyl-β-D-Manp 免疫调节 [51] WDOP-1 1.58×106 Man:Glc:Gal:Xyl:Ara:Rha=
35.65:44.89:13.99:12.25:9.13:1.76主链(1→4)-Manp、(1→4)-Glcp,侧链(1→3,4)-Glcp,末端Galp-(1→、Araf(1→ 降血糖 [52] WDOP-2 2.68×106 Man:Glc:Gal:Xyl:Ara:Rha=
72.34:25.87:11.02:10.09:0.5:0.09主链(1→4)-Manp、(1→4)-Glcp,侧链(1→3,4/2,4)-Manp,末端Galp(1→、Manp(1→ 降血糖 [52] 金钗石斛 DNPE6(4) 9.92×104 Ara:Glc:Gal:Man=2.5:0.9:0.3:0.8 主链(1→3,6)-L-Araf、(1→3)-D-Glcp、(1→3/4)-D-Galp、(1→6)-D-Manp,末端β-D-Manp(1→ 抗病毒 [53] DNP1 6.80×103 Man:Glc=3.14:1.0 主链(1→4)-β-D-Manp、(1→4)-β-D-Glcp、(1→4)-O-2/3-acetyl-β-D-Manp 抗炎 [54] JCS1 2.3×104 Glc:Man:Xyl:Ara=40.2:2.3:1.7:1.0 主链(1→4)-β-Manp、(1→4)-α-Glcp,侧链(1→6)-α-Xylp、(1→4)-α-Glcp,末端 α-Araf(1→ 调节神经系统 [55] DNPE6(11) 3.01×103 Man:Glc:Gal=3.0:11.0:3.0 主链(1→4)-D-Glcp、(1→6)-D-Manp、(1→4)-O-2/3-acetyl-β-D-Manp,末端D-Galp(1→ 抗病毒 [56] DNP-W2 1.80×104 Glc:Man:Gal=6.1:2.9:2.0 主链(1→4/6)-β-D-Glcp、(1→4)-β-D-Manp、(1→4)-O-2-acetyl-β-D-Manp,侧链(1→4)-β-D-Glcp、(1→4)-β-D-Manp,末端 α-D-Galp(1→ 免疫调节 [57] DNP-W3 7.1×105 Gal:Rha:Ara=3.1:1.1:1.0 主链(1→3)-β-D-Galp,侧链(1→4)-α-L-Rhap,末端β-L-Arap(1→ 免疫调节 [58] DNP-W4 5.0×105 Man:Glc:Gal:Xyl:Rha:GalA=
1.0:4.9:2.5:0.5:1.0:0.9主链(1→4/6)-β-D-Glcp、(1→6)-β-D-Galp、(1→4)-O-4/6-acetyl-β-D-Glcp、(1→6)-O-3-acetyl-β-D-Galp,侧链(1→6)-β-D-Manp、(1→3)-β-D-Glcp、(1→4)-α-D-GalAp、(1→2)-α-L-Rhap/Xylp,末端 β-D-Manp(1→ 免疫调节 [59] DNP 8.76×104 Rha:Ara:Xyl:Man:Glc:Gal=
1.0:2.8:2.2:30.76:117.96:31.76主链(1→6)-α-D-Glcp、(1→6)-α-D-Galp,侧链(1→4)-α-D-Glcp、(1→4)-α-D-Manp 抗氧化 [60] 鼓槌石斛 DCPP-I-a 6.7×104 Xyl:Glc:Gal=1.44:6.93:12.79 NA 抗增殖 [61] 密花石斛 DDP-1-D 9.44×103 Glc:Man=1.0:3.01 主链(1→4/6)-α-D-Glcp、(1→2)-α-D-Manp、(1→4)-β-D-Manp NA [62] 细茎石斛 DMP4a-1 3.04×103 Glc:Man:Rha:Ara:Gal=
2.87:2.85:1.76:1.27:1.0由1→4、1→3及1→6糖苷键组成 免疫调节 [63] DMP2-A 1.07×104 Ara:Xyl:Man:Glc:Gal=1.0:1.5:0.8:4.5:1.5 由(1→3/4/3,6)-D-Glcp、(1→3/4/3,6)-D-Galp、(1→3,5)-L-Araf、(1→3,6)-D-Manp糖残基构成,末端Xylp(1→ NA [64] 注:Man-甘露糖;Glc-葡萄糖;Gal-半乳糖;Rha-鼠李糖;Ara-阿拉伯糖;Xyl-木糖;GlcA-葡萄糖醛酸;GalA-半乳糖醛酸;acetyl-乙酰基;NA-文献中未提及。 表 3 石斛多糖的生物活性及作用机制
Table 3 Biological activity and mechanism of action of Dendrobium polysaccharides
生物活性 动物模型/体外实验 剂量 药效/机制 文献 抗肿瘤 肝癌HepG-2细胞 50、100、200、400 μg/mL 减少抗凋亡蛋白Bcl-2表达,增加促凋亡蛋白Bax表达 [65] 胃癌MFC细胞 0.025、2.5 mg/mL 上调抑癌基因p53的表达,下调原癌基因c-myc的表达 [40] 子宫颈癌Hela细胞 25、400 μg/mL 上调JNK、ERK和p38丝裂原活化蛋白激酶表达 [66] AOM/DSS诱导的小鼠结直肠癌模型 50、100、200 mg/kg 减少CTL上PD-1表达,增强T细胞在TME中抗肿瘤免疫反应 [67] AOM/DSS诱导的小鼠结直肠癌模型 400、800 μg/mL 激活AMPK-mTOR信号通路,促进CT26细胞过度自噬凋亡 [68] 免疫调节 甲氨蝶呤诱导低免疫小鼠模型 50、100、200 mg/kg 激活免疫因子IFN-γ和IL-4分泌 [69] 急性单核细胞白血病细胞系 25、50、100 μg/mL 提升NF-κB和 ERK1/2蛋白磷酸水平 [70] 雌性ICR小鼠骨髓细胞 0.05、0.1、0.2 mg/mL 促进PPs和MLN的分泌,增加AsIgA的产生 [32] 雄性C57BL/6小鼠脾脏 10~200 μg/mL 增加CD4+T亚群、B淋巴细胞亚群和NK细胞比例,降低CD8+T 亚群比例 [71] 抗炎 DSS 诱导的小鼠急性结肠炎模型 50、100、200 mg/kg 抑制TNF-α、IL-1β和IL-6等炎症因子的产生和释放 [72] DSS诱导小鼠急性结肠炎继发性肝
损伤模型50、100、200 mg/kg 上调Nrf-2、HO-1和NQO-1的mRNA或蛋白表达水平,激活Nrf-2信号通路 [73] 乙醇诱导的大鼠胃溃疡模型 100、300 mg/kg 抑制MMP信号通路的激活 [74] Ⅱ型胶原诱导关节炎CIA小鼠模型 0.1095、0.438 g/kg 抑制NF-κB、MAPKs、PI3K/Akt和JAK1/STAT3信号通路的激活 [75] 香烟诱导的小鼠肺炎模型 100、200、400 mg/kg 降低MAPKs信号转导中的血清AP-1的活性 [76] 抗氧化 体外抗氧化实验 0~3.0 mg/mL 对·OH和ABTS+·均具有一定的清除作用 [78] 体外抗氧化实验 1.0~10.0 mg/mL 对DPPH·具有一定的清除作用 [79] 衰老小鼠、6周龄的年轻雌性小鼠 70 mg/kg 降低MDA的含量,提高SOD、CAT、GSH-Px的活性 [80] 调节肠
道菌群健康小鼠 50、200 mg/kg 调节肠道微生物菌群组成以及代谢,增强肠道物理、生化和免疫屏障功能 [81] 健康小鼠 0.4 g/kg 增加SCFA含量,提高有益微生物群属的多样性 [83] 健康小鼠 不明 代谢产生更多丁酸盐,介导肠道的免疫反应 [84] 降血糖 高脂饮食联合STZ建立的糖尿病小鼠模型 100、200、400 mg/kg 影响cAMP-PKA和Akt/FoxO1信号通路,促进肝糖原合成,抑制肝糖原降解和异生 [85] STZ诱导的糖尿病大鼠 15、25、100 mg/kg 增加血清胰岛素和GLP-1的分泌水平 [48] 棕榈酸诱导的高糖细胞胰岛素抵抗 100、200、400 μg/mL 增加PPAR-γ表达水平,调节细胞胰岛素敏感性 [86] 抗白内障 H2O2 诱导小鼠晶状体损伤 2、4 mg/mL 提高晶状体水溶性蛋白含量及GSH-Px和SOD活性 [87] 腹腔注射链脲菌诱导的糖尿病大鼠模型 25、50、100 mg/kg 抑制介导氧化应激的ERK/Raf信号通路激活 [88] 抗病毒 含有CMV的外壳蛋
白基因质粒的大肠杆菌125、500 μg/mL 对农作物PVY和CMV病毒有较好的杀灭效果 [56] 抗疲劳 负重游泳小鼠疲劳模型 50 mg/kg 减弱疲劳相关代谢物LDH、CK、TG和MDA的水平 [89] 保肝 酒精诱导肝损伤小鼠模型 100、200、400 mg/mL 增加肝脏中GSH-Px水平,平衡酒精诱导下氧化还原 [90] 表 4 石斛多糖的化学修饰
Table 4 Chemical modification of Dendrobium polysaccharides
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