Research Progress on Extraction, Structure Determination, Chemical Modification and Biological Activity of Garlic Polysaccharides
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摘要: 作为大蒜的主要活性成分之一,大蒜多糖具有增强免疫力、抗菌、抗病毒、抗氧化、保肝、降血脂、降血糖等多种生物活性,应用前景广阔。大蒜多糖的提取方法以热水法、酶法和超声辅助法最为常见,大蒜多糖是由果糖、葡萄糖、半乳糖、甘露糖、半乳糖醛酸等组成的杂多糖,乙酰化、硒化和磷酸化等化学修饰可以增加大蒜多糖抗氧化等生物活性。本文从大蒜多糖的提取、结构测定、化学修饰及生物活性的角度出发,系统总结了大蒜多糖的研究现状,未来应关注多糖结构与生物活性的构效关系,深入探讨大蒜多糖的功效机理,以期为大蒜多糖作为功能性产品的开发利用提供理论参考。Abstract: As one of the main active ingredients of garlic, garlic polysaccharide has many biological activities, such as enhancing immunity, antibacterial, antiviral, antioxidant, hepatoprotective, hypolipidemic, hypoglycemic, etc., and has a broad application prospect. The extraction methods of garlic polysaccharides are most common by hot water method, enzyme method and ultrasound-assisted method. It is found by structural determination that garlic polysaccharide is a heteropolysaccharide composed of fructose, glucose, galactose, mannose, galacturonic acid, etc. Chemical modifications such as acetylation, selenization and phosphorylation can increase the antioxidant and other biological activities of garlic polysaccharide. This paper systematically summarizes the current research status of garlic polysaccharides from the perspectives of extraction, structure determination, chemical modification and bioactivity of garlic polysaccharides, and in the future, attention should be paid to the conformational relationship between polysaccharide structure and bioactivity, and the efficacy mechanism of garlic polysaccharides should be explored in depth, with a view to providing theoretical references for the development and utilization of garlic polysaccharides as functional products.
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大蒜,为百合科葱属植物蒜(Allium sativum L.)的地下鳞茎[1],又名胡蒜、蒜头等[2],性温味辛,是日常生活中最常见的调味品。大蒜原产于中亚,目前许多国家都有种植[3]。自汉代起,大蒜种植技术引入我国,之后扩展到全国各地[4]。目前,我国是大蒜的主要生产国。作为一种药食两用资源,大蒜因其独特的风味及增强健康的特性受到广泛关注,目前常用于食物、保健品和膳食补充剂[5]。研究表明,新鲜大蒜所含化学成分十分复杂,除含有特有的活性物质有机硫化合物外,还含有丰富的碳水化合物、蛋白质、氨基酸、脂肪酸、维生素、矿质元素、黄酮和多酚类化合物等[6−7]。这些复杂的成分是大蒜药理活性的物质基础,赋予了大蒜及其分解产物抗氧化[8]、抗炎抑菌[9−10]、保肝抗癌[11−12]、保护心血管[13]、免疫调节[14]等生物学活性,能够预防和治疗多种身体疾病。
多糖也称多聚糖,是10个以上单糖残基通过糖苷键相连而成的聚合体,是构成生命的四大基本物质之一[15]。研究表明,多糖具有抗炎[16]、抑菌[17]、抗氧化[18]、抗肿瘤[19]、降血脂[20]、降血压[21]等多种药理作用。近年来,从天然植物中提取的多糖因具有多种生物活性且安全无毒而备受各界青睐。大蒜多糖是一种水溶性植物多糖[22],约占大蒜鲜重的26%~30%[23]。作为大蒜主要的活性成分之一,大蒜多糖生物学活性广泛,在医药、保健品、食品领域应用前景广阔。然而,目前对于大蒜多糖的研究多见于分离提取和生物活性的理论研究,其应用研究较为局限,不利于大蒜多糖精深产品的开发利用。本文系统总结了大蒜多糖在提取分离、结构组成、化学修饰、药理活性等方面的研究现状,以期为大蒜多糖作为功能性产品的进一步开发应用提供理论参考。
1. 大蒜多糖的提取
不同方法提取大蒜多糖的研究见表1,其中以热水提取法、酶法提取和超声辅助提取法最为常见。
表 1 大蒜多糖的提取方法Table 1. Extraction methods of garlic polysaccharides序号 提取方法 提取条件 得率(%) 来源 参考
文献温度(℃) 时间(min) 料液比 功率(W) 酶类型及比例 酶添加量 1 热水浸提法 81 84 1:14 / / / 16.60 江苏扬州 [24] 2 热水浸提法 90 254 1:35 / / / 4.04 吉林长春 [25] 3 热水浸提法 80 150 1:7 / / / 6.29 重庆沙坪坝 [26] 4 酶法提取 50 160 1:25 / 纤维素酶、果胶酶(1:1) 2% 34.76 江苏徐州 [27] 5 酶法提取 67.2 60 1:30 / 纤维素酶、果胶酶(5:1) 1.5% 33.60 江苏邳州 [28] 6 酶法提取 55 90 1:30 / 木瓜蛋白酶 2% 5.51 吉林长春 [29] 7 酶法提取 45 80 / / 纤维素酶 800 U/g 35.34 江苏新浦 [30] 8 超声波辅助水提法 / 8 1:30 150 / / 31.48 江苏徐州 [31] 9 超声波辅助水提法 50 40 1:40 350 / / 25.12 陕西汉中 [32] 10 超声波辅助复合酶法 50 90 1:20 350 果胶酶、酸性蛋白酶(3:1) 1.5% 35.25 河南商丘 [33] 11 超声波辅助复合酶法 45 30 1:6 400 果胶酶、纤维素酶、木瓜蛋白酶(1:7:4) / 25.80 山东青岛 [34] 12 超声波辅助复合酶法 49.5 16 1:7.6 400 果胶酶、纤维素酶、木瓜蛋白酶(1:7:4) / 26.12 山东青岛 [35] 13 微波辅助水提法 65 6 1:7 200 / / 19.2 湖南永州 [36] 14 电场辅助提取法 70 60 1:20 / / / 9.73 江苏无锡 [37] 注:/表示未提到;表2、表3同。 近年来,学者们利用超声、微波等手段对大蒜多糖进行辅助提取,并探索了影响大蒜多糖得率的主要因素,相关试验研究进一步提高了大蒜多糖的得率。董玉玮等[27]发现酶解时间是影响大蒜多糖得率最重要的因素,其次是酶添加量和酶解温度,在酶解时间160 min,纤维素酶和果胶酶添加量2%,酶解温度50 ℃时,大蒜多糖得率达34.76%;席椿凯等[31]发现超声辅助提取法影响多糖得率最主要的因素是料液比,在料液比1:30,超声功率为150 W,超声时间8 min的条件下,大蒜多糖得率为31.48%。
如表1所示,热水提取法时间长且温度高,提取的多糖得率较低,而酶法和超声辅助提取法提取时间短且温度低,多糖得率普遍提高。热水提取法较为传统,存在能耗高、得率低的问题;超声波提取法效率高、得率高、无污染且操作安全简单,其通过空化作用和高频振动破坏细胞,加速细胞内有效物质的溶出,达到增产的效果[38−39];酶法反应条件温和,能高效地促进细胞壁破裂,加快多糖的浸出,所得多糖产物性质稳定,活性较高[28,40]。由此可见,大蒜多糖的得率与提取方法有着密切的联系,不同的提取方法原理不同,提取效果不同,多糖得率不同。因此,采用合适的提取方法至关重要。
尽管超声波提取法和酶法在提高多糖得率方面效果显著,但其仍是一种辅助手段,需依赖传统的热水提取方法,过高的温度会造成样品的降解。Yang等[37]发现激发电压、频率、pH和温度均能影响多糖的提取,在激发电压200 V、频率20 kHz、pH2、温度70 °C的条件下,采用感应电场法提取的大蒜多糖得率可达9.73%,且具有较强的DPPH自由基清除活性和铁还原能力。尽管和上述酶法相比,多糖得率有所下降,但其构建的变压装置为多糖的提取提供了一种新的可行思路,未来仍需寻找更为合适的技术来提高大蒜多糖的得率。
2. 大蒜多糖的结构测定
多糖的生物功能取决于其化学结构,因此,阐明大蒜多糖的结构组成是研究开发的前提。作为大蒜鳞茎中极为重要的生物活性物质,大蒜多糖结构多样,品种不同,所含多糖不同,因此测定的结构也不同。目前,对大蒜多糖结构的研究主要集中在分子量、单糖组成及糖苷键构型等方面[41−44]。高效液相凝胶色谱法和高效空间排阻色谱常用于多糖分子量的测定;高效液相色谱法、离子色谱、气相色谱法用于单糖组成的测定;红外光谱和核磁共振主要对多糖的主链构型和糖链分支进行结构分析。
Chen等[45]发现大蒜多糖是以果糖为主的杂多糖,通过对其结构表征发现,大蒜的果聚糖结构主要由β-糖苷键连接,且不含糖醛酸。刘平香等[46]通过研究发现,大蒜多糖为小分子杂多糖,平均分子量为9~10 kDa。邵鑫[47]经提取纯化得到一种小分子水溶性的大蒜多糖,是一种同时含有α和β构型糖苷键且以β-糖苷键为主的果聚糖,经结构鉴定和解析发现其由2.60%的岩藻糖、32.50%的葡萄糖和64.90%的果糖组成。李瑞瑞[48]通过薄层色谱鉴别发现大蒜中主要存在果糖蔗糖、蔗果三糖、蔗果四糖和多糖等物质,并建立了测定不同海拔大蒜单糖和低聚糖含量的新方法。Li等[49]通过HPTLC分析了新鲜大蒜和黑蒜的单糖组分,结果表明,大蒜多糖主要由果糖、半乳糖和半乳糖醛酸组成,摩尔比为307:25:32,而黑蒜在发酵过程中果聚糖被分解,黑蒜多糖主要由半乳糖和半乳糖酸组成,摩尔比为63:20。
综上,大蒜多糖的单糖组成种类丰富,是以果糖为主的由果糖、葡萄糖、半乳糖、甘露糖、阿拉伯糖、葡萄糖醛酸、半乳糖醛酸等组成的杂多糖[50]。由于大蒜品种及提取测定方法存在差异,因此学者所获得的大蒜多糖结构及其组成也存在一定程度的差异[51],大蒜多糖分离纯化及结构测定的相关研究见表2。
表 2 大蒜多糖分离纯化及结构测定Table 2. Separation, purification and structure determination of garlic polysaccharide序号 多糖类型 提取方法 纯化方法 分子量(kDa) 单糖组成 糖苷键/结构 结构表征方法 参考文献 1 中性大蒜多糖GPS 水提醇沉法 DEAE52纤维素柱+超滤 6.57 果糖、葡萄糖 β-糖苷键 傅里叶变换红外光谱 [41] 2 酸性大蒜多糖GPS 水提醇沉法 三相分割法(TPP)+梯度乙醇沉淀法(GEP)+透析 8.93~10.62 岩藻糖、鼠李糖、半乳糖、葡萄糖、果糖 存在果糖呋喃基,具有果糖的结构特征 傅里叶变换红外光谱 [42] 3 大蒜多糖GPS 热水浸提法 SephadexG200柱层析 7.35 果糖、葡萄糖、半乳糖 β-糖苷键 傅里叶变换红外光谱 [43] 4 大蒜多糖
GBP-1a水提醇沉法 DEAESepharose色谱柱+
Sephacryl™200凝胶柱15 半乳糖、葡萄糖和阿拉伯糖 β-糖苷键 傅立叶变换红外光谱+
1H 13C核磁共振波谱[52] 5 大蒜低聚果糖LMWF / DEAE-纤维素色谱柱+
Sephadex G-25色谱柱1.77 D -果糖、D-葡萄糖 (2,1)连接的β-D-Fruf主链和(2,6)连接的β-D-Fruf侧链,具有菊糖的结构特征 傅里叶变换红外光谱+
1H 13C核磁共振波谱[53] 6 大蒜低聚糖GOs 热水提取法 SephadexG-75 凝胶柱+
DEAE-Toyopearl色谱柱1.8 葡萄糖、蔗糖 十个果糖分子通过β1-2键与末端葡萄糖相连 1H 13C核磁共振波谱 [54] 7 水溶性大蒜多糖WSGP 热水浸提法 DEAE-52纤维素柱+
Sephadex G-100色谱柱1.853 果糖、葡萄糖、焦糖 β-d-聚呋喃苷、β-(1,2)键 傅里叶变换红外光谱+
1H 13C核磁共振波谱[55] 8 大蒜果聚糖GF 水提醇沉法 DEAE52纤维素柱+
Sephadex G-100色谱柱4.54 果糖、葡萄糖 β-糖苷键/(2→1)-d-果糖吡喃糖 傅里叶变换红外光谱+
1H 13C核磁共振波谱[56] 9 大蒜多糖PSG 热水浸提法 乙醇分级沉淀+透析 8.242 半乳糖、葡萄糖、阿拉伯糖 β-糖苷键 傅里叶变换红外光谱 [57] 3. 大蒜多糖的化学修饰
实践证明,多糖活性与分子结构有着密切的联系[50]。通过适当的化学修饰,多糖的生物活性大大增加,毒副作用明显减少,且赋予了多糖许多新的药用价值[26]。目前,常用的修饰多糖方法有羧甲基化、硫酸化、磷酸化、硒化等,极大改变了多糖的抗氧化等生物活性,扩大了多糖在食品医药等领域的应用。
作为最常用的化学改性方法之一,乙酰化可使多糖单元的羟基转化成乙酰基,不仅改变多糖的物化性质,还能影响多糖的活性。Chen等[45]通过化学改性大蒜多糖,并通过羟胺量热法和酸碱滴定法测定,成功合成了乙酰基大蒜多糖,并制备得到锌含量为8.8%的大蒜多糖锌络合物,通过体外抗氧化试验证明了乙酰化是增强大蒜多糖抗氧化活性的有利途径。此外,Chen等[58]以大蒜多糖和三氯化铁为原料,通过共热法合成了大蒜多糖-铁(Ⅲ)配合物,经过鉴定发现其属于果聚糖,糖苷键由β-糖苷键连接,尽管稳定性略有下降,但其抗氧化效果明显增强,该复合物不仅保留了多糖和金属离子的活性,还可避免游离态离子所引起的不良反应,副作用小,配位稳定,易被吸收和利用,在调节免疫、抗病毒等生物活性方面发挥作用,可以作为一种新型的金属元素补充剂。
近年来,硒化多糖倍受关注。与亚硒酸钠等无机硒相比,硒多糖的生物活性高于硒和多糖,且易于吸收,毒性小[59]。研究表明,元素硒在大蒜抗菌、抗病毒、抗癌等功能中起到了重要作用[60]。邱树磊等[59]采用HNO3-Na2SeO3法得到9个硒化大蒜多糖,发现硒化修饰能提高大蒜多糖抗新城疫病毒的活性。此外,经HNO3-Na2SeO3法硒化得到的大蒜多糖能够促进细胞因子TNF-α、IL-1β和IL-6的分泌[61]。Gao等[62]发现亚硒化修饰可以增强大蒜多糖的免疫活性,HNO3-Na2SeO3法效率最高,且成本低、制备简单、产品回收方便。硒化修饰能够显著提高大蒜多糖的抗氧化活性,可能的原因是硒作为生命活动中的必需元素,参与了辅酶Q的生物合成、谷胱氨肽过氧化物酶的激活、抗体合成的刺激等过程,通过分子修饰和结构重建使多糖产生了新的活性或者增强了活性[63]。
多糖的磷酸化修饰是将磷酸键引入到多糖中,由于磷酸根三个负电荷的存在,增加了电负性,影响了多糖的某些活性。Chen等[50]发现磷酸化大蒜多糖清除羟基自由基的能力明显强于大蒜多糖,推测原因可能是大蒜多糖被磷酸化导致其分子结构发生变化,提高了其阻止•OH链式反应的能力。Cheng等[26]对羧甲基化大蒜多糖进行了硫酸化和磷酸化修饰,发现单基团和双基团的化学修饰影响了多糖的抗氧化活性,磷酸基的引入增强了羟基自由基的清除能力,羧甲基的引入增强了超氧化物阴离子的清除能力。程浩[60]通过化学修饰改变了大蒜多糖的抗氧化活性,磷酸化及羧甲基磷酸化提高了羟自由基清除能力,磷酸基团的引入加强了多糖的抗脂质过氧化能力。单基团的化学修饰和双基团的化学修饰对多糖的抗氧化活性有不同程度的影响。因此,对大蒜多糖进行不同的化学修饰可产生多种不同的药用价值,具有良好的应用前景。
4. 大蒜多糖的生物活性
4.1 抗炎抑菌作用
作为植物类广谱抗生素,大蒜对多种细菌和真菌具有良好的抑制作用,甚至强于一些化学抑菌剂及抗生素,是替代抗生素、抑菌剂、农药兽药的最佳选择[64]。顾林等[65]利用蒜渣提取多糖,发现水提多糖和碱提多糖可以抑制大肠杆菌、沙门氏菌、金黄色葡萄球菌的生长,其中碱提多糖抑菌效果更佳。
当受到外界环境的刺激时,机体会触发炎症反应,引发免疫系统的防御功能,反应过强时会对机体造成严重危害[66]。研究表明,大蒜多糖能够激活免疫器官,促进巨噬细胞和淋巴细胞的增殖分化,刺激免疫因子分泌,从而起到较好的抗炎作用[5]。Shao等[55]经分析发现大蒜多糖能够改善肠黏膜屏障,抑制白细胞介素和肿瘤坏血因子的表达,促进短链脂肪酸的产生,调节肠道菌群的组成,减轻了葡聚糖硫酸钠诱导的小鼠结肠炎。邵鑫[47]发现大蒜多糖改善了急性溃疡性结肠炎小鼠的功能障碍,通过调控代谢通路减轻了肠道菌群失衡,较好地发挥了抗炎作用。
4.2 抗氧化活性
多糖的还原能力与羟基数目有关,通过充当电子供体与自由基进行反应,将其转化为更稳定的产物来终止自由基链式反应[67]。一方面,多糖分子可直接作用于酶,通过提高过氧化氢酶、超氧化物歧化酶及谷胱甘肽过氧化物酶的活性来体现抗氧化能力;另一方面,多糖的羟基基团可以与金属离子结合,阻止金属离子催化产生羟基自由基[68]。
Bo等[67]采用HNO3-Na2SeO3法制备硒化大蒜多糖,发现硒化修饰可以提高多糖的抗氧化活性,推测其原因是硒从硒酸盐中提供一个氢原子或电子给羟基自由基,从而形成水,阻止了自由基链反应,达到抗氧化的效果[69]。Chen等[45]制备了乙酰化大蒜多糖和大蒜多糖锌复合物,发现乙酰化是提高大蒜多糖抗氧化活性的有力途径,大蒜多糖锌复合物可以作为抗氧化剂和锌补充剂的潜在候选物。研究发现,大蒜多糖铁复合物也有类似的抗氧化效果,与大蒜多糖相比,复合物抗氧化活性最高,协同作用更明显[58]。
4.3 护肝作用
作为天然的植物多糖,大蒜多糖安全低毒,来源广泛,提取工艺简单。重要的是,大蒜多糖既具有良好的抗病毒作用,又对肝功能有较好的保护作用,是一种较为理想的护肝物质[70]。
孔祥槐等[70]发现大蒜多糖对卡介苗加脂多糖诱发的免疫性肝损伤有保护作用,600 mg/(kg·d)剂量组可有效降低免疫性肝损伤小鼠血清谷丙转氨酶活性,提高肝组织转氨酶水平,同时降低血清NO水平,改善肝细胞水肿及变性程度。李瑞瑞[48]发现完整的大蒜经高温、胃酸等灭酶后,灭酶体系中的多糖仍具有抗氧化活性,能降低血清和肝脏中转氨酶活力,提高肝脏中谷胱甘肽过氧化物酶活力,降低肿瘤坏血因子水平,对小鼠化学性肝损伤有保护作用。Wang等[71]通过研究发现,大蒜多糖具有类似益生元的作用,可通过诱导微生物竞争来减少有害肠道菌群的数量,酒精性肝纤维化小鼠经大蒜多糖预处理后,螺菌科和乳杆菌属增加,费克蓝姆氏菌属和厚壁菌门减少,肝保护作用可能与抑制转化生长因子、肿瘤坏血因子的表达和促进核心蛋白的表达有关。范颖等[72]通过实验发现25 mg/mL的大蒜多糖预防给药能够调节急性酒精性肝损伤导致的肠道菌群失调,肠道中优杆菌属和乳酸菌属数量增多,肠道菌群结构多样性和均匀度增加,其效果与长春海外制药集团有限公司生产的护肝片(批号:Z22020994)相似。
由于“肠—肝”轴的存在,肠道菌群与肝损伤的发生有着密切的联系,包括大蒜多糖在内的许多天然产物都具有调节肠道菌群平衡的作用,赋予了其改善肝脏功能的生物活性。
4.4 增强免疫力
作为大蒜的主要成分之一,多糖具有广泛的免疫调节活性。Li等[49]通过比较新鲜大蒜和黑蒜多糖的免疫调节作用,发现新鲜大蒜多糖的免疫调节活性更强,可以促进巨噬细胞吞噬、NO生成和细胞因子的表达,而黑蒜多糖基本没有免疫调节作用,可能原因是在发酵过程中,果聚糖成分发生了降解。Qiu等[73]发现具有较强免疫增强活性的硒化多糖需要较高硒含量和较高碳水化合物含量的组合,大蒜多糖通过HNO3-Na2SeO3法改性后能促进鸡外周血淋巴细胞增殖,提高血清抗体滴度、干扰素和白细胞介素的含量。刘宽辉等[74]通过体外试验和体内试验,筛选出了新型免疫增强剂的复方,发现大蒜多糖-硒化党参多糖促进T淋巴细胞增殖的作用最强,血清抗体效价和IL-2、IL-6及IFN-γ含量均为最高,且显著高于免疫对照组。纪迅[75]采用HNO3-Na2SeO3法得到了硒含量为38.27 mg/g的大蒜硒化多糖,研究发现其可以促进生长,且可以提高新城疫鸡的血清抗体效价,促进空肠、气管IgA、IFN-γ和IL-2的分泌而提高黏膜免疫。Bo等[67]采用HNO3-Na2SeO3法制备了硒化大蒜多糖,通过体内研究发现其能显著提高抗新城疫病毒的血清凝集抑制的抗体滴度,增强空肠和气管灌洗液IgA、IFN-γ、IL-2的分泌,硒化改变了大蒜多糖的聚集体外观,其形态为球形,这可能是硒化多糖效果较好的原因之一。
4.5 增强记忆力
随着年龄的不断增加,机体抗氧化能力逐渐递减,氧化自由基产生逐渐增多。研究表明,氧化自由基损伤参与脑组织衰老的过程[76]。植物多糖具有抗氧化的生物活性,不仅能增强免疫、抑制自由基,而且能改善东莨菪碱、亚硝酸钠、D-半乳糖等所导致的小鼠多种学习记忆障碍[77−78]。
陈红光等[79]通过跳台实验证实大蒜多糖具有改善东莨菪碱致小鼠学习记忆障碍的作用,600 mg/kg大蒜多糖组小鼠跳台错误次数显著降低,触电潜伏期明显延长,被动回避反应的记忆获得作用明显改善。陈翠桃等[80]发现海马区细胞损伤导致了小鼠学习记忆神经障碍,而大蒜多糖可以改善因持续过量饮酒导致的学习记忆能力下降和脑组织损伤,其机制与调节改善乙酰胆碱酶和单胺氧化酶等神经系统的功能有关。
4.6 其他
大蒜多糖是大蒜中一类重要的活性成分,也是大蒜功能活性的物质基础之一,除具有抗氧化、保护肝脏、提高免疫力功能外,大蒜多糖还具有抗疲劳、防治便秘、降血糖、抗凝血、抗肿瘤、益生等多种生物活性,且安全性高、毒副作用小,可药食两用,近年来受到了学者的广泛关注。
大蒜多糖相关生物活性及作用机理研究见表3。
表 3 大蒜多糖的生物活性及机理研究Table 3. Studies on the biological activity and mechanism of garlic polysaccharides序号 提取方法 纯化方法 生物活性 试验类型 作用剂量 作用效果 作用机理 参考文献 1 热水浸提法 Sevage法除蛋白+
活性炭脱色抗疲劳 SPF级,雄性,昆明小鼠 800 mg/kg 延长力竭游泳时间 提高抗氧化酶活力,清除脂质过氧化产物,降低生物膜受损程度,抑制能量代谢紊乱,延缓疲劳 [81] 2 / / 防治便秘 CL级,雄性,昆明小鼠 6.6 g/kg 促进便秘模型小鼠小肠推进作用 多糖组成为果聚糖,能促进双歧杆菌增殖,促进肠道蠕动,增加粪便水分,防止便秘 [82] 3 热水浸提法 活性炭脱色+阴离子层析柱、阳离子层析柱纯化 降血糖 雄性,
昆明小鼠5 g/kg 血糖较糖尿病模型组
降低42%调节葡萄糖激酶、糖原合成酶和磷酸烯醇丙酮酸羧激酶来调控肝糖原代谢 [83] 4 碱提法 Sevage法除蛋白+DEAE-52纤维素柱层析+SephadexG-100凝胶层析 抗凝血 体外 4 mg/moL 活化部分凝血活酶时间延长 半乳糖是粘附分子凝集素家族识别的配体,与凝集素结合,阻断了凝集素在血栓形成中的粘附作用 [84] 5 热水浸提法 Sevage法除蛋白除蛋白、醇沉 抗肿瘤 体外,人HepG2细胞系 1 mg/mL 抑制肿瘤细胞增殖,
48 h抑制率26.18%可诱导Hep G2细胞停滞于G2/M期,通过诱导癌细胞凋亡
发挥抗肿瘤作用[85] 6 / / 益生 SPF级,雌性,昆明小鼠 9 g/kg 双岐杆菌、乳酸杆菌数量增加 具有益生元的功效 [86] 7 热水浸提法 除蛋白,醇沉 提高免疫力 SPF级,雌性,昆明小鼠 800 mg/kg 脾中CD4+T细胞百分比明显升高 提高小鼠的免疫功能,从而提高机体的抗病能力和抵抗力 [87] 8 热水浸提法 Sevage法除蛋白、醇沉、DEAE-52纤维素柱、Sephadex G-100柱纯化 抗炎 C57BL/6,
雄性,小鼠400 mg/kg 改善结肠炎症状,减轻结肠组织损伤 抑制促炎细胞因子IL-6、IL-1β和TNF-α的产生和基因表达 [55] 9 超声辅助提取法 TCA法除蛋白、醇沉、3500 Da膜透析 护肝 C57BL/6J,
雄性,小鼠300 mg/kg 抑制炎症性肠病,缓解继发性肝损伤 逆转过氧化物酶、二胺氧化酶活性、iNOS水平,增加ZO1、occludin和MUC2的表达 [57] 10 热水浸提法 Sevage法除蛋白、
醇沉、柱层析抗氧化 体外 1.0 mg/mL 抑制羟基自由基、超氧阴离子、脂质过氧化 乙酰基的引入影响大蒜多糖的
极性、构象或电荷密度[45] 11 热水浸提法 乙醇分级沉淀、DEAE-52纤维素
柱纯化护肝 雄性,
昆明小鼠250 mg/kg 对血清血浆中酶活、脂蛋白和肝脏指数的影响与阳性药物护肝片相当 调节脂质过氧化和氧化应激,
调节TGF-b1、TNF-a和核心蛋
白聚糖信号通路[71] 12 / / 降血脂 SPF级,雄性,昆明小鼠 5.0 g/kg 降低糖尿病小鼠的甘油三酯和低密度脂蛋白胆固醇水平 改善正常人群肠道菌群,缓解糖尿病引起的脂质代谢紊乱 [88] 5. 结论与展望
大蒜多糖属于杂多糖,各种糖的组成和构成比例不同,实验方法和操作条件会影响多糖的纯度和组分,从而影响其生物活性。因此,在不影响活性的前提下提高多糖得率及寻求新的提取纯化方法是将来研究中需要关注的问题。结构特征与多糖的生物活性联系紧密,单糖组成、糖链构型等初级结构是目前研究多糖主要的方向,其高级结构的研究较少,多糖结构与生物活性的构效关系目前尚不清楚。未来可以从多糖结构与生物活性关系的角度出发,明确多糖生物活性所处的分子量范围及单糖组成,以优化大蒜多糖在食品及其他领域中的应用。
当前市场上的大蒜仅作为调味品出现在大众视野中,精深加工产品极为罕见。经化学改性、发酵等工艺,大蒜多糖可加工成黑蒜、磷酸化、硫酸、硒化大蒜多糖及大蒜多糖锌复合物等新型产品,附加值较高,可用于免疫增强剂、膳食补充剂等食品药品的生产,在保健食品及功能性药品上具有广阔的应用价值。虽然大蒜多糖的药理作用报道较多,但多见于体外试验,有关进一步的作用机制研究较少,未来应注重从人体健康和医学的角度深刻阐明其作用机理,从而有助于其作为辅助类药物应用于临床治疗。除应用于医药卫生领域外,大蒜多糖还具有抗氧化、抑菌等功效,可考虑作为添加剂应用于肉制品、奶制品及饮料等产品中,也可作为饲料添加剂防治细菌感染,提高畜禽免疫力,改善肉制品品质。
综合而言,大蒜多糖的研究仍处于初级阶段,关于其结构组成、作用机理及精深产品的研究还需学者们进行更深入的探索。
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表 1 大蒜多糖的提取方法
Table 1 Extraction methods of garlic polysaccharides
序号 提取方法 提取条件 得率(%) 来源 参考
文献温度(℃) 时间(min) 料液比 功率(W) 酶类型及比例 酶添加量 1 热水浸提法 81 84 1:14 / / / 16.60 江苏扬州 [24] 2 热水浸提法 90 254 1:35 / / / 4.04 吉林长春 [25] 3 热水浸提法 80 150 1:7 / / / 6.29 重庆沙坪坝 [26] 4 酶法提取 50 160 1:25 / 纤维素酶、果胶酶(1:1) 2% 34.76 江苏徐州 [27] 5 酶法提取 67.2 60 1:30 / 纤维素酶、果胶酶(5:1) 1.5% 33.60 江苏邳州 [28] 6 酶法提取 55 90 1:30 / 木瓜蛋白酶 2% 5.51 吉林长春 [29] 7 酶法提取 45 80 / / 纤维素酶 800 U/g 35.34 江苏新浦 [30] 8 超声波辅助水提法 / 8 1:30 150 / / 31.48 江苏徐州 [31] 9 超声波辅助水提法 50 40 1:40 350 / / 25.12 陕西汉中 [32] 10 超声波辅助复合酶法 50 90 1:20 350 果胶酶、酸性蛋白酶(3:1) 1.5% 35.25 河南商丘 [33] 11 超声波辅助复合酶法 45 30 1:6 400 果胶酶、纤维素酶、木瓜蛋白酶(1:7:4) / 25.80 山东青岛 [34] 12 超声波辅助复合酶法 49.5 16 1:7.6 400 果胶酶、纤维素酶、木瓜蛋白酶(1:7:4) / 26.12 山东青岛 [35] 13 微波辅助水提法 65 6 1:7 200 / / 19.2 湖南永州 [36] 14 电场辅助提取法 70 60 1:20 / / / 9.73 江苏无锡 [37] 注:/表示未提到;表2、表3同。 表 2 大蒜多糖分离纯化及结构测定
Table 2 Separation, purification and structure determination of garlic polysaccharide
序号 多糖类型 提取方法 纯化方法 分子量(kDa) 单糖组成 糖苷键/结构 结构表征方法 参考文献 1 中性大蒜多糖GPS 水提醇沉法 DEAE52纤维素柱+超滤 6.57 果糖、葡萄糖 β-糖苷键 傅里叶变换红外光谱 [41] 2 酸性大蒜多糖GPS 水提醇沉法 三相分割法(TPP)+梯度乙醇沉淀法(GEP)+透析 8.93~10.62 岩藻糖、鼠李糖、半乳糖、葡萄糖、果糖 存在果糖呋喃基,具有果糖的结构特征 傅里叶变换红外光谱 [42] 3 大蒜多糖GPS 热水浸提法 SephadexG200柱层析 7.35 果糖、葡萄糖、半乳糖 β-糖苷键 傅里叶变换红外光谱 [43] 4 大蒜多糖
GBP-1a水提醇沉法 DEAESepharose色谱柱+
Sephacryl™200凝胶柱15 半乳糖、葡萄糖和阿拉伯糖 β-糖苷键 傅立叶变换红外光谱+
1H 13C核磁共振波谱[52] 5 大蒜低聚果糖LMWF / DEAE-纤维素色谱柱+
Sephadex G-25色谱柱1.77 D -果糖、D-葡萄糖 (2,1)连接的β-D-Fruf主链和(2,6)连接的β-D-Fruf侧链,具有菊糖的结构特征 傅里叶变换红外光谱+
1H 13C核磁共振波谱[53] 6 大蒜低聚糖GOs 热水提取法 SephadexG-75 凝胶柱+
DEAE-Toyopearl色谱柱1.8 葡萄糖、蔗糖 十个果糖分子通过β1-2键与末端葡萄糖相连 1H 13C核磁共振波谱 [54] 7 水溶性大蒜多糖WSGP 热水浸提法 DEAE-52纤维素柱+
Sephadex G-100色谱柱1.853 果糖、葡萄糖、焦糖 β-d-聚呋喃苷、β-(1,2)键 傅里叶变换红外光谱+
1H 13C核磁共振波谱[55] 8 大蒜果聚糖GF 水提醇沉法 DEAE52纤维素柱+
Sephadex G-100色谱柱4.54 果糖、葡萄糖 β-糖苷键/(2→1)-d-果糖吡喃糖 傅里叶变换红外光谱+
1H 13C核磁共振波谱[56] 9 大蒜多糖PSG 热水浸提法 乙醇分级沉淀+透析 8.242 半乳糖、葡萄糖、阿拉伯糖 β-糖苷键 傅里叶变换红外光谱 [57] 表 3 大蒜多糖的生物活性及机理研究
Table 3 Studies on the biological activity and mechanism of garlic polysaccharides
序号 提取方法 纯化方法 生物活性 试验类型 作用剂量 作用效果 作用机理 参考文献 1 热水浸提法 Sevage法除蛋白+
活性炭脱色抗疲劳 SPF级,雄性,昆明小鼠 800 mg/kg 延长力竭游泳时间 提高抗氧化酶活力,清除脂质过氧化产物,降低生物膜受损程度,抑制能量代谢紊乱,延缓疲劳 [81] 2 / / 防治便秘 CL级,雄性,昆明小鼠 6.6 g/kg 促进便秘模型小鼠小肠推进作用 多糖组成为果聚糖,能促进双歧杆菌增殖,促进肠道蠕动,增加粪便水分,防止便秘 [82] 3 热水浸提法 活性炭脱色+阴离子层析柱、阳离子层析柱纯化 降血糖 雄性,
昆明小鼠5 g/kg 血糖较糖尿病模型组
降低42%调节葡萄糖激酶、糖原合成酶和磷酸烯醇丙酮酸羧激酶来调控肝糖原代谢 [83] 4 碱提法 Sevage法除蛋白+DEAE-52纤维素柱层析+SephadexG-100凝胶层析 抗凝血 体外 4 mg/moL 活化部分凝血活酶时间延长 半乳糖是粘附分子凝集素家族识别的配体,与凝集素结合,阻断了凝集素在血栓形成中的粘附作用 [84] 5 热水浸提法 Sevage法除蛋白除蛋白、醇沉 抗肿瘤 体外,人HepG2细胞系 1 mg/mL 抑制肿瘤细胞增殖,
48 h抑制率26.18%可诱导Hep G2细胞停滞于G2/M期,通过诱导癌细胞凋亡
发挥抗肿瘤作用[85] 6 / / 益生 SPF级,雌性,昆明小鼠 9 g/kg 双岐杆菌、乳酸杆菌数量增加 具有益生元的功效 [86] 7 热水浸提法 除蛋白,醇沉 提高免疫力 SPF级,雌性,昆明小鼠 800 mg/kg 脾中CD4+T细胞百分比明显升高 提高小鼠的免疫功能,从而提高机体的抗病能力和抵抗力 [87] 8 热水浸提法 Sevage法除蛋白、醇沉、DEAE-52纤维素柱、Sephadex G-100柱纯化 抗炎 C57BL/6,
雄性,小鼠400 mg/kg 改善结肠炎症状,减轻结肠组织损伤 抑制促炎细胞因子IL-6、IL-1β和TNF-α的产生和基因表达 [55] 9 超声辅助提取法 TCA法除蛋白、醇沉、3500 Da膜透析 护肝 C57BL/6J,
雄性,小鼠300 mg/kg 抑制炎症性肠病,缓解继发性肝损伤 逆转过氧化物酶、二胺氧化酶活性、iNOS水平,增加ZO1、occludin和MUC2的表达 [57] 10 热水浸提法 Sevage法除蛋白、
醇沉、柱层析抗氧化 体外 1.0 mg/mL 抑制羟基自由基、超氧阴离子、脂质过氧化 乙酰基的引入影响大蒜多糖的
极性、构象或电荷密度[45] 11 热水浸提法 乙醇分级沉淀、DEAE-52纤维素
柱纯化护肝 雄性,
昆明小鼠250 mg/kg 对血清血浆中酶活、脂蛋白和肝脏指数的影响与阳性药物护肝片相当 调节脂质过氧化和氧化应激,
调节TGF-b1、TNF-a和核心蛋
白聚糖信号通路[71] 12 / / 降血脂 SPF级,雄性,昆明小鼠 5.0 g/kg 降低糖尿病小鼠的甘油三酯和低密度脂蛋白胆固醇水平 改善正常人群肠道菌群,缓解糖尿病引起的脂质代谢紊乱 [88] -
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