CN114031693B - Chemical degradation method of dendrobium officinale polysaccharide - Google Patents
Chemical degradation method of dendrobium officinale polysaccharide Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
本发明属于多糖制备领域,具体涉及一种铁皮石斛多糖的化学降解方法。采用二乙烯三胺五乙酸螯合亚铁离子与过氧化氢反应形成Fenton体系,产生的羟基自由基氧化降解铁皮石斛多糖。本发明提供的降解方法工艺简单,采用该方法处理铁皮石斛多糖后能够有效降低其分子量,利于后续铁皮石斛多糖的利用。
The invention belongs to the field of polysaccharide preparation, in particular to a chemical degradation method of Dendrobium officinale polysaccharide. Diethylenetriaminepentaacetic acid was used to chelate ferrous ions and hydrogen peroxide to form a Fenton system, and the generated hydroxyl radicals were oxidized to degrade Dendrobium officinale polysaccharides. The degradation method provided by the invention has a simple process, and the method can effectively reduce the molecular weight of the Dendrobium officinale polysaccharide after processing the method, which is beneficial to the subsequent utilization of the Dendrobium officinale polysaccharide.
Description
技术领域technical field
本发明属于多糖制备领域,具体涉及一种铁皮石斛多糖的化学降解方法。The invention belongs to the field of polysaccharide preparation, in particular to a chemical degradation method of Dendrobium officinale polysaccharide.
背景技术Background technique
铁皮石斛(学名:Dendrobium officinale)为兰科石斛属植物,其叶为两列长圆状披针形,茎不分支,具多节。铁皮石斛常以新鲜或干燥茎入药,具有滋阴清热、生津益胃、润肺止咳、明目强身等功效,是传统名贵中药材。Dendrobium officinale (scientific name: Dendrobium officinale) is a plant of the orchid family Dendrobium. Dendrobium officinale is often used as medicine with fresh or dried stems. It has the functions of nourishing yin and clearing heat, promoting body fluid and benefiting the stomach, moistening the lungs and relieving cough, improving eyesight and strengthening the body. It is a traditional and precious Chinese medicinal material.
铁皮石斛多糖是铁皮石斛中的主要活性成分,含量范围多在10-49%内,为一种水溶性杂多糖,多以α、β-(1→4)糖苷键连接葡萄糖和半乳糖构成。铁皮石斛多糖具有增强免疫、抗肿瘤、降血糖、抗氧化等生理活性,具有重要的食用及药用价值。Dendrobium officinale polysaccharide is the main active ingredient in Dendrobium officinale, and its content is mostly in the range of 10-49%. Dendrobium officinale polysaccharide has physiological activities such as enhancing immunity, anti-tumor, lowering blood sugar, and anti-oxidation, and has important edible and medicinal value.
但是,多糖的生物吸收率很低,经口摄取后肠胃吸收率低于5%。当前的研究很多是基于细胞实验,忽略了吸收率低这一问题,而注射方式有诸多的安全性问题。所以,降低多糖的分子量、提高消化系统吸收率、提高或保持其生理活性,是多糖研究的热点。However, the bioabsorption rate of polysaccharides is very low, and the gastrointestinal absorption rate is less than 5% after oral ingestion. Many current studies are based on cell experiments, ignoring the low absorption rate, and the injection method has many safety issues. Therefore, reducing the molecular weight of polysaccharides, improving the absorption rate of the digestive system, and improving or maintaining their physiological activities are the hotspots of polysaccharide research.
目前针对铁皮石斛多糖的降解主要有物理降解法、化学降解法和生物降解法。物理降解法主要包括超声降解法和微波降解法,无需引入其他反应物,操作简单,但反应时间长、降解效率较低,若要提高降解效率可通过与酸降解等其他降解方法相结合(专利号:CN200510008708.2)。生物降解法主要为酶降解法,其反应条件温和,专一性强,例如氧化水解酶可有效降解纤维素及壳聚糖(专利号:CN201710735196.2)。化学降解法主要包括酸降解法和过氧化氢降解法,反应体系简单,时间短;但是其应用限制因素在于某些制剂有毒有害,限制了其在食品中的应用。酸降解法一般采用盐酸或硫酸水解多糖分子糖苷键,致使多糖分子量降低,其反应简便易行,如用三氟乙酸水解猴头菌多糖可使其分子量降低一个数量级(专利号:CN201711088514.7)。过氧化氢降解法利用氧化还原反应氧化降解多糖,成本较低。曲爱琴等人(专利号:CN1459455A)发明的多糖的自由基降解方法是将多糖置于有机酸、过氧化氢与维生素C的复合体系中,适用于绝大多数来源的多糖,但反应时间为2-72小时,溶液浓度均在1-100mmol/L范围内,反应时间长所需浓度高。At present, the degradation of Dendrobium officinale polysaccharide mainly includes physical degradation, chemical degradation and biodegradation. The physical degradation method mainly includes ultrasonic degradation method and microwave degradation method. It does not need to introduce other reactants. The operation is simple, but the reaction time is long and the degradation efficiency is low. To improve the degradation efficiency, it can be combined with other degradation methods such as acid degradation (patent). No.: CN200510008708.2). The biodegradation method is mainly an enzymatic degradation method with mild reaction conditions and strong specificity. For example, oxidohydrolase can effectively degrade cellulose and chitosan (patent number: CN201710735196.2). The chemical degradation method mainly includes acid degradation method and hydrogen peroxide degradation method. The reaction system is simple and the time is short; however, its application limitation is that some preparations are toxic and harmful, which limits its application in food. The acid degradation method generally uses hydrochloric acid or sulfuric acid to hydrolyze the glycosidic bond of the polysaccharide molecule, which reduces the molecular weight of the polysaccharide, and the reaction is simple and easy. . The hydrogen peroxide degradation method utilizes redox reactions to oxidatively degrade polysaccharides, and the cost is low. The free radical degradation method of polysaccharide invented by Qu Aiqin et al. (patent number: CN1459455A) is to place the polysaccharide in the composite system of organic acid, hydrogen peroxide and vitamin C, which is suitable for most polysaccharides from sources, but the reaction time is limited. For 2-72 hours, the concentration of the solution is in the range of 1-100 mmol/L, and the concentration required for a long reaction time is high.
发明内容SUMMARY OF THE INVENTION
针对上述技术问题,本发明的目的在于提供一种铁皮石斛多糖的化学降解方法,采用二乙烯三胺五乙酸(DTPA)螯合亚铁离子与过氧化氢反应形成Fenton体系,产生的羟基自由基氧化降解铁皮石斛多糖,本发明提供的降解方法工艺简单,且采用该方法处理铁皮石斛多糖后能够降低其分子量,提高其抗氧化能力,利于后续铁皮石斛多糖的利用。In view of the above-mentioned technical problems, the object of the present invention is to provide a kind of chemical degradation method of Dendrobium officinale polysaccharide, adopt diethylenetriaminepentaacetic acid (DTPA) chelated ferrous ion and hydrogen peroxide to react to form Fenton system, the hydroxyl radical that produces For the oxidative degradation of Dendrobium officinale polysaccharide, the degradation method provided by the invention is simple in process, and the method can reduce the molecular weight of the Dendrobium officinale polysaccharide after processing the polysaccharide, improve its antioxidant capacity, and facilitate the subsequent utilization of the Dendrobium officinale polysaccharide.
本发明是通过以下技术方案实现的:The present invention is achieved through the following technical solutions:
一种铁皮石斛多糖的化学降解方法,以二乙烯三胺五乙酸螯合的亚铁离子和过氧化氢反应形成Fenton体系,利用所述Fenton体系产生的羟基自由基氧化降解铁皮石斛多糖,得到降解粗产物溶液,对所述降解粗产物溶液调整pH,以沉淀去除铁离子,醇沉烘干后获得铁皮石斛降解多糖。A method for chemical degradation of Dendrobium officinale polysaccharide, wherein a Fenton system is formed by reacting ferrous ions chelated with diethylenetriaminepentaacetic acid and hydrogen peroxide, and the hydroxyl radicals generated by the Fenton system are used to oxidatively degrade Dendrobium officinale polysaccharide to obtain degradation For the crude product solution, the pH of the degraded crude product solution is adjusted to remove iron ions by precipitation, and after alcohol precipitation and drying, the degraded polysaccharide of Dendrobium officinale is obtained.
进一步地,所述方法具体以下步骤:Further, the method specifically follows the steps:
(1)将铁皮石斛多糖溶于水,得到铁皮石斛多糖溶液;(1) dissolve Dendrobium officinale polysaccharide in water to obtain Dendrobium officinale polysaccharide solution;
(2)向所述铁皮石斛多糖溶液中加入二乙烯三胺五乙酸螯合的亚铁离子(即DTPA-亚铁离子),然后再加入过氧化氢溶液,形成Fenton体系,室温下进行氧化反应降解铁皮石斛多糖,得到降解粗产物溶液;(2) in described Dendrobium officinale polysaccharide solution, add the ferrous ion (being DTPA-ferrous ion) that diethylenetriaminepentaacetic acid is chelated, then add hydrogen peroxide solution again, form Fenton system, carry out oxidation reaction at room temperature Degrading Dendrobium officinale polysaccharide to obtain a solution of degraded crude product;
(3)调节所述降解粗产物溶液的pH,弃沉淀,得到铁皮石斛多糖降解液;(3) adjusting the pH of the degraded crude product solution, discarding the precipitation to obtain the dendrobium officinale polysaccharide degradation solution;
(4)所述铁皮石斛多糖降解液进行醇沉,弃液相,烘干沉淀,得到铁皮石斛降解多糖。(4) alcohol precipitation is carried out in the dendrobium officinale polysaccharide degradation solution, the liquid phase is discarded, and the precipitation is dried to obtain the dendrobium officinale degraded polysaccharide.
进一步地,所述铁皮石斛多糖的分子量在2×107-1.5×105Da范围内;所述铁皮石斛降解多糖分子量在5×104-1.5×104Da之间。Further, the molecular weight of the Dendrobium officinale polysaccharide is in the range of 2×10 7 -1.5×10 5 Da; the molecular weight of the degraded polysaccharide of Dendrobium officinale is in the range of 5×10 4 -1.5×10 4 Da.
进一步地,步骤(1)中,所述铁皮石斛多糖溶液的浓度为1-10mg/mL。Further, in step (1), the concentration of the Dendrobium officinale polysaccharide solution is 1-10 mg/mL.
进一步地,步骤(2)具体为:Further, step (2) is specifically:
向铁皮石斛多糖溶液中加入二乙烯三胺五乙酸螯合的亚铁离子,直至铁皮石斛多糖溶液中DTPA-亚铁离子浓度为5-20μm/L;再加入过氧化氢溶液至铁皮石斛粗多糖水溶液中过氧化氢的体积比浓度为0.05-0.5%;室温下进行氧化反应降解铁皮石斛多糖,反应一定时间后得到降解粗产物溶液。Add diethylenetriaminepentaacetic acid chelated ferrous ion to Dendrobium officinale polysaccharide solution until the concentration of DTPA-ferrous ion in Dendrobium officinale polysaccharide solution is 5-20 μm/L; then add hydrogen peroxide solution to Dendrobium officinale crude polysaccharide The volume ratio concentration of hydrogen peroxide in the aqueous solution is 0.05-0.5%; the oxidation reaction is carried out at room temperature to degrade Dendrobium officinale polysaccharide, and the degraded crude product solution is obtained after a certain time of reaction.
在该步骤中,需要严格控制DTPA-亚铁离子浓度为5-20μm/L、过氧化氢的体积比浓度为0.05-0.5%,正是通过控制Fenton体系中DTPA-亚铁离子浓度和过氧化氢的体积比浓度,才能够获得分子量在5×104-1.5×104Da之间的铁皮石斛降解多糖,同时能够确保铁皮石斛多糖不被过度降解为单糖。In this step, it is necessary to strictly control the concentration of DTPA-ferrous ions to be 5-20 μm/L and the volume ratio of hydrogen peroxide to be 0.05-0.5%. It is precisely by controlling the concentration of DTPA-ferrous ions in the Fenton system and peroxide The volume ratio of hydrogen concentration can obtain the degradable polysaccharides of Dendrobium officinale with molecular weight between 5×10 4 -1.5×10 4 Da, and at the same time, it can ensure that the polysaccharides of Dendrobium officinale are not excessively degraded into monosaccharides.
优选地,选用的铁皮石斛多糖浓度为2mg/mL,铁皮石斛多糖溶液中DTPA-亚铁离子浓度为5μm/L,过氧化氢溶液的体积比浓度为0.1%。Preferably, the selected Dendrobium officinale polysaccharide concentration is 2 mg/mL, the DTPA-ferrous ion concentration in the Dendrobium officinale polysaccharide solution is 5 μm/L, and the volume ratio concentration of the hydrogen peroxide solution is 0.1%.
本发明所述方法采用二乙烯三胺五乙酸(DTPA)为一种重要的氨羧螯合剂,对金属离子尤其是高价态显色金属离子的配位能力极强,广泛应用于医用药剂中,螯合必需微量元素后可促进体内重金属毒物的排出。DTPA对过渡金属形成的螯合物的稳定性及抗氧化作用均优于乙二胺四乙酸(EDTA),可有效络合Fenton反应中使用的亚铁离子,以降低反应过程中亚铁离子被氧化为三价铁离子的可能性。The method of the invention adopts diethylenetriaminepentaacetic acid (DTPA) as an important ammonia carboxyl chelating agent, which has extremely strong coordination ability to metal ions, especially high-valence chromogenic metal ions, and is widely used in medical pharmaceuticals. After chelating essential trace elements, it can promote the excretion of heavy metal poisons from the body. The stability and antioxidant effect of DTPA on the chelates formed by transition metals are better than those of ethylenediaminetetraacetic acid (EDTA). Possibility of oxidation to ferric ions.
进一步地,步骤(2)中,步骤(2)中,调节铁皮石斛多糖溶液pH至3.0-4.0后再加入二乙烯三胺五乙酸螯合的亚铁离子。Further, in step (2), in step (2), the pH of the Dendrobium officinale polysaccharide solution is adjusted to 3.0-4.0, and then the ferrous ions chelated by diethylenetriaminepentaacetic acid are added.
进一步地,步骤(2)中,降解时间为降解时间为10-20min。Further, in step (2), the degradation time is 10-20min.
进一步地,步骤(3)中,调节所述降解粗产物溶液的pH为9.0-10.0。Further, in step (3), the pH of the crude degradation product solution is adjusted to be 9.0-10.0.
进一步地,步骤(4)中,步骤(4)中,所述醇沉的温度为3-5℃、醇沉的时间为6-18h。Further, in step (4), in step (4), the temperature of the alcohol precipitation is 3-5°C, and the time of alcohol precipitation is 6-18h.
本发明的有益技术效果:Beneficial technical effects of the present invention:
1)本发明所提供的降解方法采用Fenton体系降解铁皮石斛多糖,具有降解时间短、成本低等优势,且可以显著降低铁皮石斛多糖分子量。本发明中采用的Fenton体系包括二乙烯三胺五乙酸螯合的亚铁离子和过氧化氢,其中,过氧化氢分解作为反应底物,产生的羟基自由基攻击铁皮石斛多糖分子以达到降低多糖分子量的效果;亚铁离子作为催化剂参与过氧化氢分解,以达到快速降解的效果;DTPA作为金属离子螯合剂螯合亚铁离子以增强其稳定性,降低被氧化的可能,同时螯合氧化产生的三价铁离子,避免干扰反应。1) The degradation method provided by the present invention adopts the Fenton system to degrade Dendrobium officinale polysaccharide, which has the advantages of short degradation time and low cost, and can significantly reduce the molecular weight of Dendrobium officinale polysaccharide. The Fenton system adopted in the present invention includes ferrous ions chelated by diethylenetriaminepentaacetic acid and hydrogen peroxide, wherein, the hydrogen peroxide is decomposed as a reaction substrate, and the generated hydroxyl radicals attack the Dendrobium officinale polysaccharide molecules to reduce polysaccharide The effect of molecular weight; ferrous ion is used as a catalyst to participate in the decomposition of hydrogen peroxide to achieve the effect of rapid degradation; DTPA is used as a metal ion chelator to chelate ferrous ions to enhance its stability and reduce the possibility of being oxidized. ferric ions to avoid interfering with the reaction.
2)本发明所提供降解方法制备获得的铁皮石斛降解多糖的抗氧化活性相较于传统水提醇沉法多糖明显提高。2) The antioxidant activity of the degraded polysaccharide of Dendrobium officinale prepared by the degradation method provided by the present invention is obviously improved compared with that of the polysaccharide by the traditional water extraction and alcohol precipitation method.
附图说明Description of drawings
图1为本发明实施例中铁皮石斛多糖DP的及铁皮石斛降解多糖DP-1、DP-2、DP-3的高效液相色谱图。Fig. 1 is the high performance liquid chromatogram of Dendrobium officinale polysaccharide DP and dendrobium officinale degraded polysaccharides DP-1, DP-2, DP-3 in the embodiment of the present invention.
图2a为本发明实施例中铁皮石斛多糖DP及铁皮石斛降解多糖DP-1对三价铁离子的还原能力图;图2b为本发明实施例中铁皮石斛多糖DP及铁皮石斛降解多糖DP-1的DPPH自由基清除能力图;图2c为本发明实施例中铁皮石斛多糖DP及铁皮石斛降解多糖DP-1的羟基自由基清除能力图;图2d为为本发明实施例中铁皮石斛多糖DP及铁皮石斛降解多糖DP-1的总抗氧化能力图。Fig. 2a is a diagram showing the reducing ability of Dendrobium officinale polysaccharide DP and Dendrobium officinale degraded polysaccharide DP-1 to ferric ions in the embodiment of the present invention; Fig. 2b is a graph of the reduction ability of Dendrobium officinale polysaccharide DP and Dendrobium officinale degraded polysaccharide DP-1 in the embodiment of the present invention. DPPH free radical scavenging ability diagram; Figure 2c is the hydroxyl radical scavenging ability diagram of Dendrobium officinale polysaccharide DP and Dendrobium officinale polysaccharide DP-1 in the embodiment of the present invention; Figure 2d is the Dendrobium officinale polysaccharide DP and Total antioxidant capacity of Dendrobium officinale degrading polysaccharide DP-1.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细描述。应当理解,此处所描述的具体实施例仅仅用于解释本发明,并不用于限定本发明。In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.
相反,本发明涵盖任何由权利要求定义的在本发明的精髓和范围上做的替代、修改、等效方法以及方案。进一步,为了使公众对本发明有更好的了解,在下文对本发明的细节描述中,详尽描述了一些特定的细节部分。对本领域技术人员来说没有这些细节部分的描述也可以完全理解本发明。On the contrary, the present invention covers any alternatives, modifications, equivalents and arrangements within the spirit and scope of the present invention as defined by the appended claims. Further, in order to give the public a better understanding of the present invention, some specific details are described in detail in the following detailed description of the present invention. The present invention can be fully understood by those skilled in the art without the description of these detailed parts.
本发明提供一种铁皮石斛多糖的化学降解方法,以二乙烯三胺五乙酸螯合的亚铁离子和过氧化氢反应形成Fenton体系,利用所述Fenton体系产生的羟基自由基氧化降解铁皮石斛多糖,得到降解粗产物溶液,对所述降解粗产物溶液调整pH,以沉淀去除铁离子,醇沉烘干后获得铁皮石斛降解多糖。所述方法具体包括以下步骤:The invention provides a chemical degradation method of Dendrobium officinale polysaccharide, which comprises the reaction of ferrous ions chelated by diethylenetriaminepentaacetic acid and hydrogen peroxide to form a Fenton system, and the hydroxyl radicals generated by the Fenton system are utilized to oxidize and degrade Dendrobium officinale polysaccharides. , obtain a degraded crude product solution, adjust the pH of the degraded crude product solution, remove iron ions by precipitation, and obtain dendrobium officinale degraded polysaccharide after alcohol precipitation and drying. The method specifically includes the following steps:
(1)将铁皮石斛多糖溶于水,得到铁皮石斛多糖溶液;所述铁皮石斛多糖溶液的浓度为1-10mg/mL,优选浓度为2mg/mL。(1) Dissolving Dendrobium officinale polysaccharide in water to obtain a Dendrobium officinale polysaccharide solution; the concentration of the Dendrobium officinale polysaccharide solution is 1-10 mg/mL, preferably 2 mg/mL.
(2)向铁皮石斛多糖溶液中加入二乙烯三胺五乙酸螯合的亚铁离子,直至铁皮石斛多糖溶液中DTPA-亚铁离子浓度为5-20μm/L;再加入过氧化氢溶液至铁皮石斛粗多糖水溶液中过氧化氢的体积浓度比为0.05-0.5%;室温下进行氧化反应降解铁皮石斛多糖,反应一定时间后得到降解粗产物溶液;降解时间为10-20min;(2) Add ferrous ion chelated by diethylenetriaminepentaacetic acid to Dendrobium officinale polysaccharide solution until the concentration of DTPA-ferrous ion in Dendrobium officinale polysaccharide solution is 5-20 μm/L; then add hydrogen peroxide solution to The volume concentration ratio of hydrogen peroxide in the aqueous solution of Dendrobium crude polysaccharide is 0.05-0.5%; the oxidation reaction is carried out at room temperature to degrade Dendrobium officinale polysaccharide, and the degraded crude product solution is obtained after a certain time of reaction; the degradation time is 10-20min;
优选地,选用的铁皮石斛多糖浓度为2mg/mL,DTPA-亚铁离子浓度为5μm/L,过氧化氢溶液的体积比浓度为0.1%。Preferably, the selected Dendrobium officinale polysaccharide concentration is 2 mg/mL, the DTPA-ferrous ion concentration is 5 μm/L, and the volume ratio concentration of the hydrogen peroxide solution is 0.1%.
(3)调节所述降解粗产物溶液的pH为9-10,弃沉淀,得到铁皮石斛多糖降解液;(3) adjusting the pH of the degraded crude product solution to be 9-10, discard the precipitation, and obtain the dendrobium officinale polysaccharide degradation solution;
(4)所述铁皮石斛多糖降解液进行醇沉,弃液相,烘干沉淀,得到铁皮石斛降解多糖。在本实施例中,所述醇沉的温度为3-5℃、醇沉的时间为6-18h。(4) alcohol precipitation is carried out in the dendrobium officinale polysaccharide degradation solution, the liquid phase is discarded, and the precipitation is dried to obtain the dendrobium officinale degraded polysaccharide. In this embodiment, the temperature of the alcohol precipitation is 3-5° C., and the time of the alcohol precipitation is 6-18 h.
下面以具体实施例进行说明:Described below with specific embodiments:
对照实施例Comparative Example
本对照实施例提供一种热水浸提法提取铁皮石斛多糖的方法:The present comparative example provides a method for extracting Dendrobium officinale polysaccharide by hot water extraction method:
称取烘干、粉碎、过60目筛的铁皮石斛粉末0.500g,加入25mL80%乙醇,涡旋震荡混匀,150W超声提取30min,6000r/min离心,取沉淀,用10mL80%乙醇洗涤离心2-3次,至上清无色;将沉淀以料液比1:100条件沸水提取2h,抽滤,减压浓缩,加入4倍体积乙醇过夜醇沉,离心,烘干,即得铁皮石斛多糖DP。Weigh 0.500 g of Dendrobium officinale powder that was dried, crushed and passed through a 60-mesh sieve, add 25 mL of 80% ethanol, vortex and mix, extract by 150 W ultrasonic for 30 min, centrifuge at 6000 r/min, take the precipitate, wash with 10 mL of 80% ethanol and centrifuge for 2- 3 times until the supernatant is colorless; the precipitate is extracted with boiling water for 2 hours at a material-to-liquid ratio of 1:100, suction filtered, concentrated under reduced pressure, added with 4 times the volume of ethanol for overnight alcohol precipitation, centrifuged, and dried to obtain Dendrobium officinale polysaccharide DP.
实施例1Example 1
本实施例提供一种铁皮石斛多糖的化学降解方法,所述方法包括:The present embodiment provides a chemical degradation method of Dendrobium officinale polysaccharide, the method comprising:
a)按照对照实施例的方法制备铁皮石斛多糖DP;a) prepare Dendrobium officinale polysaccharide DP according to the method of Comparative Example;
b)采用二乙烯三胺五乙酸(DTPA)溶液螯合亚铁离子,获得二乙烯三胺五乙酸螯合的亚铁离子,即DTPA-亚铁离子;b) adopting diethylenetriaminepentaacetic acid (DTPA) solution to chelate ferrous ion to obtain ferrous ion chelated by diethylenetriaminepentaacetic acid, namely DTPA-ferrous ion;
c)将步骤a)制备的铁皮石斛粗多糖DP配制为浓度为2mg/mL的铁皮石斛多糖水溶液,调节pH至3.0;再加入DTPA-亚铁离子,至铁皮石斛多糖水溶液中DTPA-亚铁离子浓度为5μm/L;再加入1%过氧化氢溶液至铁皮石斛多糖水溶液中过氧化氢浓度为0.1%;室温下进行氧化反应;c) The crude polysaccharide DP of Dendrobium officinale prepared in step a) is prepared into an aqueous solution of Dendrobium officinale polysaccharide with a concentration of 2 mg/mL, and the pH is adjusted to 3.0; The concentration is 5 μm/L; 1% hydrogen peroxide solution is added to the Dendrobium officinale polysaccharide aqueous solution to the hydrogen peroxide concentration of 0.1%; the oxidation reaction is carried out at room temperature;
d)反应20分钟后,调反应液pH值至9.0,离心除去沉淀;d) after 20 minutes of reaction, adjust the pH value of the reaction solution to 9.0, and remove the precipitate by centrifugation;
e)将上清液调pH至中性,减压浓缩,烘干,得铁皮石斛多糖自由基降解产物DP-1。e) adjusting the pH of the supernatant to neutral, concentrating under reduced pressure, and drying to obtain DP-1, a free radical degradation product of Dendrobium officinale polysaccharide.
采用超纯水复溶,经0.22μm Nylon膜过滤进行高效液相色谱分析。It was reconstituted in ultrapure water and filtered through a 0.22 μm Nylon membrane for high performance liquid chromatography analysis.
本实施例中,所采用色谱柱为C18柱(4.6×250mm,5μm),流动相为100%超纯水,流速1mL/min,柱温30℃,进样量20μL,运行时间15min。运行结束后依据保留时间与标准品进行比较。In this example, the chromatographic column used was a C18 column (4.6×250 mm, 5 μm), the mobile phase was 100% ultrapure water, the flow rate was 1 mL/min, the column temperature was 30 °C, the injection volume was 20 μL, and the running time was 15 min. After the run, compare with the standard according to the retention time.
实施例2Example 2
本实施例提供一种铁皮石斛多糖的化学降解方法,与实施例1基本相同,唯不同之处在于:步骤d)中,所述反应时间为10min;得铁皮石斛多糖降解产物DP-2。This embodiment provides a chemical degradation method of Dendrobium officinale polysaccharide, which is basically the same as
实施例3Example 3
本实施例提供一种铁皮石斛多糖的化学降解方法,与实施例1基本相同,唯一不同之处在于:步骤c)中,所述DTPA-亚铁离子替换为亚铁离子:加入亚铁离子,至铁皮石斛多糖水溶液中亚铁离子浓度为5μm/L。最终得到铁皮石斛多糖降解产物DP-3。This embodiment provides a chemical degradation method of Dendrobium officinale polysaccharide, which is basically the same as
图1为铁皮石斛多糖DP及铁皮石斛降解多糖DP-1、DP-2和DP-3的高效液相色谱图,由图1可知:采用高效液相色谱分析DP、DP-1、DP-2、DP-3的分子量,确定DP主要由四种分子量的多糖组成,分别为2.4×107Da、1.9×106Da、1.3×106Da、1.5×105Da;DP-1主要由两种分子量多糖组成,分别为4.7×104Da和1.6×104Da,降解后多糖分子量显著低于降解前多糖分子量,且分子量分布更为集中。DP-2与DP-1分子量基本一致,但相较于DP-1,其低分子量多糖比例较低。DP-3主要由两种分子量的多糖组成,分别为8.5×105Da、2.0×104Da。降解后铁皮石斛多糖分子量显著低于降解前多糖分子量,但相比于螯合亚铁离子和过氧化氢氧化降解铁皮石斛多糖,降解后分子量较高,低分子量多糖占比较少。Fig. 1 is the high performance liquid chromatogram of Dendrobium officinale polysaccharide DP and the degraded polysaccharides DP-1, DP-2 and DP-3 of Dendrobium officinale. It can be seen from Fig. 1 that: DP, DP-1, DP-2 were analyzed by high performance liquid chromatography. , DP-3 molecular weight, it is determined that DP is mainly composed of four molecular weight polysaccharides, which are 2.4 × 10 7 Da, 1.9 × 10 6 Da, 1.3 × 10 6 Da, 1.5 × 10 5 Da; DP-1 is mainly composed of two The molecular weight of polysaccharides after degradation was significantly lower than that before degradation, and the molecular weight distribution was more concentrated. The molecular weights of DP-2 and DP-1 are basically the same, but compared with DP-1, the proportion of low molecular weight polysaccharides is lower. DP-3 is mainly composed of two molecular weight polysaccharides, 8.5×10 5 Da and 2.0×10 4 Da, respectively. The molecular weight of Dendrobium officinale polysaccharide after degradation was significantly lower than that of polysaccharide before degradation, but compared with the degradation of Dendrobium officinale polysaccharide by chelating ferrous ions and hydrogen peroxide, the molecular weight after degradation was higher, and the proportion of low molecular weight polysaccharides was less.
图2为铁皮石斛多糖DP及铁皮石斛降解多糖DP-1的抗氧化活性图,图2a为对三价铁离子的还原能力图,图2b为DPPH自由基清除能力图,图2c为羟基自由基清除能力图,图2d为总抗氧化能力图。由图2a-d可知,降解后的铁皮石斛多糖DP-1的抗氧化能力均高于未降解的铁皮石斛多糖DP,在3.0mg/mL浓度下,DP-1的总抗氧化能力约为DP的2.6倍;其中抗氧化能力提高最明显的为DPPH自由基清除能力,在0.5mg/mL浓度下,DP-1的总抗氧化能力约为DP的5.7倍。Figure 2 is a graph showing the antioxidant activity of Dendrobium officinale polysaccharide DP and Dendrobium officinale degraded polysaccharide DP-1, Figure 2a is a graph of the reducing ability to ferric ions, Figure 2b is a graph of DPPH free radical scavenging ability, and Figure 2c is a graph of hydroxyl radicals The scavenging capacity map, Figure 2d is the total antioxidant capacity map. It can be seen from Figure 2a-d that the antioxidant capacity of the degraded Dendrobium officinale polysaccharide DP-1 is higher than that of the undegraded Dendrobium officinale polysaccharide DP. At the concentration of 3.0 mg/mL, the total antioxidant capacity of DP-1 is about DP. 2.6 times that of DP-1; the most obvious improvement in antioxidant capacity is the scavenging capacity of DPPH radicals. At the concentration of 0.5 mg/mL, the total antioxidant capacity of DP-1 is about 5.7 times that of DP.
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