摘要
花粉是植物的精细胞,素有“完全营养素”和“微型营养库”之美誉。近20年来,国内外对花粉有益成分、药理作用和生理功能进行了大量的研究,证实其对人体六十多种疾病具有良好的防治效果,在医药、保健品、功能性食品以及日用化妆品领域有着广阔的开发前景。松花粉是我国资源蕴藏量巨大的风媒花粉,药用和食用历史悠久,1997年被批准为新资源食品,现已列作普通食品管理。但目前松花粉的开发利用整体技术水平仍然偏低,基础研究匮乏,产品形式单一,亟待展开深层次的系统研究,为资源的有序开发和高效利用奠定基础。
本硕士论文研究课题立足传统医学对松花粉“久服令轻身”的功效记载、以及近期对松花粉抗氧化、降血脂、增加脂质排泄等的实验研究报道,选用我国资源量最大、最具开发利用价值的三个主要松花粉品种(马尾松、油松和云南松),采用70%的乙醇分别对其破壁和不破壁试样进行提取,对醇提物中主要次生代谢产物(黄酮、酚酸和甾醇)的含量进行分析比较,并对油松花粉醇提物进行“四部位法”分级处理。然后,依据天然产物的减肥功效与其抗氧化功能因子之间存在一定耦合度的背景资料,采用多种不同的抗自由基和抗氧化评价体系,对六个松花粉醇提物以及油松花粉的四个分级部位进行分析和比较,测试其对脂肪酸合酶(FAS)的抑制活性,观察破壁处理对松花粉表面形态、持水力和吸油性等的影响,比较其在模拟胃液和肠液条件下吸附胆固醇和胆酸钠能力的大小,并采用小鼠预防性减肥试验模型,探索松花粉及其提取物的体重控制效果及可能的作用机制。主要的研究结论如下:
(1)不同品种松花粉试样的常规组分对比分析
选取蛋白质、游离脂肪、总糖和膳食纤维含量作为常规组分的代表。分析结果表明,云南松的蛋白质和游离脂肪含量都明显低于马尾松和油松,而可溶性糖和膳食纤维的含量则明显高于后者;油松的蛋白质和游离脂肪含量显著高于其他两种。
(2)不同品种、破壁与否松花粉试样的非营养素对比分析
以松花粉70%醇提物为测试对象,重点考察其总黄酮、酚酸和甾醇三类次生代谢产物的含量。结果表明,三个不同品种间总黄酮含量差异较为明显,以马尾松为高(破壁与不破壁的醇提物中分别达到17.38mg/g与23.74mg/g)、油松为低(12.23mg/g与15.15mg/g),三者的酚酸和甾醇含量差异不明显;未经破壁的原花粉的各项非营养素指标均高于对应的破壁试样,尤以马尾松总黄酮的差异最为显著(Δ=6.36mg/g)。
(3)松花粉醇提物抗氧化性能的比较研究
采用多种抗氧化评价体系,其中包括活性氧自由基(O_2~-、OH、HOO、DPPH、ABTS~+)清除、全血总抗氧化能力评价和抗油脂氧化能力测定,比较了三个品种、破壁处理与否的六个醇提试样和油松醇提物四个分级部位(石油醚相、乙酸乙酯相、正丁醇相和水相)的抗氧化性能强弱。结果显示,各试样均表现出一定的抗氧活性,破壁后提取物明显优于未破壁的,品种不同对抗氧活性亦带来一定差异;油松醇提物的四个分级部位中,中等极性组分(乙酸乙酯和正丁醇相)的抗油脂氧化能力显著高于其他部位。然而,试样抗氧化能力的大小与其总黄酮和酚酸含量之间的关联度不强。
(4)松花粉及其提取物轻身减肥功效及其作用机理研究
体外实验:测试了油松花粉醇提物及其不同分级部位对脂肪酸合酶(FAS)的抑制能力,考察了六个松花粉样品颗粒的显微结构、比表面积、持水力、吸油性,以及在模拟胃液和肠液条件下对胆固醇和胆酸钠的吸附能力;动物试验:选用ICR小鼠的预防性减肥模型,进行了为期6周的动物试验,全面考察油松破壁花粉、70%乙醇提取物、提取后残渣对小鼠体重、体脂、肝脏FAS酶活性及其与脂质代谢相关的血生化指标的影响。主要实验结果如下:
①油松花粉醇提物的石油醚相和乙酸乙酯相在体外均表现出很强的FAS抑制活性,其作用强于阳性对照绿原酸和表没食子儿茶素没食子酸酯(EGCG),正丁醇相与总的醇提物活性接近,而萃余后的水相几乎无FAS抑制作用,说明松花粉中具有FAS抑制能力的物质主要是中低极性的化合物,与其强抗氧活性组分(中极性部位)存在一定的交集。
②松花粉是一种优质的人体膳食纤维补充剂,其持水力、吸油性、对胆固醇和胆酸钠吸附能力均强于大豆膳食纤维;破壁处理能够大大增加颗粒的比表面积,破壁后花粉试样在持水力(WHC)、吸油性(OHC)以及模拟胃液(pH=2.0)条件下对胆固醇的吸附能力略优于原花粉;而在模拟肠液(pH=7.0)条件下,原花粉对胆固醇和胆酸钠的吸附能力显著优于破壁试样,认为可能与松花粉颗粒的表面性质有关。
③在小鼠预防性减肥试验中,油松原花粉(1.2g/kgBW.d,未破壁)与破壁油松花粉醇提物的中剂量(0.3g/kgBW.d)均表现出可与阳性对照药物(赛尼可,有效成分为奥利司他,0.05g/kgBW.d)相媲美的体重增长抑制效果,且油松原花粉显著降低了实验小鼠的血清TC和TG水平;油松花粉醇提物高剂量(0.7g/kgBW.d)能显著降低小鼠血清TG水平;各松花粉试样(包括提取后的残渣)均能显著抑制小鼠肝脏的FAS活性,其中醇提物高剂量(0.7g/kgBW.d)对FAS酶活的抑制率超过50%。
综合上述成分分析和体内外实验的结果,证实了松花粉具有一定的“轻身减肥”功效,作用机制可能与其抗氧化活性、FAS抑制作用、松花粉结构形态和膳食纤维的行为特征有关,关于松花粉控制体重的关键组分及其作用途径有待进一步的深入研究。
Pollen is the sperm cell of plant. It is well known as "full nutrients library" and "micro nutrients base". Researches in and abroad China within 20 years showed that pollen of different species can cure or prevent more than 60 kinds of diseases of human beings. It has promising prospects in medicine, healthy products, and functional cosmetics. Pine is a kind of anemophilous plant with and extreme abundant resources in China. Pine pollen is Chinese traditional medicine-food. China National Ministry of Public Health has defined pine pollen as a kind of new food resource and supervised it as a normal food science 1997. However, there are only a few basic researches, a small range of product divisions, and much lower utilization level on this special precious resource. It is in urgent need to research and develop deeply and systematically, in order to lay a foundation for the efficient utilization of pine pollen.
This subject is conducted according to the introduction of pine pollen's weight lose function written in Chinese traditional medical books, and advanced scientific evidences about its antioxidant activity, adjustment capacity on blood lipids, and promotion effect on lipid excrete. Pollens of Pinus massoniana, Pinus tabulaeformis Carr, Pinus yunnanensis Franch were chosen as experimental samples due to their abundant recourses and high economic value. This subject investigated and compared the contents of 3 main secondary metabolites (flavonoid, phenolic acid and sterol) in their 70% ethanol extracts. The 70% ethanol extract of Pinus tabulaeformis Carr was separated into 4 polar parts by organic solvents. According to the information that there may be some intersections between antioxidant components and weight control factors within natural products, we tested and compared the antioxidant ability of 6 different pine pollen extracts and 4 polar fractions of Pinus tabulaeformis Carr using by 7 evaluation systems in vitro, the inhibition capacity on fatty acid synthase (FAS) of ethanol extract and its 4 polar fractions of Pinus tabulaeformis Carr pollen in vitro.
The influence of wall-breaking pre-treatment on pine pollen's surface character and adsorption capacity of water, oil, cholesterol and bile sodium in vitro were investigated. Finally, an animal test of preventive weight control was carried out to verify the weight control function using by mice model.
The main results and conclusions are as follows:
1) Fundamental components in 3 species of pine pollen Protein, fat, soluble sugar and dietary fiber were inspected. The results showed that the pollen of Pinus yunnanensis Franch is much lower in protein and fat levels, but higher in total soluble sugar and dietary fiber. Pollen of Pinus tabulaeformis Carr has the highest protein and fat contents.
2) Secondary metabolites in 6 extract samples of pine pollen (3 species, including wall-broken and original) Contents of total flavonoids, phenolic acids and total sterols in all ethanol extracts were tested. The results showed that there is significant difference in the contents of flavonoids. Among those, the extract of Pinus massoniana has the highest values, 17.78mg/g in wall-broken sample and 23.74mg/g in original sample; the extract of Pinus tabulaeformis Carr has the lowest, 12.23mg/g in wall-broken sample and 15.15mg/g in original sample. The contents of phenolic acids and sterols are similar in all 6 samples. Generally, whole pollen extracts are of higher level than wall-broken counterparts about all 3 tested indexes, especially the content gap of total flavonoids in Pinus massoniana (Δ=6.36mg/g).
3) The antioxidant ability of pine pollen extracts Antioxidant ability of 6 different ethanol extracts of 3 species and 4 polar fractions of Pinus massonian extract were evaluated using by 7 bio-assess systems including scavenging on O·2~-,·OH, HOO·, DPPH·、ABTS·~+, and total antioxidant ability in blood and in oil. The results showed that all tested samples have certain antioxidant ability. Extracts of pine pollen with wall-broking pre-treatment are better than those of whole pollen, and the species of pollen can also affect the antioxidant activity. The medium polarity phases (n-butanol phase and ethyl acetate phase) of Pinus massonian extract are better than other polar parts. However, the relationship between antioxidant capacity and contents of total flavonoids and phenolic acids in extracts are not clear.
4) Weight control effect of pine pollen and the mechanisms In vitro: the inhibition abilities to fatty acid synthase of Pinus tabulaeformis Carr pollen extracts and its 4 polar fractions were tested; the influence of wail-breaking pre-treatment on surface character and adsorption capacity of water, oil, cholesterol and bile sodium were investigated; In vivo: the subject chose a ICR mice's preventive model of weight control lasting 6wk to investigate the effects of pine pollen, ethanol extract and extracted waste on mice's body weight, body fat, liver FAS activity and blood lipids.
The main results are as follows:
①Two phases of petrol ether and ethyl acetate of Pinus tabulaeformis Carr pollen have high FAS inhibition activities in vitro, better than the positive controls of EGCG and chlorogenic acid; the inhibition abilities of ethanol extract and its n-butanol phase are close; and remained water phase is very weak. It may illustrate that the key components of FAS inhibition are focused on the low and middle polar phases. It maybe explained as that there are some component intersections between high FAS inhibition and high antioxidation.
②Pine pollen is a good dietary fiber supplement. Absorption abilities of pine pollen on water, fat, cholesterol, bile sodium in vitro are all better than those of soybean dietary fiber. Wall-broken pollen has higher specific surface area than the whole pollen. Wall-broken pollen has better water holding capacity, oil, cholesterol (under simulated human gastric, pH=2.0) absorption capacity than whole pollen, the results are opposite in the absorption of bile acid and cholesterol (under simulated human intestine, pH=7.0). It might because of the granule character of pine pollen surface.③In the preventative experiment of mice's weight control, the inhibition effects on weight increase of whole pollen (1.2g/kgBW.d) and ethanol extract (0.3g/kgBW.d) are close to the effect of positive control, a widely used weight lose medicine of Orlistat (0.05g/kgBW.d). Whole pine pollen (1.2g/kgBW.d) can significantly reduce TC and TG in mice's blood serum; ethanol extract (0.7g/kgBW.d) can significantly reduce TG. Whole pollen, wall-broken pollen, ethanol extract and extracted waste can significantly inhibit the activity of FAS in mice's liver. Ethanol extract at the dosage of 0.7g/kgBW.d inhibits the mice's liver FAS by 50%.
All experimental results showed that pine pollen has weight control effect both in vitro and in vivo. The mechanism may be related to its antioxidant ability, FAS inhibition activity, the physical character of pollen and the function of dietary fiber. More researches on the key compounds of pine pollen and the action sites on weight control are required.
引文
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