虫草发酵及其代谢产物的分离与生物活性研究
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摘要
虫草是一类珍稀的真菌药材资源,具有代谢产物和生物活性多样的特点,在功能性食品和生物医药领域展现出巨大的应用前景。本学位论文主要以我国特有的药用虫草新资源——戴氏虫草Cordyceps taii Z.Q. Liang & A.Y. Liu (1991) (= Metacordyceps taii)和江西虫草Cordyceps jiangxiensis Z.Q. Liang, A.Y. Liu & Y.C. Jiang (2001)为研究对象,立足于虫草可持续开发利用尚待解决的一些科学技术问题,在前期研究的基础上,重点就虫草菌丝体及其多糖的发酵生产、生物活性、菌丝体的化学组成分析及其代谢产物的分离纯化等展开研究。
虫草及其代谢产物的发酵生产是虫草研究领域的重要课题。率先把非线性数学模型——期望函数法引入到真菌发酵过程优化,针对江西虫草发酵菌丝体及其多糖两个目标的发酵生产条件进行优化。优化后的发酵条件使虫草菌丝体产量和多糖产量分别高达24.5g/L和8.9g/L,均比优化前增加了2倍多。这为发酵过程复数个目标的优化提供了成功的事例,也奠定了江西虫草可持续开发利用的基础。同时,还采用一种组合的优化实验设计策略,即单次单因子法与Plackett-Burman设计、最陡爬坡法、中心组合设计等响应面法联用的策略,对戴氏虫草胞外多糖的发酵生产条件进行优化,建立了预测戴氏虫草多糖发酵生产的二次多元回归数学模型,并获得了最适的发酵生产条件。优化后的条件使多糖产量从0.77g／L提高到40g/L以上,这为戴氏虫草活性多糖的进一步研发提供了有力的保障。
在上述发酵条件的研究基础上,对戴氏虫草和江西虫草发酵菌丝体多糖组分的生物活性及作用机制进行了探索。观察到这两种虫草的菌丝体多糖组分均具有较强的抗氧化新功效。尤其是大部分多糖组分清除超氧阴离子自由基的活性明显强于硫脲和丁基羟基茴香醚两个阳性对照组；有两个戴氏虫草发酵菌丝体多糖组分,在中低剂量时螯合Fe离子的活性,明显强于EDTA阳性对照组；戴氏虫草多糖的抗氧化作用机制与显著提高内源性抗氧化酶SOD、CAT和GSH-Px的活性以及明显降低脂质过氧化代谢产物MDA的水平有关。另外,观察到江西虫草发酵菌丝体多糖组分MPCJ4和MPCJ5存在非免疫应答的抗肿瘤作用机制,即,作用于肿瘤细胞G1／S期,阻断细胞进入G2／M期,并诱导G1期细胞凋亡,且其与Caspase依赖的凋亡途径有关。
除了多糖这一虫草的主要活性成分之外,江西虫草和戴氏虫草发酵菌丝体中还有其它具有重要价值的营养成分和生物活性物质。为此,对戴氏虫草、江西虫草和古尼虫草Cordyceps gunnii (Berk.) Berk. (1859)发酵菌丝体的营养成分、生物活性物质及潜在的有毒化学物等进行了系统的定性定量分析。古尼虫草、戴氏虫草和江西虫草发酵菌丝体中多糖、虫草酸和核苷类化合物的水平均为江西虫草>戴氏虫草>古尼虫草；古尼虫草、戴氏虫草和江西虫草的发酵菌丝体中富含不饱和脂肪酸和氨基酸,不饱和脂肪酸分别占总脂肪酸中的78.7%,66.9%和79.0%；古尼虫草、戴氏虫草和江西虫草虫草发酵菌丝体中总氨基酸的含量在146.0-168.3mg/g范围,其中必需氨基酸分别占总氨基酸中的37.05%、39.39%和39.37%；古尼虫草、戴氏虫草和江西虫草发酵菌丝体均富含Fe、Zn、Se, Cr和Co等微量元素,Al、Sn、Pb、Cd、As和Hg等潜在毒性物质含量均低于国家的限制水平。综上,江西虫草、戴氏虫草和古尼虫草发酵菌丝体富含较高利用价值的营养成分和生物活性物质,所含潜在毒性物质均在安全范围内,具有作为食药品新资源的潜力。同时,研究结果为建立这些虫草菌丝体生产的相应质控体系提供了实验依据。
在虫草菌丝体营养成分及生物活性物质的全面分析过程,本文还建立了虫草菌丝体中作为重要质控指标的虫草酸和核苷的检测方法。确立了酶标仪比色法检测虫草酸,该方法可替代常规分光光度比色法,其灵敏度、精确度和稳定性良好,准确度较高,并具高通量检测特点；还建立了同时检测7种主要核苷与碱基的高效液相色谱法(HPLC-PDA)。该方法有良好的精密度、稳定性和重复性,比已报道的HPLC方法的分析时间缩短了一半(12min),并能更全面反映虫草及其发酵菌丝体产品中的核苷及碱基；采用HPLC-PDA法检测到江西虫草发酵菌丝体含有尿嘧啶、尿苷、鸟苷、腺嘌呤、腺苷和虫草菌素,其中尿苷(7802.14gg／g)和腺苷(2705.92μg/g)是主要的核苷成分。
众所周知,虫草的研发价值除氨基酸等营养成分以及多糖、虫草酸、核苷等生物活性物质外,还有其它许多有用的次生代谢产物,这些是功能食品研发和新药创制的源泉,也是真正实现虫草高附加值二次化学成分开发利用的基础。在前期抗肿瘤活性检测的基础上,首次从江西虫草发酵菌丝体抗肿瘤有效部位中分离出30多种单体,其中鉴定出18种化合物,即二十四烷酸(2)、十八烷酸-α-单甘油酯(5)、江西烯酮酯jiangxienone(7)、菜油甾醇-二十二烷酸酯(8)、麦角甾醇-十六烷酸酯(9)、尿嘧啶核苷(14)、麦角甾醇(21)、5a-麦角甾-7,22-二烯-3β-醇(22)、丁二酸(23)、尿嘧啶(24)、腺嘌呤(25)、腺嘌呤核苷(26)、烟酸(27)、3’-甲氧基尿嘧啶核苷(28)、2’-甲氧基腺嘌呤核苷(31)、2’-脱氧尿嘧啶核苷(32)、L-焦谷氨酸甲酯(33)和龙脑香醇酮(34)。在这18种化合物中,化合物7-9为新化合物,属于甾体类。其中化合物7(江西烯酮酯)的氧化度高,抑制肿瘤细胞增殖活性明显强于一线化疗药物顺铂,具有创制抗肿瘤新药的潜力。此外,还有10个已知化合物(2,5,22-23,27-28,31-34)为首次从虫草属真菌中获得。这些研究结果丰富了虫草天然产物化合物库,为新药创制增添了化合物资源,也为江西虫草菌丝体的二次化学成分研发奠定了基础。
Cordyceps, an entomopathogenic macrofungus, has been used for centuries as a rare traditional Chinese medicinal herb. It contains various known and untapped bioactive compounds, and is recognized as an important natural resource for the development of health-promoting functional food and for new drug discovery related to immunomodulatory, antitumor, hypoglycemic and hypocholesterolemic activities, etc. The fungi of Cordyceps therefore receive increasing attention around the world as an interesting natural drug resource. Two new and folk medicinal species of Cordyceps, namely Cordyceps taii Z.Q. Liang & A.Y. Liu (1991) (=Metacordyceps taii) and Cordyceps jiangxiensis Z.Q. Liang, A.Y. Liu & Y. C. Jiang (2001), which were discovered in China, were used in the present study. Fermentation for production of mycelial biomass and polysaccharide, evaluation of polysaccharide bioactivity, analysis of Cordyceps mycelia chemical composition, and separation and purification of metabolites from the fermented Cordyceps were performed on the basis of previous work.
The production of Cordyceps spp. and its metabolites using fermentation technology is seen as an important project on research yield of Cordyceps. Desirability function, a nonlinear mathematical model, was applied to optimize the fermentation conditions for simultaneous hyper-production of biomass and intracellular polysaccharide by C. jiangxiensis in submerged cultivation for the first time. Under optimal fermentation conditions obtained by desirability function method, the production titer of mycelia and intracellular polysaccharide reached 24.45 and 8.93 g/1, respectively, after a 9-day submerged cultivation, which was 2 folds higher than that of un-optimized conditions. The use of desirability function was proved effective in optimizing multi-response fermentation processes and it is readily applicable to other bioprocesses.
A combinatorial experimental design strategy involving both the one-variable-at-a-time method and response surface methodology (Plackett-Burman, steepest ascent, and central combination designs) was employed for the optimization of mycelial growth and exopolysaccharides (EPS) production by C. taii. A quadratic multivariate regression model accurately predicting EPS yield was established through the above optimization process. This optimization gave a surprising increase in EPS yield (>40g/L) compared to the level (0.77g/L) obtained in basal medium in shake-flask culture. The improved production condition will be helpful for the development of EPS in the fields of health foods and drug discovery.
Further works such as bioactivity of mycelial polysaccharide from both C. taii and C. jiangxiensis and their mechanism of action were also performed. The antioxidant potency of polysaccharides from C. taii and C. jiangxiensis were evaluated using five different in vitro assays after the fermentation conditions of biomass and polysaccharide production by both C. jiangxiensis and C. taii were established. Among these assays, polysaccharide from C. taii and C. jiangxiensis showed a strong capability of scavenging free radicals and the ferrous ion chelating activity, however, there was a certain variation among different fractions of polysaccharide from the same Cordyceps species. Especially most polysaccharide fractions from two cultured Cordyceps species possessed more potent scavenging superoxide anion radical activity than positive control, and two polysaccharide fractions of C. taii exhibited more significant chelating capability on ferrous ion than positive control. The antioxidant effect of C. taii polysaccharide fraction on D-galactose induced aging mice is associated with enhancing markedly the activities of endogenous antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, and decreasing evidently maleic dialdehyde level. The results suggest that C. taii and C. jiangxiensis are promising sources for development of natural antioxidant. In additional, MPCJ4 and MPCJ5, two enriched-polysaccharide fractions of cultured C. jiangxiensis, exerted antitumor effects at different dose range from 5 to 500μg/mL while had no cytotoxicity. Further investigations indicated that MPCJ4 and MPCJ5 markedly induced cell cycle arrest in the S phase, and resulted in tumor cell death of G1 phase via apoptotic biochemical pathway. These findings thus suggest that MPCJ4 and MPCJ5 might be a promising candidate for application in cancer therapy, and Cordyceps-derivated polysaccharides possess a new antitumor mechanism of action with non-immunologic response.
Besides polysaccharide, the mycelial biomass of Cordceps spp. including both C. jiangxiensis and C. taii possess many high-value nutritional ingredient and bioactive compounds. To evaluate the nutritional and health-promoting value of three edible and medicinal mushrooms C. jiangxiensis, C. taii, and Cordyceps gunnii (Berk.) Berk.(1859) and simultaneously get a considerable data for establishing the quality control standard of Cordyceps preparations, their proximate compositions, bioactive ingredients and potential toxic constituents were investigated by using various chromatographic and spectrographic assays such as HPLC, GC/MS, amino acid high speed chromatography, ICP atomic emission spectrometer etc. and routine physical and chemical analysis. The contents of polysaccharide, cordycepic acid, and nucleosides were C. jiangxiensis>C. taii>C. gunnii. The three fungi contained high levels of unsaturated fatty acids (UFA) and essential amino acids. The percent of UFA relative to total fatty acids was 78.7% for C. gunnii,66.9% for C. taii, and 79.0% for C. jiangxiensis. The Cordyceps species displayed similar amino acid profiles and a high level of total amino acids ranging from 146.0 to 168.3 mg/g, where glutamic acid and aspartic acid were the two principal amino acids with over 10% of the total amino acid contents. Other essential amino acids made up 37.05%,39.39%,39.37% of the total amino acids of C. gunnii, C. taii, and C. jiangxiensis, respectively. Abundant amounts of Fe、Zn、Se、Cr and Co were found in these species, while toxic microelements such as Al、Sn、Pb、Cd、As and Hg were presented at very low amounts and could reach the related quality standard of SFDA in China. Taken together, our findings suggest that the three medicinal Cordyceps species can be regarded as a promising source of nutriceuticals and drugs without any health risk.
During the above process of detecting nutritional ingredients and bioactive compounds of Cordyceps spp, a new universal and high-throughput method was also developed for quantitative determination of cordycepic acid in Cordyceps species using microplate reader, detected at 412 nm. The linearity of the method was found within 6.3-60μg/ml concentration ranges of cordycepic acid, and the linear correlation coefficient was good (R2> 0.9997) within the test ranges. The limits of detection (LOD) and quantification (LOQ) were 1.8μg/mL and 6.3μg/ml, respectively. The recoveries were in the range of 96.71 to 104.61%. Taken together, compared to conventional method, the new method showed good accuracy, less error, and high-throughput ability, and it is applicable in Cordyceps bioproduct. Besides, a simple high-performance liquid chromatography with photo diode array (HPLC-PDA) method for the separation and quantitative determination of nucleosides and nucleobases in cultured C. jiangxiensis has also been developed. The use of mobile phase with ultrapure water and methanol enabled the efficient separation and determination of seven nucleosides and nucleobases within a 12 min.Validation of the method was done with its linearity, precision, and stability, where a good linear correlation (R2>0.9900) for all analytes was obtaied within the test range, the corresponding LOD and LOQ were 0.01-0.30μg/mL and 0.04-0.96μg/mL, respectively, and the relative standard deviations (RSD) for precision and stability were all within 3%. Under the optimized conditions, six nucleosides and nucleobases were detected in cultured C. jiangxiensis, of which uridnine (7802.14μg/g) and adenosine (2705.92μg/g) were principal nucleosides compounds. In short, the developed HPLC-PDA method could be efficiently applied for the simultaneous analysis of nucleosides and nucleobases during fermentation of C. jiangxiensis.
Polysaccharides, cordycepic acid and nucleosides are some major bioactive compounds of Cordyceps spp., but the most valuable compounds should be attributed to some secondary metabolites possessing different pharmcological effects, which are true sources of new drug discovery. Therefore it is necessary to develop the separation and purification of metabolites from the fermented Cordyceps and their bioactivity evaluation, which is useful pathway to discover new compounds and new functions for the development of high-value-added products. With the bioactivity-oriented separation, over 30 compounds were separated from cultured C. jiangxiensis by repeated liquid column chromatography, preparative thin layer chromatography and recrystallzation. The chemical structures of thirteen compounds were elucidated on the basis of spectroscopic methods including UV, IR, ESIMS, HRESIMS, 1D-NMR (1H-NMR,13C-NMR, and DEPT) and 2D-NMR ('H-'H COSY, HMQC, HMBC, and NOESY). Eighteen compounds were identified as tetracosanoic acid (2),1-monostearin (5), jiangxienone (7), docosanoic acid campesterol-ester (8), hexadecanoic acid ergosterol-ester (9), uridine (14), ergosterol (21),5a-Ergosta-7,22-dien-3β-ol (22), succinic acid (23), uracil(24), adenine (25), adenosine (26), nicotinic acid (27), and 3'-methoxyuridine (28),2'-methoxyadenosine (31),2'-deoxyuridine (32), methyl L-pyroglutamate (33), and dipterocarpol (34), of which compounds 7-9 were of new chemical structure belonging to steroides. Ten known compounds (2,5,22-23,27-28,31-34) were obtained from Cordyceps species for the first time. Jiangxienone, a high oxidation degree compound, showed more significant inhibition capability versus first-line antitumor drug cisplatin for inhibiting tumor cell proliferation. Jiangxienone therefore possesses a great potential as a potential new antitumor drug candidate. These findings contributed to a compound source for new drug discovery in Cordyceps-derived natural compound library.
虫草及其代谢产物的发酵生产是虫草研究领域的重要课题。率先把非线性数学模型——期望函数法引入到真菌发酵过程优化,针对江西虫草发酵菌丝体及其多糖两个目标的发酵生产条件进行优化。优化后的发酵条件使虫草菌丝体产量和多糖产量分别高达24.5g/L和8.9g/L,均比优化前增加了2倍多。这为发酵过程复数个目标的优化提供了成功的事例,也奠定了江西虫草可持续开发利用的基础。同时,还采用一种组合的优化实验设计策略,即单次单因子法与Plackett-Burman设计、最陡爬坡法、中心组合设计等响应面法联用的策略,对戴氏虫草胞外多糖的发酵生产条件进行优化,建立了预测戴氏虫草多糖发酵生产的二次多元回归数学模型,并获得了最适的发酵生产条件。优化后的条件使多糖产量从0.77g／L提高到40g/L以上,这为戴氏虫草活性多糖的进一步研发提供了有力的保障。
在上述发酵条件的研究基础上,对戴氏虫草和江西虫草发酵菌丝体多糖组分的生物活性及作用机制进行了探索。观察到这两种虫草的菌丝体多糖组分均具有较强的抗氧化新功效。尤其是大部分多糖组分清除超氧阴离子自由基的活性明显强于硫脲和丁基羟基茴香醚两个阳性对照组；有两个戴氏虫草发酵菌丝体多糖组分,在中低剂量时螯合Fe离子的活性,明显强于EDTA阳性对照组；戴氏虫草多糖的抗氧化作用机制与显著提高内源性抗氧化酶SOD、CAT和GSH-Px的活性以及明显降低脂质过氧化代谢产物MDA的水平有关。另外,观察到江西虫草发酵菌丝体多糖组分MPCJ4和MPCJ5存在非免疫应答的抗肿瘤作用机制,即,作用于肿瘤细胞G1／S期,阻断细胞进入G2／M期,并诱导G1期细胞凋亡,且其与Caspase依赖的凋亡途径有关。
除了多糖这一虫草的主要活性成分之外,江西虫草和戴氏虫草发酵菌丝体中还有其它具有重要价值的营养成分和生物活性物质。为此,对戴氏虫草、江西虫草和古尼虫草Cordyceps gunnii (Berk.) Berk. (1859)发酵菌丝体的营养成分、生物活性物质及潜在的有毒化学物等进行了系统的定性定量分析。古尼虫草、戴氏虫草和江西虫草发酵菌丝体中多糖、虫草酸和核苷类化合物的水平均为江西虫草>戴氏虫草>古尼虫草；古尼虫草、戴氏虫草和江西虫草的发酵菌丝体中富含不饱和脂肪酸和氨基酸,不饱和脂肪酸分别占总脂肪酸中的78.7%,66.9%和79.0%；古尼虫草、戴氏虫草和江西虫草虫草发酵菌丝体中总氨基酸的含量在146.0-168.3mg/g范围,其中必需氨基酸分别占总氨基酸中的37.05%、39.39%和39.37%；古尼虫草、戴氏虫草和江西虫草发酵菌丝体均富含Fe、Zn、Se, Cr和Co等微量元素,Al、Sn、Pb、Cd、As和Hg等潜在毒性物质含量均低于国家的限制水平。综上,江西虫草、戴氏虫草和古尼虫草发酵菌丝体富含较高利用价值的营养成分和生物活性物质,所含潜在毒性物质均在安全范围内,具有作为食药品新资源的潜力。同时,研究结果为建立这些虫草菌丝体生产的相应质控体系提供了实验依据。
在虫草菌丝体营养成分及生物活性物质的全面分析过程,本文还建立了虫草菌丝体中作为重要质控指标的虫草酸和核苷的检测方法。确立了酶标仪比色法检测虫草酸,该方法可替代常规分光光度比色法,其灵敏度、精确度和稳定性良好,准确度较高,并具高通量检测特点；还建立了同时检测7种主要核苷与碱基的高效液相色谱法(HPLC-PDA)。该方法有良好的精密度、稳定性和重复性,比已报道的HPLC方法的分析时间缩短了一半(12min),并能更全面反映虫草及其发酵菌丝体产品中的核苷及碱基；采用HPLC-PDA法检测到江西虫草发酵菌丝体含有尿嘧啶、尿苷、鸟苷、腺嘌呤、腺苷和虫草菌素,其中尿苷(7802.14gg／g)和腺苷(2705.92μg/g)是主要的核苷成分。
众所周知,虫草的研发价值除氨基酸等营养成分以及多糖、虫草酸、核苷等生物活性物质外,还有其它许多有用的次生代谢产物,这些是功能食品研发和新药创制的源泉,也是真正实现虫草高附加值二次化学成分开发利用的基础。在前期抗肿瘤活性检测的基础上,首次从江西虫草发酵菌丝体抗肿瘤有效部位中分离出30多种单体,其中鉴定出18种化合物,即二十四烷酸(2)、十八烷酸-α-单甘油酯(5)、江西烯酮酯jiangxienone(7)、菜油甾醇-二十二烷酸酯(8)、麦角甾醇-十六烷酸酯(9)、尿嘧啶核苷(14)、麦角甾醇(21)、5a-麦角甾-7,22-二烯-3β-醇(22)、丁二酸(23)、尿嘧啶(24)、腺嘌呤(25)、腺嘌呤核苷(26)、烟酸(27)、3’-甲氧基尿嘧啶核苷(28)、2’-甲氧基腺嘌呤核苷(31)、2’-脱氧尿嘧啶核苷(32)、L-焦谷氨酸甲酯(33)和龙脑香醇酮(34)。在这18种化合物中,化合物7-9为新化合物,属于甾体类。其中化合物7(江西烯酮酯)的氧化度高,抑制肿瘤细胞增殖活性明显强于一线化疗药物顺铂,具有创制抗肿瘤新药的潜力。此外,还有10个已知化合物(2,5,22-23,27-28,31-34)为首次从虫草属真菌中获得。这些研究结果丰富了虫草天然产物化合物库,为新药创制增添了化合物资源,也为江西虫草菌丝体的二次化学成分研发奠定了基础。
Cordyceps, an entomopathogenic macrofungus, has been used for centuries as a rare traditional Chinese medicinal herb. It contains various known and untapped bioactive compounds, and is recognized as an important natural resource for the development of health-promoting functional food and for new drug discovery related to immunomodulatory, antitumor, hypoglycemic and hypocholesterolemic activities, etc. The fungi of Cordyceps therefore receive increasing attention around the world as an interesting natural drug resource. Two new and folk medicinal species of Cordyceps, namely Cordyceps taii Z.Q. Liang & A.Y. Liu (1991) (=Metacordyceps taii) and Cordyceps jiangxiensis Z.Q. Liang, A.Y. Liu & Y. C. Jiang (2001), which were discovered in China, were used in the present study. Fermentation for production of mycelial biomass and polysaccharide, evaluation of polysaccharide bioactivity, analysis of Cordyceps mycelia chemical composition, and separation and purification of metabolites from the fermented Cordyceps were performed on the basis of previous work.
The production of Cordyceps spp. and its metabolites using fermentation technology is seen as an important project on research yield of Cordyceps. Desirability function, a nonlinear mathematical model, was applied to optimize the fermentation conditions for simultaneous hyper-production of biomass and intracellular polysaccharide by C. jiangxiensis in submerged cultivation for the first time. Under optimal fermentation conditions obtained by desirability function method, the production titer of mycelia and intracellular polysaccharide reached 24.45 and 8.93 g/1, respectively, after a 9-day submerged cultivation, which was 2 folds higher than that of un-optimized conditions. The use of desirability function was proved effective in optimizing multi-response fermentation processes and it is readily applicable to other bioprocesses.
A combinatorial experimental design strategy involving both the one-variable-at-a-time method and response surface methodology (Plackett-Burman, steepest ascent, and central combination designs) was employed for the optimization of mycelial growth and exopolysaccharides (EPS) production by C. taii. A quadratic multivariate regression model accurately predicting EPS yield was established through the above optimization process. This optimization gave a surprising increase in EPS yield (>40g/L) compared to the level (0.77g/L) obtained in basal medium in shake-flask culture. The improved production condition will be helpful for the development of EPS in the fields of health foods and drug discovery.
Further works such as bioactivity of mycelial polysaccharide from both C. taii and C. jiangxiensis and their mechanism of action were also performed. The antioxidant potency of polysaccharides from C. taii and C. jiangxiensis were evaluated using five different in vitro assays after the fermentation conditions of biomass and polysaccharide production by both C. jiangxiensis and C. taii were established. Among these assays, polysaccharide from C. taii and C. jiangxiensis showed a strong capability of scavenging free radicals and the ferrous ion chelating activity, however, there was a certain variation among different fractions of polysaccharide from the same Cordyceps species. Especially most polysaccharide fractions from two cultured Cordyceps species possessed more potent scavenging superoxide anion radical activity than positive control, and two polysaccharide fractions of C. taii exhibited more significant chelating capability on ferrous ion than positive control. The antioxidant effect of C. taii polysaccharide fraction on D-galactose induced aging mice is associated with enhancing markedly the activities of endogenous antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, and decreasing evidently maleic dialdehyde level. The results suggest that C. taii and C. jiangxiensis are promising sources for development of natural antioxidant. In additional, MPCJ4 and MPCJ5, two enriched-polysaccharide fractions of cultured C. jiangxiensis, exerted antitumor effects at different dose range from 5 to 500μg/mL while had no cytotoxicity. Further investigations indicated that MPCJ4 and MPCJ5 markedly induced cell cycle arrest in the S phase, and resulted in tumor cell death of G1 phase via apoptotic biochemical pathway. These findings thus suggest that MPCJ4 and MPCJ5 might be a promising candidate for application in cancer therapy, and Cordyceps-derivated polysaccharides possess a new antitumor mechanism of action with non-immunologic response.
Besides polysaccharide, the mycelial biomass of Cordceps spp. including both C. jiangxiensis and C. taii possess many high-value nutritional ingredient and bioactive compounds. To evaluate the nutritional and health-promoting value of three edible and medicinal mushrooms C. jiangxiensis, C. taii, and Cordyceps gunnii (Berk.) Berk.(1859) and simultaneously get a considerable data for establishing the quality control standard of Cordyceps preparations, their proximate compositions, bioactive ingredients and potential toxic constituents were investigated by using various chromatographic and spectrographic assays such as HPLC, GC/MS, amino acid high speed chromatography, ICP atomic emission spectrometer etc. and routine physical and chemical analysis. The contents of polysaccharide, cordycepic acid, and nucleosides were C. jiangxiensis>C. taii>C. gunnii. The three fungi contained high levels of unsaturated fatty acids (UFA) and essential amino acids. The percent of UFA relative to total fatty acids was 78.7% for C. gunnii,66.9% for C. taii, and 79.0% for C. jiangxiensis. The Cordyceps species displayed similar amino acid profiles and a high level of total amino acids ranging from 146.0 to 168.3 mg/g, where glutamic acid and aspartic acid were the two principal amino acids with over 10% of the total amino acid contents. Other essential amino acids made up 37.05%,39.39%,39.37% of the total amino acids of C. gunnii, C. taii, and C. jiangxiensis, respectively. Abundant amounts of Fe、Zn、Se、Cr and Co were found in these species, while toxic microelements such as Al、Sn、Pb、Cd、As and Hg were presented at very low amounts and could reach the related quality standard of SFDA in China. Taken together, our findings suggest that the three medicinal Cordyceps species can be regarded as a promising source of nutriceuticals and drugs without any health risk.
During the above process of detecting nutritional ingredients and bioactive compounds of Cordyceps spp, a new universal and high-throughput method was also developed for quantitative determination of cordycepic acid in Cordyceps species using microplate reader, detected at 412 nm. The linearity of the method was found within 6.3-60μg/ml concentration ranges of cordycepic acid, and the linear correlation coefficient was good (R2> 0.9997) within the test ranges. The limits of detection (LOD) and quantification (LOQ) were 1.8μg/mL and 6.3μg/ml, respectively. The recoveries were in the range of 96.71 to 104.61%. Taken together, compared to conventional method, the new method showed good accuracy, less error, and high-throughput ability, and it is applicable in Cordyceps bioproduct. Besides, a simple high-performance liquid chromatography with photo diode array (HPLC-PDA) method for the separation and quantitative determination of nucleosides and nucleobases in cultured C. jiangxiensis has also been developed. The use of mobile phase with ultrapure water and methanol enabled the efficient separation and determination of seven nucleosides and nucleobases within a 12 min.Validation of the method was done with its linearity, precision, and stability, where a good linear correlation (R2>0.9900) for all analytes was obtaied within the test range, the corresponding LOD and LOQ were 0.01-0.30μg/mL and 0.04-0.96μg/mL, respectively, and the relative standard deviations (RSD) for precision and stability were all within 3%. Under the optimized conditions, six nucleosides and nucleobases were detected in cultured C. jiangxiensis, of which uridnine (7802.14μg/g) and adenosine (2705.92μg/g) were principal nucleosides compounds. In short, the developed HPLC-PDA method could be efficiently applied for the simultaneous analysis of nucleosides and nucleobases during fermentation of C. jiangxiensis.
Polysaccharides, cordycepic acid and nucleosides are some major bioactive compounds of Cordyceps spp., but the most valuable compounds should be attributed to some secondary metabolites possessing different pharmcological effects, which are true sources of new drug discovery. Therefore it is necessary to develop the separation and purification of metabolites from the fermented Cordyceps and their bioactivity evaluation, which is useful pathway to discover new compounds and new functions for the development of high-value-added products. With the bioactivity-oriented separation, over 30 compounds were separated from cultured C. jiangxiensis by repeated liquid column chromatography, preparative thin layer chromatography and recrystallzation. The chemical structures of thirteen compounds were elucidated on the basis of spectroscopic methods including UV, IR, ESIMS, HRESIMS, 1D-NMR (1H-NMR,13C-NMR, and DEPT) and 2D-NMR ('H-'H COSY, HMQC, HMBC, and NOESY). Eighteen compounds were identified as tetracosanoic acid (2),1-monostearin (5), jiangxienone (7), docosanoic acid campesterol-ester (8), hexadecanoic acid ergosterol-ester (9), uridine (14), ergosterol (21),5a-Ergosta-7,22-dien-3β-ol (22), succinic acid (23), uracil(24), adenine (25), adenosine (26), nicotinic acid (27), and 3'-methoxyuridine (28),2'-methoxyadenosine (31),2'-deoxyuridine (32), methyl L-pyroglutamate (33), and dipterocarpol (34), of which compounds 7-9 were of new chemical structure belonging to steroides. Ten known compounds (2,5,22-23,27-28,31-34) were obtained from Cordyceps species for the first time. Jiangxienone, a high oxidation degree compound, showed more significant inhibition capability versus first-line antitumor drug cisplatin for inhibiting tumor cell proliferation. Jiangxienone therefore possesses a great potential as a potential new antitumor drug candidate. These findings contributed to a compound source for new drug discovery in Cordyceps-derived natural compound library.
引文
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