赤灵芝的营养成分、生物活性及其应用研究进展

赤灵芝具有抗肿瘤、抗氧化、提高免疫力等功效。文章概括了近年来赤灵芝中主要营养成分及其生物活性的最新研究进展,总结了赤灵芝在医疗保健等领域的综合利用现状,分析了存在的问题,并展望了赤灵芝的未来发展方向。     

Bistage control of pH for improving exopolysaccharide production from mycelia of Ganoderma lucidum in an air-lift fermentor

It was found that pH control definitely affects mycelial cell growth and exopolysaccharide (EPS) production of the mycelial cultivation of Ganoderma lucidum. Compared to the case of uncontrolled pH cultivation, a culture system whose pH was kept constant at 3 and 6 exhibited improved mycelial cell growth and EPS production, respectively. The bistage pH control technique, that is, shifting the pH from 3 to 6 at the initial phase of the exponential growth, is introduced to improve cell growth and EPS production. This technique can greatly increase EPS production to 20.1 g/l from 4.1 g/l in the case of uncontrolled pH cultivation, without adverse effects on cell growth as in the case of constant maintenance of a high pH. It was also proved that bistage pH control retained the desirable morphologies of the mycelia during cultivation and resulted in low viscosity and yield stress of the culture broth. It will be useful for the application of the culture process to mycelial growth in a large-scale fermentor.     

继代培养对灵芝胞外酶活力及品质的影响

为探究菌种继代培养对灵芝栽培过程中胞外酶活力和灵芝子实体品质的影响,采用比色法、高效液相色谱法等研究了不同传代次数菌种对灵芝胞外酶活力、子实体干基生物学效率、浸出物、多糖、三萜及甾醇、灵芝酸类成分的影响,并通过相关性分析探讨胞外酶活力和灵芝品质之间的相关性。结果表明,随着菌种继代培养次数的增加,灵芝漆酶、滤纸酶、纤维素酶和淀粉酶酶活力呈下降趋势;相关性分析表明,漆酶、滤纸酶、纤维素酶和淀粉酶酶活与子实体干基生物学效率、浸出物、多糖质量分数及灵芝酸A、灵芝酸D、灵芝酸F、灵芝烯酸A和灵芝烯酸D质量浓度呈极显著正相关;进一步通径分析发现滤纸酶是影响灵芝干基生物学效率及多糖、灵芝酸F质量浓度的关键胞外酶,纤维素酶是影响灵芝烯酸A质量浓度的关键胞外酶。因此,在实际生产中,应将灵芝菌种继代培养次数控制在5代以内,基质中主要胞外酶具有高活力,灵芝干基生物学效率高且品质最好。     

Preparative isolation of triterpenoids from Ganoderma lucidum by counter-current chromatography combined with pH-zone-refining

Ganoderma lucidum is a famous fungus. The triterpenoids are the main bioactive components and exhibit various pharmacological activities. However, the scarcity of the pure triterpenoid has greatly restrained the modern research of G. lucidum. The present paper first describes a convenient method to separate the main triterpenoids from G. lucidum using counter-current chromatography (CCC) technique. Ganoderic acid C6 (38 mg), E (76 mg), F (32 mg) were successfully separated from the crude extract in 1 day with the HPLC purity of above 90% using stepwise CCC. Ganoderic acid G (36 mg), A (64 mg), B (25 mg), D (28 mg) and ganoderenic acid D (11 mg) were separated in 2 days with the HPLC purity of above 90% using a combination of stepwise CCC and pH-zone-refining CCC. The method presented in the paper is much quicker and easier than the previous methods.     

Effect of mycelial culture broth of Ganoderma lucidum on the growth characteristics of human cell lines

Two types of purified samples, water-soluble (sample A; M. W, 1.2×10⁶ dalton) and water-insoluble (sample C; M. W., 1.0×10⁶ dalton) samples, were obtained through consecutive separation processes from the culture broth of Ganoderma lucidia mycelium. It was found that both samples from the culture broth were very effective in inhibiting the growth of several human cancer cell lines, having a 93-85% growth inhibition on Hep3B, AGS and A549 with the least cytotoxicity on the normal human lung cell line, WRL68 of less than 25% the highest supplementation concentration of 1.0 mg/l. In general, the sample C showed greater inhibition of cancer cell growth than the sample A. The same trend was also observed in antimutagenicity using the Chinese hamster ovary cell line (CHO test) or Salmonella typhimurium (Ames test). The CHO test showed that sample C had higher antimutagenicity on mutagens 4NQO or MMNG than sample A (approximately 40% vs approximately 25%). The percentage of antimutagenicity from the Ames test was lower than that from the CHO test, possibly due to the difference in the sensitivity of mutagens. The water-insoluble sample greatly enhanced the growth of the human T cell line (H9) up to 1×10⁵ with sample supplementation at 1.0 mg/l concentration from 4.3×10⁴ without sample supplementation as well as improved the secretion level of both IL-6 and TNF-α up to 100 pg/ml from approximately 40 pg/ml without sample supplementation. The kinetics of response to the immune cell growth was illustrated by the response time obtained when the sample concentration was increased. The water-insoluble sample can be used for effectively treating cancer in that it accelerated apoptosis of human carcinoma cells up to 70% compared to less than 50% for the control. The sample also increased the differentiation ratio of HL-60 cells up to 58% after four days of cultivation, compared to 18% in the case of no sample supplementation. These results can be used in implying that the insoluble part of G. lucidium mycelium culture broth must be related to controlling signal transduction, resulting in the regulation of cancer cell growth.     

Quantification of total polysaccharides and triterpenoids in Ganoderma lucidum and Ganoderma atrum by near infrared spectroscopy and chemometrics

Two chemometrics, the partial least-squares (PLS) and radial basis function (RBF) network were performed to develop a quantification method for total polysaccharides and triterpenoids in Ganoderma lucidum and Ganoderma atrum from different origins based on near infrared reflectance spectroscopy (NIR). The influences of spectral window and spectral pre-treatments were initially studied in the construction of PLS model. The best result was obtained when the standard normal transformation (SNV) +1st derivative spectrum over 4100–7750 cm⁻¹ was used for the modelling. Then based on each principle, both of the two models were optimised respectively. The final results with high determination coefficient (R²) (higher than 0.973, 0.989 for PLS and RBF, respectively) and low root mean square errors of prediction (RMSEP) (low to 0.1109 and 0.01298 for polysaccharides and triterpenoids, respectively) confirm the good predictability of the two models. The overall results show that NIR spectroscopy combined with chemometrics can be efficiently utilised for accurate analysis of routine chemical compositions in G. lucidum and G. atrum.     

灵芝总皂苷对结肠癌HCT116细胞的抑制作用

为探究灵芝总皂苷提取物对结肠癌HCT116细胞的抑制作用和相关机制,以灵芝子实体为原料,醇提并纯化总皂苷,通过CCK8法测定灵芝总皂苷提取物对结肠癌HCT116细胞的抑制效果,同时采用流式细胞术检测细胞凋亡,荧光分光光度计检测细胞内线粒体膜电位及细胞内活性氧含量,实时荧光定量PCR检测凋亡相关基因p53、noax、bax、bcl-2、caspase-9和caspase-3的表达。结果显示,与空白组相比,灵芝总皂苷质量浓度为40 mg/L以上时,可极显著地抑制HCT116细胞增殖,且抑制效果与时间呈正相关。经灵芝总皂苷干预后,细胞凋亡率显著增加,当加入灵芝总皂苷的质量浓度为150 mg/L时,其凋亡率达到50.2%,且灵芝总皂苷可显著降低细胞内线粒体膜电位,增加细胞内活性氧含量,增加促凋亡基因p53、noax、bax、caspase-9和caspase-3的表达,降低抗凋亡基因bcl-2的表达。综上,灵芝总皂苷对于结肠癌细胞具有显著抑制作用,且其抑制效果可通过线粒体凋亡途径实现,是值得进一步研究的抑结肠癌候选药物和食品添加剂。     

双水相法分离纯化灵芝中三萜的工艺优化

基于亲水性醇-无机盐双水相体系,建立高效分离纯化灵芝三萜的新方法。考察亲水性有机试剂种类、无机盐(种类、浓度、pH)、样品加入量、分相后下相溶液pH对三萜得率的影响。在单因素实验的基础上,选择K₃PO₄溶液浓度、样品加入量、分相后下相溶液pH为实验因素,采用L₉(3⁴)正交实验优化灵芝中三萜分离纯化的最佳条件。最佳萃取条件为:10.6% K₃PO₄、42.5%甲醇、5%灵芝粗提液组成的双水相体系,成相后调节下相萃取液pH至 4.0,灵芝三萜提取量可达(10.13±0.19) mg/g,较碱提酸化法(7.42±0.09) mg/g提高了36.5%。甲醇/K₃PO₄双水相体系是一种简单、高效分离纯化灵芝三萜的新方法。     

Bioactive dammarane-type triterpenoids derived from the acid hydrolysate of Gynostemma pentaphyllum saponins

From an acid hydrolysate of the crude saponins of Gynostemma pentaphyllum, three triterpenes, including a new compound (23S)-3β-hydroxydammar-20,24-dien-21-oic acid 21,23-lactone (1) and two known aglycons (20S, 23R)-3β,20β-dihydroxydamma-24-dien-21-oic acid 21,23-lactone (2) and (20S, 24S)-20,24-epoxydammarane-3β,12β,25-triol (3), were isolated. Their structures were established on the basis of extensive spectral evidence (HR-ESI-MS, IR, 1D and 2D-NMR experiments). In bioactive assays in vitro, compound 1 was found to have potent cytotoxicity against the human breast cancer cells MDA-MB-435, whereas compounds 2 and 3 exhibited modest inhibitory activity toward porcine pancreatic lipase. The results indicated that acid treatment of G. pentaphyllum extract could produce diverse structures with interesting bioactivity.     

Changes in some components of soymilk during fermentation with the basidiomycete Ganoderma lucidum

Soymilk was fermented with the basidiomycete Ganoderma lucidum WZ02 and the changes in the contents of polysaccharide, sugars, crude protein, B-vitamins, free amino acids and isoflavones were analyzed. Polysaccharide and crude protein were increased by the fermentation of G. lucidum while most free amino acids were reduced. The flatulence factor (e.g. stachyose and raffinose) was significantly decreased and stachyose was not detected after 72 h of fermentation. The contents of thiamin, riboflavin, and niacin were increased during the fermentation. Most of isoflavone glycosides were converted to aglycones and the contents of daidzein and genistein were increased by the fermentation of G. lucidum. The results suggested that fermentation by G. lucidum could improve the acceptability and health properties of soymilk.     

Effect of fermentation time on antioxidative activities of Ganoderma lucidum broth using leguminous plants as part of the liquid fermentation medium

Oxidative damage plays an important role in the pathology of human diseases. Ganoderma lucidum, a medicinal fungus, has been used for thousands of years in traditional Oriental medicine. It is reported to have antioxidant functions such as inhibition of lipid peroxidation. The objective of the present study was to investigate the effect of fermentation time on the antioxidative activities of G. lucidum broth filtrate using leguminous plants as part of the liquid fermentation medium. Inhibition of Cu²⁺-induced oxidation of human low-density lipoprotein (LDL), DPPH radical-scavenging activity, total phenolic compounds, isoflavones and protocatechuic acid were measured to evaluate the antioxidant activity of G. lucidum fermentation broth filtrate. Our results showed that black soybean and Astragalus membranaceus improved the antioxidant activity of the G. lucidum fermentation broth filtrate. Protocatechuic acid was identified by LC–MS as the antioxidant compounds whose relative potency of inhibiting LDL oxidation to Trolox is 1.55. Protocatechuic acid showed positive correlation with the antioxidant activity of the fermentation broth filtrate while isoflavones did not contribute to antioxidant activity.     

Biotransformation of ginsenoside Rd in the ginseng extraction residue by fermentation with lingzhi (Ganoderma lucidum)

Ginseng and lingzhi (Ganoderma lucidum) both are valuable traditional Chinese medicines and have been extensively utilised in functional foods and traditional medicines in many Asian countries. However, massive quantity of ginseng residue is produced after extraction of ginseng which still contains a lot of bioactive compounds such as ginsenosides. The goal of this study was to reuse the American ginseng extraction residue as the fermentation medium of G. lucidum to produce bioactive ginsenoside enriched biotransformation products. The changes of ginsenosides in the fermentation products were analysed during fermentation. Our results showed that after 30 days of fermentation, ginsenoside Rg1, Rd, and compound K (CK) significantly increased, especially Rd, while other ginsenosides (Re, Rb1 and Rc) decreased during fermentation. Ginsenoside Rd is the major ginsenoside in the final fermentation product. Furthermore, the biotransformation of ginsenosides was the major reaction in this fermentation process.     

Biochemical characterization of a proteoglycan complex from an edible mushroom Ganoderma lucidum fruiting bodies and its immunoregulatory activity

A fraction, LZ-D-7, was isolated from the fruiting body of the edible mushroom Ganoderma lucidum using a series of chromatographic steps. Biochemical experiments showed that the complex is a proteoglycan and has a carbohydrate percentage of 95.3%, along with a protein percentage of 3.3%. Methylation analysis showed the polysaccharide moiety of LZ-D-7 had a backbone of 1,4-disubsituted-glucopyranosyl and 1,2,6-trisubstituted-glucopyranosyl residues. The branches were mainly composed of 1-substituted-glucopyranosyl residue, which was attached to 1,2,6-trisubstituted-glucopyranosyl residue via 2 or 6 position. Investigation of modulation of LZ-D-7 on BALB/c mouse spleen lymphocytes cells resulted in a three to four-fold increase of MSLs percentage. Analysis of activation and proliferation of MSLs indicated that LZ-D-7 activating most of the activated cells were B cells. The B cells were enlarged, expressed CD⁷¹⁺ and CD²⁵⁺ on the cell surface.     

Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus

Eight strains of Lactobacillus plantarum, 6 strains of Lactobacillus casei, Lactobacillus acidophilus LA-5, and Bifidobacterium lactis BB-12 were screened for β-glucosidase activity. We then proceeded to investigate the enzymatic potential of selected strains for bioconversion of delphinidin and malvidin glycosides to their metabolites. L. plantarum and L. casei strains showed the highest cell-envelope associated β-glucosidase activity. Intracellular β-glucosidase activity from B. lactis BB-12 was up to 287-fold higher than that of the other strains. The L. acidophilus strain showed low β-glucosidase activity, both, intra and extracellularly. No aglycons were detected in bacterial extract reactions with anthocyanin glycosides. Delphinidin-3-glucoside underwent chemical degradation to form mainly gallic acid, although delphinidin-3-glucoside degradation due to B. lactis BB-12 and enzymatic activity towards chemically-formed metabolites due to L. casei LC-01 were observed. Incubation of malvidin-3-glucoside with B. lactis BB-12, L. plantarum IFPL722, and L. casei LC-01 cell-free extracts led to different patterns of gallic, homogentisic and syringic acid formation.     

Antioxidant activities from the aerial parts of Platycodon grandiflorum

In order to obtain basic data necessary for the utilisation of aerial parts from Platycodon grandiflorum as a functional substance in Korea, the antioxidant activities of solvent fractions from the ethanol extract of P. grandiflorum aerial parts were examined. The butanol fraction from P. grandiflorum showed the most potent antioxidant activities in each assay, showing 91.31% in the DPPH radical scavenging method, 99.62% in the ABTS radical scavenging method, 7.84% in the reducing power method, and 1.29% in the FRAP method at a concentration of 10 mg/ml. The DPPH, ABTS, reducing power, and FRAP assay indicated that the butanol fraction of aerial parts of P. grandiflorum was the most potent radical-scavengers and reducing agents compared to the other two extracts. Therefore, our study verified that the butanol fraction has strong antioxidant activities which are correlated with its high level of phenolics, particularly luteolin-7-O-glucoside and apigenin-7-O-glucoside. This extract of P. grandiflorum aerial parts can be utilised as an effective and safe source of functional food materials such as natural antioxidants.     

Evaluation of in vivo antioxidant activities of Ganoderma lucidum polysaccharides in STZ-diabetic rats

Effect of Ganoderma lucidum polysaccharides treatment on blood glucose, serum insulin level, lipid peroxidation, nonenzymic and enzymic antioxidants in the plasma and liver of streptozotocin (STZ)-induced diabetic rats was studied. Adult male rats of Wistar strain, weighing 195 to 250 g, were randomized into control and experimental groups. Experiment group rats were induced diabetes by administration of STZ (45 mg/kg b.wt.) intraperitoneally. The diabetic rats were treated with G. lucidum polysaccharides (60, 120, 180 mg/kg b.wt.) dissolved in 15% dimethyl sulphoxide (DMSO) for 30 days. The normal control rats were treated with 15% DMSO for 30 days. Streptozotocin treatment elevated the levels of lipid peroxidation markers (thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes), and reduced nonenzymic antioxidants (vitamin C and reduced glutathione, vitamin E) levels, and enzymic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) activities in the plasma and liver of untreated diabetic control rats. Decreased level of serum insulin and increased level of blood glucose (BG) were observed in the plasma of untreated diabetic control rats. G. lucidum polysaccharides treatment significantly and dose-dependently increased nonenzymic and enzymic antioxidants, serum insulin level and reduced lipid peroxidation, blood glucose levels in STZ-diabetic rats. From the present study, it can be concluded that G. lucidum polysaccharides can be considered as a potent antioxidant.     

Production of -aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods

Lactobacillus strains that accumulated γ-aminobutyric acid (GABA) in culture medium were screened to determine strains with high GABA-producing ability. One strain, NFRI 7415, which was isolated from a Japanese traditional fermented fish (funa-sushi), showed the highest GABA-producing ability among the screened strains. Identification tests (i.e., 16S rDNA sequencing and sugar assimilation ability) indicated that NFRI 7415 belongs to Lb. paracasei. The GABA production was further improved by the addition of pyridoxal phosphate to the culture medium and pH regulation of culture medium at pH 5.0. Under optimal cultivation conditions, strain NFRI 7415 produced GABA at a concentration of 302 mm when the glutamate concentration in the culture medium was 500 mm.     

Protective effects of triterpenoids from Ganoderma resinaceum on H2O2-induced toxicity in HepG2 cells

Ganoderma resinaceum Boud. (Polyporeseae) has long been used for antioxidant, immunoregulation and liver protection. From the fruiting bodies of G. resinaceum, eight new lanostanoids, lucidones D–G (1–4), 7-oxo-ganoderic acid Z₂ (5), 7-oxo-ganoderic acid Z₃ (6), ganoderesin A (7), and ganoderesin B (8), together with six known lanostanoids (9–14) were isolated. The structures of new compounds were elucidated through extensive spectroscopic analysis. In an in vitro model, ganoderesin B (8), ganoderol B (10) and lucidone A (11) showed inhibitory effects against the increase of ALT and AST levels in HepG2 cells induced by H₂O₂ compared to a control group in the range of their maximum non-toxic concentration (MNTC). However, compounds 8, 10 and 11 displayed no anti-oxidant activities by DPPH assay. Meanwhile, activation for PXR (Pregnane X Receptor) of ganoderesin B (8), ganoderol B (10) and lucidone A (11) was evaluated; ganoderol (10) exhibited a vital activation for PXR-induced CYP3A4 expression. These results suggested that GTs (Ganoderma triterpenoids) exhibited hepatoprotective activities by lowering ALT and AST levels.     

Fumigant properties of physical preparations from mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) beetle for stored grain insects

Vapors released from foliage of mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) Beetle, through a patented process, were hypothesized to have an insecticidal time of action (24 h or less after time of exposure) similar to the fumigant methyl bromide. Patented preparations were more effective from plants harvested from a relatively wet site in mid to late summer (5 July to 11 September). Bioassays with the lesser grain borer, Rhyzopertha dominica (F.), 0–3 days after adult emergence indicated an LT₅₀ of 7.0±1.2 h for the volatiles generated from only 30 mg dry processed plant material (=0.56 mg active ingredients) per ml headspace. Hatching of eggs of the Indian meal moth, Plodia interpunctella (Hübner), was completely suppressed when exposed 4–20 h after oviposition to a concentration of 7 mg processed plant material per ml headspace (=0.14 mg active ingredients) in a container that allowed passive diffusion and from which the terpenes disappeared by 48 h. Adult red flour beetle, Tribolium castaneum (Herbst), had an LT₅₀ of 40.7±1.2 h when exposed to 29 mg processed plant material per ml headspace. Gas chromatography/mass spectrometry (GC/MS) analyses of the headspace above this processed plant material revealed five major peaks, all non-chlorinated and non-brominated. The two main volatiles, 1,8-cineole and camphor, occurred initially in a mean ratio of 1:3.2, gradually shifting to 1:2.4 over 24 h. The μg/ml headspace of each detectable compound in a sealed container was followed intensely (0.25, 1, 2, 12, 24, 48, and 72 h) for 72 h and at less frequent intervals for 60 days. The active compounds released by the plant material in a closed, but not airtight container, were no longer detectable after 24 h based on GC/MS analysis. Fumigative studies with the same ratio of the two main compounds generated synthetically indicated that embryos of P. interpunctella and adults of R. dominica were as sensitive to the synthetic mixture as they were to the processed plant material. Although one could apply the precise commercial terpenes in the same ratio, the plant material provides a natural formulation that is conveniently diluted (formulated) to levels safe for handling. Therefore, this preparation method and plant material shows good potential as an alternative to methyl bromide for protection of stored grain, commodity, and space fumigations. No residues are detectable in the headspace of aerated commodity, milled product, or in fumigated space.