Ling Zhi Research Articles

Trifolirhizin from Sophora flavescens as a Promising Medicinal Agent: An Update on Biological Significance, Pharmacological Activity, and Analytical Applications

Sophora flavescens is an important medicinal plant found in India and East Asia, primarily in China, Japan, Korea and Russia. Trifolirhizin is an active phytochemical of Sophora flavescens and other Chinese medications such as Zeng-Sheng-Ping, Xian-Lian-Ke-Li, ASHMI, Ling Zhi, Ku Shen, and Gan Cao, Ku Shen injection and Zeng Sheng Ping tablets. Sophora flavescens have numerous therapeutic benefits against hematochezia, jaundice, liver disorders, dysentery, oliguria, asthma, eczema, vulvar swelling, and inflammatory complications. The purpose of the present review is to summarize the scientific information on trifolirhizin for their biological potential, pharmacological activities, and analytical aspects of medicine. Scientific information on trifolirhizin has been collected from various scientific databases such as Scopus, Science Direct, PubMed, and Google Scholar. Further, the present review summarized the pharmacological activities of trifolirhizin and their different molecular mechanisms and analytical aspects. The scientific data on trifolirhizin signified the biological potential and therapeutic effectiveness of trifolirhizin in medicine. Present review signified the presence of trifolirhizin in the Sophora flavescens. Sophora flavescens has been shown to be effective against ulcers, chronic bronchitis, acne, atopic dermatitis, chronic hepatitis, enteritis, pyogenic infection and other inflammatory diseases. Furthermore, the present review also summarized the biological potential of trifolirhizin in gastric cancer, hepatocellular carcinoma, colorectal cancer, apoptosis, ulcerative colitis, and asthma. However, hepatoprotective effects, anti-inflammatory effects, wound healing potential, and efficiency of melanin synthesis were also revealed. In addition, this review also summarizes the molecular mechanisms of trifolirhizin and their pharmacokinetic and metabolic parameters. This review also describes the analytical aspects of trifolirhizin in medicine. This review will be useful to the entire research community exploring the biological potential of trifolirhizin in medicine. However, scientific studies require comprehensive and detailed data on trifolirhizin evaluation, quality control, toxicity, and clinical efficacy.

Preclinical Safety Evaluation of Engineered Lactococcus lactis NZ3900 Biotherapeutics Using a 13-Week Repeated Dose Oral Toxicity Study in Rabbits

ABSTRACT Lactococcus lactis is one of the most important and safe microorganisms used in the dairy industry. Previously, we orally fed cholesterol-fed rabbits with recombinant Ling Zhi 8 (LZ8) protein from L. lactis NZ3900/pNZ8149 and showed anti-inflammatory effects during the development of fatty liver and atheroma. In order to generate a clinically effective oral immunotherapy for the treatment of allergy, L. lactis clones for Der p 2, Per a 2, and Cla c 14 were introduced into L. lactis NZ3900/pNZ8149 to obtain recombinant allergen-based vaccines. In this study, we developed a triple-antigen vaccine formulation, Lactis- IL-1Ra, Lactis-LZ8, Lactis-Triple, and evaluated their preclinical safety in rabbits by oral administration with repeated doses for 90 days. All of the rabbits survived the challenge without any adverse clinical signs or abnormal changes. A notable advantage of this vaccine is that it can be given orally, and thus self-administration at home would be feasible.

Comparative study on chitin content of Bangladeshi edible and medicinal mushrooms

Chitin provides immense beneficial roles to the humanity and environment. Most of the chitin extracted worldwide are from the shell of the crustaceans. An alternative source of chitin has been observed as the number of crustaceans has been dilapidating. Here, extraction of chitin from the edible and medicinal macrofungi, mushrooms, have been described. This is a novel approach to meet the ever increasing chitin demand worldwide. Among four mushroom species (Pleurotus ostreatus, Agaricus bisporus, Lentinula edodes and Ganoderma lucidum), the reishi mushroom or ling zhi (G. lucidum) had been found containing the highest amount of chitin on percentage basis of dried mushroom powder. Chitin content trend observed in this study was: G. lucidum (44%) > L. edodes (35%) > A. bisporus (18%) > P. ostreatus (10%). Thus, mushrooms could be considered as important source of chitin and its deacetylated product, chitosan. In this way, mushrooms could aid in biomedical and environmental intervention through suppliance of chitin and chitosan.

Neuroprotective, neurogenic, and anticholinergic evidence of Ganoderma lucidum cognitive effects: Crucial knowledge is still lacking.

Ganoderma lucidum is a mushroom that has been widely used for centuries in Asian countries for its antiaging properties. It is popularly known as"Ling Zhi,""Reishi," and "Youngzhi,"and because of its benefits, it is known as the "immortality mushroom." Pharmacological assays have revealed that G. lucidum ameliorates cognitive impairments through inhibition of β-amyloid and neurofibrillary tangleformation, antioxidant effect, reduction of inflammatory cytokine release and apoptosis, genic expression modulation, among other activities.Chemical investigations onG. lucidumhaverevealed the presence of metabolites such as triterpenes, which are the most explored in this field, as well as flavonoids, steroids, benzofurans, and alkaloids; in theliterature, thesehave also been reportedto havemnemonic activity. These properties of the mushroom make ita potential source of new drugs to prevent or reverse memory disorders,as actual medications are able toonly alleviate some symptomsbut are unable to stop the progress of cognitive impairments, with no impact on social, familiar, and personal relevance. In this review, we discuss the cognitive findings of G. lucidum reported in the literature, converging the proposed mechanisms through the several pathways that underlie memory and cognition processes. In addition, we highlight the gaps that deserveparticular attention to support future studies.

PLGA nanoparticles containing Lingzhi extracts rescue corneal epithelial cells from oxidative damage

Oxidative stress-related ocular surface epithelial damage can be initiated by ambient oxygen, UV radiation, and chemical burns. The oxidative damage to cornea can lead to inflammation and even vision loss. Lingzhi (Ganoderma lucidum) is a Chinese herbal drug and has been shown to prevent chronic diseases in clinical practices and has been proven to possess anti-oxidative and anti-inflammatory properties. In the study, we prepared poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as a sustained drug release system of Lingzhi (LZH) to improve bioavailability. The particle size of developed NPs containing LZH (LZH-NPs) was ~184 nm with narrow size distribution. The results of cellular uptake revealed that using NPs as a drug delivery system could significantly increases the intracellular retention time. The results of the cell viability and chemiluminescence assay revealed that 5 μg/ml of LZH-NPs might be the threshold concentration for cultivation of corneal epithelial cells. After treating LZH-NPs in oxidative damaged cells, the results showed that the inflammation-related gene expression and DNA fragmentation level were both significantly decreased. Post-treatment of LZH-NPs in damaged corneal epithelial cells could increase the cell survival rate. In the rabbit corneal alkali burn model, topical instillation of LZH-NPs could promote corneal wound healing and decrease the inflammation. These results suggest that LZH-NPs may have the potential to treat ocular surface diseases caused by oxidative stress.

Lingzhi and San-Miao-San with hyaluronic acid gel mitigate cartilage degeneration in anterior cruciate ligament transection induced osteoarthritis.

SummaryObjectiveTo investigate the mitigate efficacy of Chinese medicine Lingzhi (LZ) and San-Miao-San (SMS) combined with hyaluronic acid (HA)-gel in attenuating cartilage degeneration in traumatic osteoarthritis (OA).MethodsThe standardized surgery of anterior cruciate ligament transection (ACLT) was made from the medial compartment of right hind limbs of 8-week-old female SD rats and resulted in a traumatic OA. Rats (n ​= ​5/group) were treated once intra-articular injection of 50 ​μl HA-gel, 50 ​μl HA-gel+50 ​μg LZ-SMS, 50 ​μl of saline+50 ​μg LZ-SMS and null (ACLT group) respectively, except sham group. Limbs were harvested for μCT scan and histopathological staining 3-month post-treatment. Inflammatory cytokines from plasma and synovial fluid were detected using Immunology Multiplex Assay kit. The putative targets of active compounds in LZ-SMS and known therapeutic targets for OA were combined to construct protein–protein interaction network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was adopted to predict the potential targets and signaling pathway of LZ-SMS in OA through the tool of DAVID Bioinformatics.ResultsIn vivo, HA-gel ​+ ​LZ-SMS treatment resulted in a higher volume ratio of hyaline cartilage (HC)/calcified cartilage (CC) and HC/Sum (total volume of cartilage), compared to ACLT and HA-gel groups. In addition, histological results showed the elevated cartilage matrix, chondrogenic and osteoblastic signals in HA-gel ​+ ​LZ-SMS treatment. Treatment also significantly altered subchondral bone (SCB) structure including an increase in BV/TV, Tb.Th, BMD, Conn.Dn, Tb.N, and DA, as well as a significant decrease in Tb.Sp and Po(tot), which implied a protective effect on maintaining the stabilization of tibial SCB microstructure. Furthermore, there was also a down-regulated inflammatory cytokines and upregulated anti-inflammatory cytokine IL-10 in HA+LZ-SMS group. Finally, 64 shared targets from 37 active compounds in LZ-SMS related to the core genes for the development of OA. LZ-SMS has a putative role in regulating inflammatory circumstance through influencing the MAPK signaling pathway.ConclusionOur study elucidated a protective effect of HA-gel ​+ ​LZ-SMS in mitigating cartilage degradation and putative interaction with targets and signaling pathway for the development of traumatic OA.The translational potential of this articleOur results provide a biological rationale for the use of LZ-SMS as a potential candidate for OA treatment.

Ganoderma lucidum cultivation affect microbial community structure of soil, wood segments and tree roots

The popular medicinal mushroom Ganoderma lucidum (Fr.) Karst. [Ling Zhi] has been widely used for the general promotion of health and longevity in Asian countries. Continuous cultivation may affect soil microbe and soil properties. However, the effect of G. lucidum cultivation on related wood segments, soil and tree roots microbial communities and soil properties is remain unknown. In our study, the microbial communities of soils, wood segments, and tree roots before and after G. lucidum cultivation were investigated by Illumina Miseq sequencing of both ITS and 16S rDNA, and taxonomic composition of eukaryotic and prokaryotic microorganisms were observed. Indices of microbial richness, diversity and evenness significantly differed between before and after G. lucidum cultivation. Each of the investigated sampling type harbored a distinctive microbial community and differed remarkably before and after G. lucidum cultivation. Ascomycota and Basidiomycota (fungi), Proteobacteria and Actinobacteria (bacteria) showed significant differences after Ling Zhi cultivation. The soil property values also changed after cultivation. The redundancy analysis (RDA) showed that both the fungal and bacterial community structure significantly correlated with soil humus, pH, nitrogen, carbon and trace elements (Fe, Zn, Mn, Cu) contents. The results indicated that G. lucidum cultivation may have significant differed the associated microbial community structures and soil properties. The study will provide useful information for G. lucidum cultivation and under-forest economic development.

P6204Oral vaccination of Lactococcus lactis expressing Ling Zhi 8 protein prevents nonalcoholic fatty liver and early atherogenesis in cholesterol-fed rabbits

Abstract Background Atherosclerosis is an inflammatory disease characterized by lipid deposits in the subendothelial space leading to severe inflammation. Nonalcoholic fatty liver disease (NAFLD) shares several risk factors with atherosclerosis. Epidemiology studies have indicated that NAFLD may be an independent risk factor for atherosclerosis. Anti-inflammation therapy by inhibiting IL-1β led to a significantly lower cardiovascular event rate in recent clinical trial (CANTOS trial). Ling Zhi 8 (LZ8) is an immunomodulatory protein that possesses a broad range of pharmacological properties, including anti-inflammatory activities. Methods and findings In this study, we developed an oral vaccination of Lactococcus lactis expressing LZ8 protein in a nisin-controlled gene expression system and investigated its anti-inflammation properties. Experimental rabbits received commercial rabbit chow supplemented with 2% cholesterol for 5 weeks and recombinant LZ8 L. lactis vaccine once a day on weekdays. The expression of IL-1β in the aorta (Figure A) was significantly suppressed after LZ8 vaccination. Moreover, in hematoxylin and eosin staining of the aorta, the intima-medial thickness was decreased, and foam cells were significantly reduced in the sub-endothelial space (Figure C). LZ8 also inhibited the expression of IL-1β in the liver (Figure B), decreased fat droplet deposits and infiltration of inflammatory cells (Figure D), and improved liver function by decreasing liver enzymes. Figure 1 Conclusions Our results suggest that LZ8 could be used as a therapeutic tool to improve both atherosclerosis and NAFLD due to its anti-inflammatory effect. Acknowledgement/Funding TCVGH-1067317C, TCVGH-1063108C

Extraction and antioxidant activity of Ganoderma lucidum polysaccharides using electrolyzed oxidizing water

The fruiting body of Ganoderma lucidum (Ling Zhi) is a traditional medicinal mushroom for promoting health and life expectancy. G. lucidum polysaccharides (GLPs) has several biological activities, including antioxidant, antitumor, antimicrobial and increase the immunity. In order to extract GLPs, electrolyzed oxidizing water (EOW)was used to accelerate their dissolution from the cell walls. Compared to traditional extraction method, electrolyzed oxidizing water is an environment friendly and economically feasible method. The extraction conditions including time, temperature, pH and water-to-solid ratio were optimized by single factor experiments followed by an orthogonal experiment, for maximising total sugar content and antioxidant capacity. The optimal extraction time, temperature, EOW pH and water-to-solid ratio were 4 h, 100°C, pH 2.5 and 20 mL/g, respectively, which yielded 78.47 ± 2.95 mg/g of GLP. The extracted GLP exhibited strong antioxidant effects, including scavenging ofDPPH• and HO•, with IC50 values of 0.55 mg/mL and 0.76 mg/mL, respectively.

Optimal culture conditions for mycelial growth and fruiting body formation of Ling Zhimushroom Ganoderma lucidum strain GA3

The objective in this study is to investigate optimal culture conditions for mycelial growth and fruiting body formation of the Ling Zhi mushroom, Ganoderma lucidum strain GA3. The results of the study show that the optimal media and temperature for the mycelial growth are potato, glucose, and agar (PGA) supplemented with rice bran, and 25-300C, respectively. Strain GA3 is able to grow in a wide pH range, between 4 and 12.The most favourable substrate mixture for the formation and development of the fruiting body is 87% sawdust + 4% corn powder + 8% rice bran + 1% calcium carbonate (CaCO3).

Pathological Changes in APP/PS-1 Transgenic Mouse Models of Alzheimer's Disease Treated with Ganoderma Lucidum Preparation.

Objective To explore the efficacy of ganoderma lucidum preparation(Ling Zhi) in treating APP/PS-1 transgenic mouse models of Alzheimer's disease(AD).Methods APP/PS-1 transgenic mice of 4 months were randomly divided into model group,ganoderma lucidum treatment groups,including high [2250 mg/(kg·d)] and middle [750 mg/(kg·d)] dose groups,i.e.LZ-H and LZ-M groups,and the positive control group(treated with donepezil hydrochloride [2 mg/(kg·d)]).In addition,C57BL/6J wild mice were selected as normal group.The animals were administered for 4 months.Histopathological examinations including hematoxylin-eosin(HE) staining,immunohistochemistry,special staining,and electron microscopy were applied,and then the pathological morphology and structures in different groups were compared. Results The senile plaques and neurofibrillar tangles in the cerebrum and cerebellum were dissolved or disappeared in LZ-H and LZ-M groups.Decrease of amyloid angiopathy was found in LZ-H and LZ-M groups.The immature neurons appeared more in hippocampus and dentate nucleus of LZ-H and LZ-M groups than those in AD model and donepezil hydrochloride groups(hippcampus:F=1.738,P=0.016;dentate nucleus:F=1.924,P=0.026),and these immature neurons differentiated to be neurons.More Purkinje cells loss occurred in AD model mice than that in LZ-H and LZ-M groups(F=9.46,P=0.007;F=9.46,P=0.010).The LZ-H and LZ-M groups had more new neuron stem cells grown up in cerebellum.Electromicroscopic examination showed the hippocampal neurons in LZ-H and LZ-M group were integrated,the nuclear membrane was intact,and the mitochondria in the cytoplasm,endoplasmic reticulum,Golgi bodies,microtubules,and synapses were also complete.The microglial cell showed no abnormality.No toxicity appeared in the pathological specimens of mice treated with ganoderma lucidum preparation.Conclusion The ganoderma lucidum preparation can dissolve and decline or dismiss the senile plaques and neurofibrillar tangles in the brain of AD mice and also reduce the amyloid angiopathy.

Ganoderma lucidum - from tradition to modern medicine

Ganoderma lucidum has a long tradition of use in folk medicine of the Far East, which is documented in the oldest Chinese pharmacopoeia, written in the first century B.C, declaring it a superior medicine. The healing properties of G. lucidum reflected on folk names such as: Reishi, Mannentake, Ling Zhi etc., which mean ?herb of spiritual power?, ?mushroom of immortality? or ?10,000-year mushroom?, respectively. It has been known, for thousands of years, that this species extends life span, increases youthful vigour and vitality and it was used in the treatments of hepatitis, kidneys? disease, hypertension, arthritis, asthma, bronchitis, arteriosclerosis, ulcers and various types of cancer. However, Western civilisation did not discover its healing properties until the 20th century. Modern scientific researches and numerous clinical trails, conducted in recent decades, have confirmed the ancient knowledge of Eastern nations and given them a scientific basis. These studies have demonstrated many biological activities of G. lucidum extracts and compounds, including: immunomodulating, antioxidative, cytotoxic, hypoglycaemic, anti-inflammatory, antiallergic, antimicrobial, etc. It has been reported that its extracts play important role in detoxification of the body and protecton of the liver, as well as in reducing cardiovascular problems, stress and anxiety. However, its most important effect is undoubtedly immunostimulating one as it is the basis of many other positive effects. The Japanese government introduced G. lucidum on the official list of auxiliary agents for the treatments of various cancers, Alzheimer?s disease, diabetes and chronic bronchitis. Many chemical components have been isolated from G. lucidum, but polysaccharides and terpenoids are the main carriers of its bioactivities.

Platelet rich plasma from umbilical cord blood with Chinese medicine formula Ling Zhi and San-Miao-San in hyaluronic gel for articular cartilage retrieval in rat osteoarthritis model

Platelet rich plasma from umbilical cord blood with Chinese medicine formula Ling Zhi and San-Miao-San in hyaluronic gel for articular cartilage retrieval in rat osteoarthritis model

Effect of Oral Coadministration of Ascorbic Acid with Ling Zhi Preparation on Pharmacokinetics of Ganoderic Acid A in Healthy Male Subjects: A Randomized Crossover Study.

The objective of this randomized, open-label, single-dose, two-phase crossover study was to determine the effect of ascorbic acid on pharmacokinetics of ganoderic acid A, an important biologically active triterpenoid compound with anticancer activities, following oral administration of water extract of fruiting bodies of Ling Zhi in 12 healthy male subjects. Each subject was randomized to receive either one of the two regimens: (1) a single dose of 3,000 mg of the Ling Zhi preparation or (2) a single dose of 3,000 mg of the Ling Zhi preparation in combination with 2,500 mg of ascorbic acid. After a washout period of at least two weeks, subjects were switched to receive the alternate regimen. Blood samples were collected in each phase immediately before dosing and at specific time points for 8 hours after dosing. Plasma ganoderic acid A concentrations were quantified using liquid chromatography-mass spectrometry (LC-MS). The pharmacokinetic parameters analyzed were maximal plasma concentration (C max), time to reach peak concentration (T max), area under the plasma concentration-time curve (AUC), and half-life (t 1/2). An oral coadministration of ascorbic acid with Ling Zhi preparation did not significantly alter the pharmacokinetic parameters of ganoderic acid A in healthy male subjects.

Determination of Heavy Metals in Herbal Drinks Using ICP-MS

Seven groups of herbal drinks including gum karaya, Indian gooseberry, Noni Indian mulberry, cat's whiskers, prickly-leaved elephant's foot, black galingale, and Ling zhi mushroom contain a variety of bioactive compounds such as polysaccharides, terpenes, saponins, and flavonoids. The extracts of these plants have several biological activities such as analgesic, anti-inflammatory, antibacterial, antioxidant and chemoprotective activities that can promote health. The popular uses of herbal drinks in Thailand raise the question about safety of their products, especially for heavy metal contamination. The concentrations of heavy metals such as As, Pb, Cu, Zn, Fe, and Cd in twenty one samples from seven groups of herbal drinks were determined by ICP-MS. None of herbal drinks passed the permissible limits under the Notification of Ministry of Public Health (No. 214) B.E. 2543 (2000) Re: Beverages in Sealed Container by the virtue of the Food Act B.E. 2522 (1979), Thailand. Moreover, the concentrations of Pb and Zn in all analyzed samples were higher than the permissible limits. The concentrations of Fe in almost all analyzed samples as well as the concentrations of As, Cd, and Cu in some analyzed samples were higher than the permissible limits. The levels of heavy metals found in this study raise concerns, not only for the safety of Thai consumers, but also the quality of herbal drinks available in Thailand.

Ling Zhi Immuno-Modulation Protein, LZ-8 Induction of Interleukin-1 Expression in Human Monocytes via Pertussis Toxin-Sensitive G Protein-Mediated Protein Kinase Pathway

＂Ganoderma lucidum＂ (G. lucidum) (a medicinal fungus) has been known to enhance a variety of immune functions in humans and animals for centuries. An immunomodulatory protein, Ling Zhi-8 (LZ-8), is isolated from G. lucidum and exhibits potent mitogenic effects upon human immune cells. However, LZ-8-mediated signal transductions in the regulation of interleukin-1 (IL-1) gene expression within human monocytes has not been less well examined. In this study, we expressed the recombinant LZ-8 protein (rLZ) via Pichia pastoris protein expression system. We investigated the effect of rLZ on induction of IL-1 expression in human monocyte-like THP-1cells. We found that rLZ induced pro-interleukin 1 (proIL-1, a precursor of IL-1) and IL-1 protein expression in human monocytes THP-1 in time- and dose-dependent manners. We found that rLZ induced IL-1 gene expression via a pertussis toxin (PTX)-sensitive G protein-couple receptor (GPCR), phospholipase C (PLC) and various protein kinases-dependent pathways in THP-1 cells. In essence, we have established the nature of rLZ-mediated signaltransduction pathways, such as GPCR, PTK/ PI-3K/ PLC, PTK/ PLC/ PKCα/ JNK, and PTK/ PLC/ PKCδ/ θ/ ERK1/2, in the regulation of IL-1 gene expression in THP-1 cells. Our current results of the analysis of rLZ-mediated signal transduction in monocyte-like cells could provide a potential explanation and application of rLZ as a pharmacological immuno-modulating agent.

Fungal beta glucan protects radiation induced DNA damage in human lymphocytes.

Ganoderma lucidum (Ling Zhi), a basidiomycete white rot macrofungus has been used extensively for therapeutic use in China, Japan, Korea and other Asian countries for 2,000 years. The present study is an attempt to investigate its DNA protecting property in human lymphocytes. Beta glucan (BG) was isolated by standard procedure and the structure and composition were studied by infrared radiation (IR) and nuclear magnetic resonance (NMR) spectroscopy, gel filtration chromatography and paper chromatography. The radioprotective properties of BG isolated from the macro fungi Ganoderma lucidum was assessed by single cell gel electrophoresis (comet assay). Human lymphocytes were exposed to 0, 1, 2 and 4 Gy gamma radiation in the presence and absence of BG. The comet parameters were reduced by BG. The results indicate that the BG of G. lucidum possessed significant radioprotective activity with DNA repairing ability and antioxidant activity as the suggestive mechanism. The findings suggest the potential use of this mushroom for the prevention of radiation induced cellular damages.

The Sophora flavescens flavonoid compound trifolirhizin inhibits acetylcholine induced airway smooth muscle contraction.

Asthma is a serious health problem worldwide, particularly in industrialized countries. Despite a better understanding of the pathophysiology of asthma, there are still considerable gaps in knowledge as well as a need for classes of drugs. ASHMI™ (Anti-asthma Herbal Medicine Intervention) is an aqueous extract of Ganoderma lucidum (Fr.) P. Karst (Ling Zhi), Sophora flavescens Aiton (Ku Shen) and Glycyrrhiza uralensis Fisch. ex DC (Gan Cao). It prevents allergic asthma airway hyper-reactivity in mice and inhibits acetylcholine (ACh) induced airway smooth muscle (ASM) contraction in tracheal rings from allergic asthmatic mice. The purpose of this research was to identify individual herb(s) and their active compound(s) that inhibit ASM contraction. It was found that S. flavescens, but not G. lucidum or G. uralensis aqueous extracts, inhibited ASM contraction in tracheal rings from asthmatic mice. Bioassay-guided isolation and identification of flavonoid fractions/compound(s) via methylene chloride extraction, preparative HPLC fractionation, and LC-MS and NMR spectroscopic analyses showed that trifolirhizin is an active constituent that inhibits acetylcholine mediated ASM contraction or directly relaxes pre-contracted ASM independent of β2-adrenoceptors.

Antihepatoma and Liver Protective Potentials of Ganoderma Lucidum (靈芝 Ling Zhi) Fermented in a Medium Containing Black Soybean (黑豆 Hēi Dòu) and Astragalus Membranaceus (生黃耆 Shēng Huáng Qí)

The antihepatoma activity and liver protective function of the fermentation products (5 L fermenator) of Ganoderma lucidum (GL; 靈芝 Ling Zhi) cultivated in a medium containing black soybean (BS; 黑豆 Hēi Dòu) and Astragalus membranaceus (AM; 生黃耆 Shēng Huáng Qí) at different fermentation temperatures were investigated in this study. Hep 3B cells pretreated with lovastatin were used to study the antihepatoma activity, and possible active components were analyzed by reverse-phase high-performance liquid chromatography. Carbon tetrachloride (CCl4)-induced primary rat hepatocyte injury was further used to evaluate the liver protective activity of the fermentation products. While all the GL broth filtrates do not inhibit the growth of Hep 3B cells, the ethanolic extract from GL-2 mycelia (GL-2-mE), cultivated in the medium containing BS (50 g/L) and AM (20 g/L) at 24°C for 11 days showed the best antihepatoma activity (IC50 26.6 μg/mL) than the other ethanolic extracts from GL mycelia, GL fruiting body, BS, and AM did. The antihepatoma activities were correlated with some unknown active components in these samples. Furthermore, GL-2-mE (100 μg/mL) without harmful effect on the growth of normal primary rat hepatocytes significantly maintained cell viability, reduced lactate dehydrogenase leakage, lowered lipid peroxidation, and increased glutathione peroxidase and glutathione S-transferase activities in the CCl4-induced damaged primary rat hepatocytes.

High-Level Expression, Purification and Production of the Fungal Immunomodulatory Protein-Gts in Baculovirus-Infected Insect Larva

Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP(bbx) secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 10⁹ cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.