ABSTRACT
Topological cytonuclear discordance is commonly observed in plant phylogenetic and phylogeographic studies, yet few studies have attempted to detect two other forms of cytonuclear discordance (branch length and geographical) and to uncover the causes of the discordance. We used the whole nuclear and chloroplast genome data from 80 individual Asian butternuts to reveal the pattern and processes of cytonuclear discordance. Our findings indicate that the chloroplast genome had substantially deeper divergence (branch-length discordance) and a steeper cline in the contact zone (geographic discordance) compared with the nuclear genome. After various hypothesis have been tested, the results suggest that incomplete lineage sorting, positive selection and cytonuclear incompatibility are probably insufficient to explain this pattern. However, isolation-by-distance analysis and gene flow estimation point to a much higher level of gene flow by pollen compared with by seeds, which may have slowed down lineage divergence and mediated wider contact for nuclear genome compared with the chloroplast genome. Altogether, this study highlights a critical role of sex-biased dispersal in causing discordance between the nuclear and plastid genome of Asian butternuts. Given its ubiquity among plants, asymmetric gene flow should be given a high priority in future studies of cytonuclear discordance.
Subject(s)
Gene Flow , Genome, Chloroplast , Cell Nucleus/genetics , Phylogeny , Pollen/genetics , Seeds/geneticsABSTRACT
Diterpenoids jolkinolide A and B, were first isolated from Euphorbia fischeriana. In our previous research, 19-(Benzyloxy)-19-oxojolkinolide B (19-BJB), a derivative of jolkinolides, was synthesized as a novel ent -abietane diterpene diepoxide. In this study, 19-BJB showed strong in vitro activity against bladder cancer cell lines. DNA damage which was observed through the interaction of 19-BJB with nucleotide chains and affected DNA repair resulted in the activation of checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) in bladder cancer cell lines. In vivo testing in nude mice also proved that 19-BJB revealed a potential inhibitory effect on tumor growth. Additionally, the 3D-QSAR models of jolkinolides were established. Briefly, we proved that 19-BJB could potentially be used as a drug to inhibit the growth of bladder tumor.
Subject(s)
Abietanes/pharmacology , DNA Damage/drug effects , Diterpenes/pharmacology , Drugs, Chinese Herbal/pharmacology , Urinary Bladder Neoplasms/drug therapy , Abietanes/therapeutic use , Animals , Antineoplastic Agents, Phytogenic , Cell Line, Tumor , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 2/metabolism , DNA Repair , Diterpenes/therapeutic use , Drugs, Chinese Herbal/therapeutic use , Euphorbia/chemistry , Humans , Male , Mice , Molecular Docking Simulation , Urinary Bladder Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
In the clinical practice of traditional Chinese medicine, Pinelliae Rhizoma is a commonly used traditional Chinese medicinal material, with a very important medicinal value, but its raw products have certain toxicity. According to the basic theory of traditional Chinese medicine, after Pinelliae Rhizoma is processed, its toxic and side effects can be reduced, there by ensuring its medication safety in clinic. Based on the processing characteristics of Pinelliae Rhizoma replication method, this article discusses the changes before and after processing of Pinelliae Rhizoma with multiple materials and multiple processes. This method does not have a unified process. One or more materials are added, and various processes, such as dipping, soaking, bleaching, or steaming, cooking or combined methods are adopted. Then, Pinelliae Rhizoma is repeatedly processed to meet the specified quality requirements. By different processing methods, the efficacy changes accordingly, and Pinelliae Rhizoma can be used for the treatment of different diseases. The article starts in the two directions of multi-materials and multi-processes, and summarizes the effects of multi-materials or multi-processes on the chemical composition and efficacy, as well as the processing mechanism of reducing toxicity and increasing efficiency, in the expectation of reducing the toxicity and enhancing the efficacy of Pinelliae Rhizoma. The medicinal scope provides a reference and theoretical basis for further studies on the processing mechanism. An in-depth study is conducted to make Pinelliae Rhizoma more safe and effective in clinic application, and ensure its clinical efficacy. It will provide reference for future studies on quality control, active ingredients and new drug development of the processing of pinellia slices. It has laid a scientific foundation for exploring the best processing techno-logy, and provided a theoretical basis for solving the production problems of processed Pinelliae Rhizoma products, so as to improve the production efficiency and the quality of medicines.
Subject(s)
Drugs, Chinese Herbal/chemistry , Pinellia/chemistry , Rhizome/chemistry , Technology, Pharmaceutical , Medicine, Chinese TraditionalABSTRACT
The present study was designed to synthesize 2-Cyano-3, 12-dioxooleana-1, 9(11)-en-28-oate-13ß, 28-olide (1), a lactone derivative of oleanolic acid (OA) and evaluate its anti-inflammatory activity. Compound 1 significantly diminished nitric oxide (NO) production and down-regulated the mRNA expression of iNOS, COX-2, IL-6, IL-1ß, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Further in vivo studies in murine model of LPS-induced acute lung injury (ALI) showed that 1 possessed more potent protective effects than the well-known anti-inflammatory drug dexamethasone by inhibiting myeloperoxidase (MPO) activity, reducing total cells and neutrophils, and suppressing inflammatory cytokines expression, and thus ameliorating the histopathological conditions of the injured lung tissue. In conclusion, compound 1 could be developed as a promising anti-inflammatory agent for intervention of LPS-induced ALI.
Subject(s)
Acute Lung Injury/drug therapy , Anti-Inflammatory Agents/administration & dosage , Oleanolic Acid/administration & dosage , Acute Lung Injury/genetics , Acute Lung Injury/immunology , Animals , Anti-Inflammatory Agents/chemical synthesis , Bronchoalveolar Lavage Fluid/immunology , Cyclooxygenase 2/genetics , Cyclooxygenase 2/immunology , Female , Humans , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Lipopolysaccharides/adverse effects , Lung/drug effects , Lung/immunology , Macrophages/drug effects , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Neutrophils/drug effects , Neutrophils/immunology , Oleanolic Acid/analogs & derivatives , Oleanolic Acid/chemical synthesis , Peroxidase/genetics , Peroxidase/immunology , RAW 264.7 Cells , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
BACKGROUND: Acupuncture is a potential therapy for Alzheimer's disease (AD), but its clinical effects and underlying mechanisms are not fully understood. Emerging evidence suggests autophagy is involved in ß-amyloid (Aß) clearance. We hypothesised that electroacupuncture (EA) treatment of AD involves the autophagy pathway in rats. METHODS: We injected 2µl Aß1-40 bilaterally into the hippocampi of 42 rats to establish AD. Rats remained untreated (AD group, n=14) or received 24 EA treatments at GV20+BL23 over 28â days from day 7 post-injection with/without co-treatment with 3-methyladenine (3-MA), an autophagy inhibitor (AD+EA+3-MA and AD+EA groups, respectively, n=14 each). Cognitive function was evaluated by Morris water maze (MWM) testing. Hippocampi were examined by transmission electron microscopy (TEM) and stained with haematoxylin and eosin/transferase dUTP nick end labelling (TUNEL) to assess neuronal morphology/apoptosis, respectively. Protein expression of Beclin-1, LC3 and Aß1-40 was examined. RESULTS: In the MWM test, the AD+EA group showed an improvement in parameters consistent with improved learning/memory compared to untreated AD rats, and 3-MA attenuated these effects. EA mitigated cellular apoptosis resulting from Aß infusion in the CA1 region and enhanced LC3II/LC3I ratios and Beclin-1 expression. Numerous autophagosome precursors and enlarged autophagosomes were observed by TEM in the hippocampi of EA-treated rats. Reduced Aß levels, and co-localisation of Aß and LC3II, were observed following EA treatment by immunofluorescence staining. EA+3-MA treated rats had much higher TUNEL-positive neurons, lower LC3II/LC3I ratios and Beclin-1 expression, and elevated Aß levels compared with EA alone. CONCLUSIONS: EA reduces neuronal apoptosis, enhances degradation of Aß, and improves learning/memory in AD rats by upregulating the autophagy pathway.
Subject(s)
Alzheimer Disease/therapy , Autophagy/physiology , Electroacupuncture/methods , Memory Disorders/therapy , Neurons , Adenine/administration & dosage , Adenine/analogs & derivatives , Alzheimer Disease/complications , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Animals , Apoptosis/physiology , Beclin-1/metabolism , Disease Models, Animal , Hippocampus/physiopathology , Memory/physiology , Memory Disorders/etiology , Microtubule-Associated Proteins/metabolism , Peptide Fragments/metabolism , RatsABSTRACT
The aim of the present study was to evaluate the effects of Penthorum chinense Pursh (PCP), a health food and folk medicine, against acute alcohol-induced liver injury and further to elucidate its probable mechanisms. Male C57BL/6 mice were treated with an aqueous extract of PCP (5.2 and 10.3 g per kg BW) once daily for 7 consecutive days prior to ethanol gavage (4.7 g kg(-1)) every 12 h for a total of three doses. Pretreatment with PCP significantly decreased the elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic triglyceride after the last ethanol administration. PCP suppressed the elevation of the malondialdehyde (MDA) level, restored the glutathione (GSH) level and enhanced the activities of superoxide dismutase (SOD) and catalase (CAT) in both the serum and liver, which were associated with the inhibition of hepatic cytochrome P450 2E1 (CYP2E1). In addition, alcohol exposure markedly induced the lipolysis of white adipose tissue (WAT) through up-regulating protein expressions of adipose triglyceride lipase (ATGL) and phosphorylation of hormone-sensitive lipase (p-HSL), and enhancing the fatty acid uptake capacity in the liver by elevated hepatic CD36 expression, which were attenuated by PCP treatment. These data demonstrated that pre-treatment with PCP protected against acute ethanol-induced liver injury, possibly by reducing CYP2E1-dependent oxidative stress and ameliorating dysfunctional WAT derived-fatty acid influx to the liver. Our findings suggest that PCP might be a promising agent for the prevention of acute alcohol-induced liver injury.
Subject(s)
Alcohols/adverse effects , Chemical and Drug Induced Liver Injury/drug therapy , Drugs, Chinese Herbal/administration & dosage , Fatty Liver/drug therapy , Magnoliopsida/chemistry , Oxidative Stress/drug effects , Protective Agents/administration & dosage , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/metabolism , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/metabolism , Cytochrome P-450 CYP2E1/metabolism , Fatty Liver/enzymology , Fatty Liver/metabolism , Glutathione/metabolism , Humans , Liver/drug effects , Liver/enzymology , Liver/metabolism , Male , Malondialdehyde/metabolism , Mice , Mice, Inbred C57BL , Superoxide Dismutase/metabolismABSTRACT
Local adaptation has been suggested to play an important role in range expansion, particularly among invasive species. However, the extent to which local adaptation affects the success of an invasive species and the factors that contribute to local adaptation are still unclear. This study aimed to investigate a case of population divergence that may have contributed to the local adaptation of invasive populations of Ambrosia artemisiifolia in China. Common garden experiments in seven populations indicated clinal variations along latitudinal gradients, with plants from higher latitudes exhibiting earlier flowering and smaller sizes at flowering. In reciprocal transplant experiments, plants of a northern Beijing origin produced more seeds at their home site than plants of a southern Wuhan origin, and the Wuhan-origin plants had grown taller at flowering than the Beijing-origin plants in Wuhan, which is believed to facilitate pollen dispersal. These results suggest that plants of Beijing origin may be locally adapted through female fitness and plants from Wuhan possibly locally adapted through male fitness. Selection and path analysis suggested that the phenological and growth traits of both populations have been influenced by natural selection and that flowering time has played an important role through its direct and indirect effects on the relative fitness of each individual. This study evidences the life history trait differentiation and local adaptation during range expansion of invasive A. artemisiifolia in China.
Subject(s)
Adaptation, Physiological/genetics , Ambrosia/growth & development , Environment , Flowers/growth & development , Introduced Species , Phenotype , Selection, Genetic , Animals , China , Pollen , Reproduction/genetics , Seeds/growth & developmentABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Penthorum chinense Pursh (Penthoraceae) has been used as a Miao ethnomedicine for the treatment of jaundice, cholecystitis, edema, infectious hepatitis and anti-drunk hangover in China. The aim of present study is to investigate the possible protective effects of Penthorum chinense against chronic ethanol-induced liver injury. MATERIAL AND METHODS: Mice were fed a Lieber-DeCarli liquid diet containing alcohol or isocaloric maltose dextrin as control diet with or without aqueous extract of Penthorum chinense (PCP, 5.15 and 10.30 g/kg/BW) for 4 weeks. Silymarin (86 mg/kg) was used as positive control to compare the efficacy of PCP against chronic ethanol-induced hepatotoxicity. RESULTS: Treatment with PCP (10.30 g/kg) significantly reduced the increases in serum ALT and AST levels, hepatic lipid accumulation and inflammatory cytokines (i.e. TNF-α, IL-6), which were induced by chronic ethanol exposure. PCP was also found to attenuate reactive oxygen species (ROS) generation and malondialdehyde (MDA) level, restore the glutathione (GSH) depletion, and increase the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities. In addition, PCP supplementation (10.30 g/kg) inhibited the induction of hepatic cytochrome P450 2E1 (CYP2E1), a major contributor to ethanol-mediated oxidative stress, and up-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream anti-oxidant protein heme oxygenase-1 (HO-1) in ethanol-treated mice. CONCLUSIONS: These results indicate that the co-treatment with aqueous extract of Penthorum chinense (10.30 g/kg) protects against chronic ethanol-induced liver injury, possibly through suppressing CYP2E1-mediated oxidative stress and enhancing the oxidant defense systems via the activation of Nrf2/HO-1 pathway.
Subject(s)
Chemical and Drug Induced Liver Injury/drug therapy , Magnoliopsida , Phytotherapy , Protective Agents/therapeutic use , Alanine Transaminase/blood , Animals , Aspartate Aminotransferases/blood , Chemical and Drug Induced Liver Injury/blood , Chemical and Drug Induced Liver Injury/metabolism , Cytochrome P-450 CYP2E1/genetics , Cytochrome P-450 CYP2E1/metabolism , Ethanol , Female , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Liver/drug effects , Liver/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Inbred C57BL , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , Plant Components, Aerial , Protective Agents/pharmacology , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Triglycerides/metabolismABSTRACT
Plant phylogeographic studies in East Asia have provided support for the biogeographic hypothesis that the complex landforms and climate of this region have provided substantial opportunities for allopatric speciation. However, most of these studies have been based on maternally inherited chloroplast DNA (cpDNA) markers and were therefore unable to reveal the role of pollen-mediated gene flow in preventing population divergence. Here, we investigate the phylogeography of the Chinese walnut Juglans cathayensis, a temperate deciduous tree widely distributed across disjunct montane sites in subtropical China. We genotyped 19 populations using seven cpDNA fragments and ten nuclear microsatellite loci and modeled the ecological niche of J. cathayensis. CpDNA analysis identified a total of nine haplotypes, and each of the 19 sampled populations was fixed for a single haplotype, displaying a prominent phylogeographic structure. The results of ecological niche modeling indicated that J. cathayensis populations survived the last glaciation in situ, although they were probably more fragmented than today. In contrast, we detected a much weaker, but nonetheless clear, genetic structure based on nuclear microsatellite data. Our study demonstrates how extensive pollen flow can erase the genetic imprint of long-term refugial isolation in maternal lineages, effectively preventing population differentiation in temperate, particularly wind-pollinated, forest trees in subtropical China.
Subject(s)
Gene Flow , Juglans/genetics , Phylogeny , Pollen/genetics , Bayes Theorem , China , DNA, Chloroplast/genetics , DNA, Plant/genetics , Genetics, Population , Genotype , Haplotypes , Microsatellite Repeats , Models, Genetic , Phylogeography , Sequence Analysis, DNA , Trees/geneticsABSTRACT
A new flavanone, 5-methoxy-pinocembrin-7-O-ß-D-glucoside (1), together with six known compounds, quercetin-3-O-ß-D-xyloside (2), quercetin-3'-O-α-L-rhamnoside (3), quercetin (4), pinocembrin-7-O-ß-D-glucoside (5), pinocembrin (6), gallic acid (7), were isolated and identified from the aerial parts of Penthorum chinense. Compounds 2 and 3 were isolated from this plant for the first time.
Subject(s)
Drugs, Chinese Herbal/isolation & purification , Flavanones/isolation & purification , Glucosides/isolation & purification , Saxifragaceae/chemistry , Drugs, Chinese Herbal/chemistry , Flavanones/chemistry , Gallic Acid/chemistry , Gallic Acid/isolation & purification , Glucosides/chemistry , Molecular Structure , StereoisomerismABSTRACT
AIM: To discover more active and water-soluble derivatives of tetracyclic diterpenoids containing an exo-methylene cyclopentanone or an α-methylenelactone moiety. METHODS: All of the key intermediates were synthesized from stevioside, and the target compounds were obtained through glycosylation of the 4-carboxyl group. The cytotoxicity of the target compounds against six human cancer cell lines, HepG2, Bel-7402, A549, U251, MCF-7 and MDA-MB-231, were evaluated by the MTT assay. RESULTS: Compound 1b was more effective than the positive control adriamycin against the HepG2, Bel-7402, A549, MCF-7, and MDA-MB-231 cell lines with IC50 values of 0.12, 0.91, 0.35, 0.08, and 0.07 µmol·L(-1), respectively. Moreover, compound 3c exhibited the most potent and selective cytotoxic activity against the HepG2 cell line (IC50, 0.01 µmol·L(-1)). CONCLUSION: Compounds 1b and 3c could be considered as potential anticancer candidates for further study.
Subject(s)
Antineoplastic Agents/toxicity , Diterpenes, Kaurane/toxicity , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Diterpenes, Kaurane/chemistry , Drug Evaluation, Preclinical , Glycosylation , Humans , Molecular StructureABSTRACT
In hermaphroditic plants, female reproductive success often varies among different positions within an inflorescence. However, few studies have evaluated the relative importance of underlying causes such as pollen limitation, resource limitation or architectural effect, and few have compared male allocation. During a 2-year investigation, we found that female reproductive success of an acropetally flowering species, Corydalis remota Fisch. ex Maxim. var. lineariloba Maxim. was significantly lower in the upper late developing flowers when compared with the lower early flowers. Supplementation with outcross pollen did not improve female reproductive success of the upper flowers, while removal of the lower developing fruits significantly increased female reproductive success of the upper flowers in both years, evidencing resource limitation of the upper flowers. Female production in upper flowers was greatly improved by simultaneous pollen supplementation of the upper flowers and removal of the lower fruits, suggesting that, when resources are abundant, pollen may limit the female reproductive success of the upper flowers. The less seed mass in the upper flowers didn't increase in all treatments due to architecture. In the upper flowers, ovule production was significantly lower and the pollen : ovule ratio was significantly higher. These results suggest that male-biased sex allocation in the upper flowers may lead to increased male reproductive success, whereas the lower flowers have higher female reproductive success.
Subject(s)
Corydalis/physiology , Flowers/physiology , Sex Characteristics , Pollen/physiology , Reproduction , Seeds/physiologyABSTRACT
Apoptosis is an essential factor in keeping homeostasis of the organism. Apoptosis is regulated by a series of cytokines. Bcl-2 family proteins are key regulators of apoptosis. The Bcl-2 family includes both anti- and pro-apoptotic proteins with opposing biological functions. Their interaction regulates the transmission of the apoptosis signal. High expression of anti-apoptotic members such as Bcl-2 and Bcl-xL are commonly found in human cancers. In recent years, following the disclosing of the crystal structures of Bcl-2 family proteins, researchers have paid attention to the development of the small molecule inhibitors of Bcl-2 family proteins. This article reviews the progress in this field from the view of drug design.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , bcl-X Protein/antagonists & inhibitors , Antimycin A/chemistry , Antimycin A/pharmacology , Benzopyrans/chemistry , Benzopyrans/pharmacology , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Cell Line, Tumor , Drug Design , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Gossypol/chemistry , Gossypol/pharmacology , Humans , Nitriles/chemistry , Nitriles/pharmacology , Nitrophenols/chemistry , Nitrophenols/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Proto-Oncogene Proteins c-bcl-2/pharmacology , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology , Thiazoles/chemistry , Thiazoles/pharmacology , Thiazolidinediones , bcl-X Protein/pharmacologyABSTRACT
BACKGROUND AND AIMS: Heterodichogamy differs from normal dichogamy, in that it involves two mating types (protogyny and protandry) that occur at a 1 : 1 ratio in a population. Flowering phases of the two mating types are synchronized and reciprocal, which was considered to ensure between-type outcrossing. This study aims to quantify the flowering pattern and pollination efficacy in Juglans mandshurica, a wind-pollinated heterodichogamous tree. METHODS: The pattern of flowering phenology was monitored within individual trees and pollen traps were used to measure air-borne pollen loads during the spring in 2003 and 2004. Pollen longevity was determined by staining technique. Also a pollen supplementation experiment was performed in 2004 to assess pollen limitation of fruit production. KEY RESULTS: There was no overlap between sexual functions within individual trees. Flowering periods of the two mating types were reciprocal and synchronous in both 2003 and 2004. Air-borne pollen loads were large, and protogynous and protandrous individuals each produced a high pollination peak, consistent with the two blooming periods. Maximum pollen longevity was about 4 h for protandrous individuals, and 3 h for protogynous individuals. Pollen supplementation did not increase fruit production in either protogynous or protandrous individuals. CONCLUSIONS: Heterodichogamous flowering in Juglans mandshurica effectively avoids selfing, promotes between-type outcrossing, and leads to efficient pollination in a natural population.
Subject(s)
Flowers/physiology , Juglans/physiology , Breeding , Flowers/anatomy & histology , Flowers/growth & development , Juglans/anatomy & histology , Juglans/growth & development , Periodicity , Pollen/physiology , Reproduction/physiologyABSTRACT
Platycodon grandiflorum cell suspension cultures were employed to biotransform the taxane diterpenoids 2alpha,5alpha,10beta,14beta-tetraacetoxy-4(20),11-taxadiene (1) and 9alpha-hydroxy-2alpha,5alpha,10beta,14beta-tetraacetoxy-4(20),11-taxadiene (2). One product, 10beta-hydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (3) was obtained from 1 and two products, 9alpha,10beta-dihydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (4) and 10beta-hydroxy-2alpha,5alpha,9alpha,14beta-tetraacetoxy-4(20),11-taxadiene (5) were obtained from 2 incubated with Platycodon cultured cells respectively, among which 5 is characterized as a new taxoid compound. The effects of the addition stage for 1 and 2 on the biotransformation were investigated and the results revealed that: (1) the optimal addition stage for 1 was in the early logarithmic phase (6th day) of the cell growth period, in which 78% of 1 was converted and the yield for 3 reached 75%; (2) the optimal addition stage for 2 was on the mid-logarithmic phase (12th day) of the cell growth period, in which 25.3% of 2 was converted and the yields for 4 and 5 reached 18.9 and 14.5%, respectively.