Abundant Natural Compounds Research Articles

Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists

Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

In vitro Assessment of Camphor Hydrazone Derivatives as an Agent Against Leishmania amazonensis

Due to serious problems with the treatment of leishmaniasis all around the world, here is an urgent need in the search for new drugs that are more effective and safer for the treatment of the various forms of leishmaniasis. Actual therapy is limited and lacks sufficient efficacy due to incomplete elimination of the parasites form of patients. In this sense, we decided to evaluate, by first-time, a series of seventeen camphor hydrazone derivatives (2a-2p) against Leishmania amazonensis. The compounds previously synthesized from camphor, an abundant natural compound, were evaluated in vitro against the extra and intracellular forms of Leishmania amazonensis, and murine macrophages. The majority of compounds, fourteen, displayed activity against the intracellular form of the parasite (amastigote) with IC50 values ranging from 21.78 to 58.23µM, being six compounds active for both forms of the parasite. The compound 2i exhibited higher activity against the amastigote form with the value of IC50 (21.78µM) close to standard utilized miltefosine (12.74µM) and selectivity index of at least 6.9. Six compounds displayed activity against promastigote form of Leishmania amazonensis 2g, 2j-2n (41.17-69.59µM), with the compound 2m being the more active with IC50 = 41.17µM, 1.9 times less active than the reference drug (IC50 = 21.39µM). The compound 2m was the more selective to this form, with a selectivity index of at least 3.6. All the compounds were non-cytotoxic to macrophages. Most compounds showed activity against amastigote form of Leishmania amazonensis, being that they were not cytotoxic to macrophage at the maximum tested concentration, showing the selective property of these compounds. Since amastigotes are the parasite stages that cause the disease in humans, these results highlight the antileishmanial effect of the compounds. This study indicates the possible development of candidates to leishmanicidal drugs from an abundant natural compound of easy access.

The plant product quinic acid activates Ca2+‐dependent mitochondrial function and promotes insulin secretion from pancreatic beta cells

Quinic acid (QA) is an abundant natural compound from plant sources which may improve metabolic health. However, little attention has been paid to its effects on pancreatic beta-cell functions, which contribute to the control of metabolic health by lowering blood glucose. Strategies targeting beta-cell signal transduction are a new approach for diabetes treatment. This study investigated the efficacy of QA to stimulate beta-cell function by targeting the basic molecular machinery of metabolism-secretion coupling. We measured bioenergetic parameters and insulin exocytosis in a model of insulin-secreting beta-cells (INS-1E), together with Ca2+ homeostasis, using genetically encoded sensors, targeted to different subcellular compartments. Islets from mice chronically infused with QA were also assessed. QA triggered transient cytosolic Ca2+ increases in insulin-secreting cells by mobilizing Ca2+ from intracellular stores, such as endoplasmic reticulum. Following glucose stimulation, QA increased glucose-induced mitochondrial Ca2+ transients. We also observed a QA-induced rise of the NAD(P)H/NAD(P)+ ratio, augmented ATP synthase-dependent respiration, and enhanced glucose-stimulated insulin secretion. QA promoted beta-cell function in vivo as islets from mice infused with QA displayed improved glucose-induced insulin secretion. A diet containing QA improved glucose tolerance in mice. QA modulated intracellular Ca2+ homeostasis, enhancing glucose-stimulated insulin secretion in both INS-1E cells and mouse islets. By increasing mitochondrial Ca2+ , QA activated the coordinated stimulation of oxidative metabolism, mitochondrial ATP synthase-dependent respiration, and therefore insulin secretion. Bioactive agents raising mitochondrial Ca2+ in pancreatic beta-cells could be used to treat diabetes.

Microbial Functional Responses to Cholesterol Catabolism in Denitrifying Sludge.

The 2,3-seco pathway, the pathway for anaerobic cholesterol degradation, has been established in the denitrifying betaproteobacterium Sterolibacterium denitrificans. However, knowledge of how microorganisms respond to cholesterol at the community level is elusive. Here, we applied mesocosm incubation and 16S rRNA sequencing to reveal that, in denitrifying sludge communities, three betaproteobacterial operational taxonomic units (OTUs) with low (94% to 95%) 16S rRNA sequence similarity to Stl. denitrificans are cholesterol degraders and members of the rare biosphere. Metatranscriptomic and metabolite analyses show that these degraders adopt the 2,3-seco pathway to sequentially catalyze the side chain and sterane of cholesterol and that two molybdoenzymes-steroid C25 dehydrogenase and 1-testosterone dehydrogenase/hydratase-are crucial for these bioprocesses, respectively. The metatranscriptome further suggests that these betaproteobacterial degraders display chemotaxis and motility toward cholesterol and that FadL-like transporters may be the key components for substrate uptake. Also, these betaproteobacteria are capable of transporting micronutrients and synthesizing cofactors essential for cellular metabolism and cholesterol degradation; however, the required cobalamin is possibly provided by cobalamin-de novo-synthesizing gamma-, delta-, and betaproteobacteria via the salvage pathway. Overall, our results indicate that the ability to degrade cholesterol in sludge communities is reserved for certain rare biosphere members and that C25 dehydrogenase can serve as a biomarker for sterol degradation in anoxic environments. IMPORTANCE Steroids are ubiquitous and abundant natural compounds that display recalcitrance. Biodegradation via sludge communities in wastewater treatment plants is the primary removal process for steroids. To date, compared to studies for aerobic steroid degradation, the knowledge of anaerobic degradation of steroids has been based on only a few model organisms. Due to the increase of anthropogenic impacts, steroid inputs may affect microbial diversity and functioning in ecosystems. Here, we first investigated microbial functional responses to cholesterol, the most abundant steroid in sludge, at the community level. Our metagenomic and metatranscriptomic analyses revealed that the capacities for cholesterol approach, uptake, and degradation are unique traits of certain low-abundance betaproteobacteria, indicating the importance of the rare biosphere in bioremediation. Apparent expression of genes involved in cofactor de novo synthesis and salvage pathways suggests that these micronutrients play important roles for cholesterol degradation in sludge communities.

Natural constituents from Cortex Mori Radicis as new pancreatic lipase inhibitors

Pancreatic lipase (PL), a key enzyme responsible for the hydrolysis of triacylglycerides in the gastrointestinal tract, has been identified as the therapeutic target for the regulation of lipid absorption. In the present study, six major constituents from a famous Chinese herbal medicine Cortex Mori Radicis (also named sangbaipi in Chinese), have been collected and their inhibitory effects on PL have been carefully investigated and well characterized by a fluorescence-based assay. The results clearly demonstrated that all tested bioactive constituents from Cortex Mori Radicis including sanggenone C (SC), sanggenone D (SD), kuwanon C (KC), kuwanon G (KG), morin and morusin displayed strong to moderate inhibitory effects towards PL with the IC50 values ranging from 0.77 μM to 20.56 μM. Further investigations on inhibition kinetics demonstrated that SC, SD, KC and KG functioned as potent and mixed inhibitors against PL-mediated 4-MU oleate hydrolysis, with the Ki values less than 5.0 μM. Furthermore, molecular docking simulations demonstrated that SD (the most potent PL inhibitor from Cortex Mori Radicis) could create strong interaction with Ser152 (the key amino acid in the catalytic triad) of PL via hydrogen bonding. All these findings provided a new powerful evidence for explaining the hypolipidemic effect of Cortex Mori Radicis, also suggested that some abundant natural compounds in this herbal medicine could be served as lead compounds for the development of new PL inhibitors.

A single nucleotide mutation of IspF gene involved in the MEP pathway for isoprenoid biosynthesis causes yellow-green leaf phenotype in rice.

We identified IspF gene through yellow-green leaf mutant 505ys in rice. OsIspF was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. On expression levels of genes in this mutant, OsIspF itself and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase were all up-regulated, however, among eight genes associated with photosynthesis, only psaA, psaN and psbA genes for three reaction center subunits of photosystem obviously changed. Isoprenoids are the most abundant natural compounds in all organisms, which originate from the basic five-carbon units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, IPP and DMAPP are synthesized through two independent pathways, the mevalonic acid pathway in cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. The MEP pathway comprises seven enzymatic steps, in which IspF is the fifth enzyme. So far, no IspF gene has been identified in monocotyledonous plants. In this study, we isolated a leaf-color mutant, 505ys, in rice (Oryza sativa). The mutant displayed yellow-green leaf phenotype, reduced level of photosynthetic pigments, and arrested development of chloroplasts. By map-based cloning of this mutant, we identified OsIspF gene (LOC_Os02g45660) showing significant similarity to IspF gene of Arabidopsis, in which a missense mutation occurred in the mutant, resulting in an amino acid change in the encoded protein. OsIspF gene was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. Further, the mutant phenotype of 505ys was complemented by transformation with the wild-type OsIspF gene. Therefore, we successfully identified an IspF gene in monocotyledonous plants. In addition, real-time quantitative RT-PCR implied that a positive regulation could exist between the OsIspF gene and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase. At the same time, it also implied that the individual genes involved in the MEP pathway might differentially regulated expression levels of the genes associated with photosynthesis.

Profiling Volatile Natural Compounds from Flowers, Leaves and Stems of Marrubium anisodon Using Headspace Solid Phase Microextraction in Combination with Gas Chromatographic Mass Spectrometric Determination

The present study describes the potential capabilities of an efficient extraction technique, namely headspace solid-phase microextraction (HS-SPME) to isolate the volatile fractions from the flowers, leaves and stems of Marrubium anisodon in combination with the analysis by GC and GC-MS. By means of the HS-SPME method, 24, 16 and 24 components were characterized in the corresponding profiles, representing 89.6%, 97.4% and 93.6% of the total compositions, respectively. Limonene was the most abundant natural compound in all the volatile profiles. It was also observed that in all of the volatiles, monoterpene hydrocarbons constituted the main components while the next most common group was due to sesquiterpene hydrocarbons for volatile parts of flowers and leaves. However, in that of stems, oxygenated monoterpenes had the second rank from frequency point of view.

Identification and characterization of naturally occurring inhibitors against human carboxylesterase 2 in White Mulberry Root-bark

White Mulberry Root-bark (WMR) is an edible Chinese herbal used for the treatment of inflammation, nephritis and asthma. This study aimed to investigate the inhibitory effects of ethanol extract from WMR against human carboxylesterase 2 (hCE2), as well as to identity and character natural hCE2 inhibitors in this herbal. Our results demonstrated that the ethanol extract of WMR displayed potent inhibitory effects against hCE2, while three major bioactive constitutes in WMR were identified on the basis of LC fingerprinting combined with activity-based screening of LC fractions. Three bioactive compounds including SD, KG and SC were efficiently identified by comparison of LC retention times, UV and MS spectral data, with the help of authentic standards. The inhibition potentials and inhibition types of these natural compounds against hCE2 were further investigated in human liver microsomes. The results demonstrated that these bioactive compounds are potent non-competitive inhibitors against hCE2, with the Ki values ranging from 0.76μM to 1.09μM. All these findings suggested that three abundant natural compounds in WMR displayed potent inhibitory effects against hCE2, which could be used as lead compounds to develop more potent hCE2 inhibitors for the alleviation of hCE2-mediated severe delayed-onset diarrhea.

Betulin induces reactive oxygen species-dependent apoptosis in human gastric cancer SGC7901 cells.

Betulin, an abundant natural compound, significantly inhibited the cell viability of advanced human gastric cancer SGC7901 cells. Mechanism study demonstrated that betulin induced apoptosis through mitochondrial Bax and Bak accumulation-mediated intrinsic apoptosis pathway. Downregulation of the anti-apoptosis proteins Bcl-2 and XIAP was involved during betulin-induced cell apoptosis. Reactive oxygen species (ROS) was generated in cells after betulin treatment in a time- and dose-dependent manner. Addition of antioxidant N-acetyl-L-cysteine (NAC) significantly attenuated betulin-induced ROS generation as well as Bcl-2 and XIAP downregulation. The mitochondrial accumulation of Bax and Bak, as well as caspase activity, was also remarkably inhibited by NAC treatment, indicating that ROS are important signaling intermediates that lead to betulin-induced apoptosis by modulating multiple apoptosis-regulating proteins in SGC7901 cells.

Chemical Composition of the Essential Oils from Flowers and Leaves of Marsdenia erecta Using Microwave Assisted Hydrodistillation Technique

In this study, microwave assisted hydrodistillation (MAHD) technique was successfully used to separate volatile essential oils from flowers and leaves of Marsdenia erecta. The obtained oils were subsequently analyzed using the gas chromatography combined with the mass spectrometry (GC-MS) technique. A total of 19 and 15 compounds were identified in the profiles of flowers and leaves, constituting 93.5 % and 90.6 % of the chemical profiles, respectively. Our study revealed the presence of high quantities of linalool in the extracted oils. Additionally, in both oils, oxygenated monoterpenes were the most abundant natural compounds, while the second and third ranks were due to non-terpene hydrocarbons and oxygenated diterpenes, respectively.

Synthesis, characterization and polymerization of novel sugars based on methacrylate

The present study describes the synthesis and characterizations of polymerizable vinyl sugars. Glucose, mannose, galactose and fructose are abundant and sustainable natural compounds. As it is not possible to make many derivatives of sugars without using protective groups, first of all, diacetone derivatives [diacetone-d-glucose (1), diacetone-d-mannose (2), diacetone-d-galactose (3) and diacetone-d-fructose (4)] were synthesized according to the literature as starting compounds. The remaining free hydroxyl groups on C-3 (diacetone glucose), C-6 (diacetone galactose), C-1 (diacetone fructose) and C-1 (diacetone mannose), were reacted with epichlorohydrin (1-chloro-2,3-epoxypropane) to produce then “-O-(2′,3′-epoxypropane-1′-yl)” ether derivatives (5, 6, 7, and 8) which are epoxy sugars in the basic medium. Next, the epoxy rings of the ethers (5, 6, 7, and 8) were opened with methacrylic acid in DMF to produce new sugar based methacrylates (9, 10, 11, and 12). Finally, free radical polymerization of these sugar based methacrylate monomers was performed, producing related polymers (13, 14, 15 and 16). The polymerizations were carried out using AIBN as an initiator at 70 °C in DMF. All the products were characterized by FTIR, 1HNMR and 13CNMR techniques. Thermal properties of all polymers were investigated by TG, DTG and DSC. The data obtained has suggested that thermal stability of the synthesized polymers has changed with the structure of the sugar and increase in molecular weight.

Cuminaldehyde as the Major Component of Cuminum cyminum, a Natural Aldehyde with Inhibitory Effect on Alpha-Synuclein Fibrillation and Cytotoxicity.

Fibrillation of alpha-synuclein (α-SN) is a critical process in the pathophysiology of several neurodegenerative diseases, especially Parkinson's disease. Application of bioactive inhibitory compounds from herbal extracts is a potential therapeutic approach for this cytotoxic process. Here, we investigated the inhibitory effects of the Iranian Cuminum cyminum essential oil on the fibrillation of α-SN. Analysis of different fractions from the total extract identified cuminaldehyde as the active compound involved in the antifibrillation activity. In comparison with baicalein, a well-known inhibitor of α-SN fibrillation, cuminaldehyde showed the same activity in some aspects and a different activity on other parameters influencing α-SN fibrillation. The presence of spermidine, an α-SN fibrillation inducer, dominantly enforced the inhibitory effects of cuminaldehyde even more intensively than baicalein. Furthermore, the results from experiments using preformed fibrils and monobromobimane-labeled monomeric protein also suggest that cuminaldehyde prevents α-SN fibrillation even in the presence of seeds, having no disaggregating impact on the preformed fibrils. Structural studies showed that cuminaldehyde stalls protein assembly into β-structural fibrils, which might be achieved by the interaction with amine groups through its aldehyde group as a Schiff base reaction. This assumption was supported by FITC labeling efficiency assay. In addition, cytotoxicity assays on PC12 cells showed that cuminaldehyde is a nontoxic compound, treatment with cuminaldehyde throughout α-SN fibrillation showed no toxic effects on the cells. Taken together, these results show for the first time that the small abundant natural compound, cuminaldehyde, can modulate α-SN fibrillation. Hence, suggesting that such natural active aldehyde could have potential therapeutic applications.

From Hemoglobin to Porous N–S–Fe-Doped Carbon for Efficient Oxygen Electroreduction

Nitrogen–sulfur–iron-doped porous carbon material with high surface area (1026 m2 g–1) and large pore size is synthesized by the pyrolysis of hemoglobin, an abundant and inexpensive natural compound, with mesoporous silica foam (MS) as a template and thiocarbamide (TCA) as an additional sulfur source in an argon atmosphere. Our results indicated that as compared to the commercial 20% Pt/C catalyst, the synthesized catalyst exhibits not only higher current density and stability but also higher tolerance to crossover chemicals. More importantly, the synthetic method is simple and inexpensive.

Polymorphic phase transition mechanism of compressed coesite.

Silicon dioxide is one of the most abundant natural compounds. Polymorphs of SiO₂ and their phase transitions have long been a focus of great interest and intense theoretical and experimental pursuits. Here, compressing single-crystal coesite SiO₂ under hydrostatic pressures of 26-53 GPa at room temperature, we discover a new polymorphic phase transition mechanism of coesite to post-stishovite, by means of single-crystal synchrotron X-ray diffraction experiment and first-principles computational modelling. The transition features the formation of multiple previously unknown triclinic phases of SiO₂ on the transition pathway as structural intermediates. Coexistence of the low-symmetry phases results in extensive splitting of the original coesite X-ray diffraction peaks that appear as dramatic peak broadening and weakening, resembling an amorphous material. This work sheds light on the long-debated pressure-induced amorphization phenomenon of SiO₂, but also provides new insights into the densification mechanism of tetrahedrally bonded structures common in nature.

Characterisation of Kraft lignin separated by gradient acid precipitation

Lignin is considered the second most abundant organic natural compound in the world. Lignin represents a promising source of new materials based on renewable resources. Currently the lignin is used in low value-added applications and for energy production and chemical recycling for industrial process itself in production of pulp and paper. Only a small amount is being used for other applications. The aim of this study was to analyze the effects of precipitation on the composition of lignin extracted from black liquor produced in industry CMPC Celulose Riograndense. For this, lignins were precipitated at different pH conditions, with various acids. Obtained lignin samples were characterized by various analytical techniques such as FTIR, GPC, HPLC and XRD. Furthermore, the quantity of carboxyl groups was also analyzed. The antioxidant activity of the analyzed lignins was evaluated by the radical ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay. Total phenolic contain was evaluated by Folin–Ciocalteu method while the antioxidant potential of lignins was measured by DPPH. The lignins showed a variable composition according to the acid used for its precipitation; the lignin precipitated with sulphuric acid presented Na2SO4 salts while with hydrochloric acid, NaCl was found. In addition, a change in lignin composition was observed at different pH.

Ginsenoside Rg5 improves cognitive dysfunction and beta-amyloid deposition in STZ-induced memory impaired rats via attenuating neuroinflammatory responses

Neuroinflammatory responses play a crucial role in the pathogenesis of Alzheimer's disease (AD). Ginsenoside Rg5 (Rg5), an abundant natural compound in Panax ginseng, has been found to be beneficial in treating AD. In the present study, we demonstrated that Rg5 improved cognitive dysfunction and attenuated neuroinflammatory responses in streptozotocin (STZ)-induced memory impaired rats. Cognitive deficits were ameliorated with Rg5 (5, 10 and 20mg/kg) treatment in a dose-dependent manner together with decreased levels of inflammatory cytokines TNF-α and IL-1β (P<0.05) in brains of STZ rats. Acetylcholinesterase (AChE) activity was also significantly reduced by Rg5 whereas choline acetyltransferase (ChAT) activity was remarkably increased in the cortex and hippocampus of STZ-induced AD rats (P<0.05). In addition, Congo red and immunohistochemistry staining results showed that Rg5 alleviated Aβ deposition but enhanced the expressions of insulin-like growth factors 1 (IGF-1) and brain derived neurophic factor (BDNF) in the hippocampus and cerebral cortex (P<0.05). Western blot analysis also demonstrated that Rg5 increased remarkably BDNF and IGF-1 expressions whereas decreased significantly Aβ deposits (P<0.05). Furthermore, it was observed that the expressions of COX-2 and iNOS were significantly up-regulated in STZ-induced AD rats and down-regulated strongly (P<0.05) by Rg5 compared with control rats. These data demonstrated that STZ-induced learning and memory impairments in rats could be improved by Rg5, which was associated with attenuating neuroinflammatory responses. Our findings suggested that Rg5 could be a beneficial agent for the treatment of AD.

Synthesis of phosphates and phosphates–acetates hybrids of green tea polyphenol (−)- epigallocatechine-3-gallate (EGCG) and its G ring deoxy analogs as potential anticancer prodrugs

A series of phosphate or phosphate–acetate hybrid modified EGCG or EGCG G ring deoxy analogs were synthesized by a convenient semi-synthesis strategy from the abundant natural compound EGCG.

Structural Modeling and Biochemical Characterization of Flavonoid O-Methyltransferase from Rice

Natural products from plants are diverse, and might be used as novel starting materials for pharmatheutical or neutrotheutical purposes. Plant phenylpropanoids, such as lignins, flavonoids, and anthocyanins, are a group containing abundant natural compounds that exert diverse biological effects on humans, as well as plants. 1 Phenylpropanoids derived from the amino acid phenylalanine by phenylalanine ammonia lyase (PAL) are synthesized via the phenylpropanoid pathway. 2

Significant decomposition of riverine humic-rich DOC by marine but not estuarine bacteria assessed in sequential chemostat experiments

Humic substances (HS) are the most abundant natural organic compounds in aquatic and terrestrial environments. However, the bacterial degradation of HS in the estuarine salinity gra- dient and in coastal regions as a sink for HS, entering the open sea, is not well understood. Therefore, we studied the bacterial degradation of humic-rich dissolved organic carbon (DOC) at increasing salinities between 1 and 30 in an estuary of the southern North Sea (Weser). Three-stage chemostats, inoculated with natural brackish (salinity 5 and 15) and coastal marine (salinity 30) bacterial commu- nities fed 0.1 µm filtered natural humic-rich freshwater adjusted to the respective salinity, were run for up to 51 d at dilution rates of 0.1 and 0.15 d -1 . DOC concentrations, bacterial numbers, and pro- duction were assessed in each stage over the incubation time. In addition, the fulvic acid (FA), humic acid (HA), and hydrophilic acid (HPA) fractions of the HS were determined by XAD fractionation. At a salinity of 30, the humic-rich DOC was decomposed to more than 60%, whereas in the other 2 experiments at salinities of 15 and 5, no detectable decomposition occurred. At the highest salinity condition, all 3 HS fractions were reduced substantially, whereas at a salinity of 5, only the HA and HPA fractions decreased, and at 15, only the HA fraction decreased. In the latter experiments, con- centrations of the FA and HPA fractions in some chemostat stages increased despite unchanged DOC concentration. Our study provides evidence that the main bacterial decomposition, i.e. conversion into bacterial biomass and remineralization, of humic-rich DOC occurs in the polyhaline estuarine region and that at lower salinities, only some transformation of the humic fractions takes place, mod- ifying the adsorptive properties of the HS, but no bacterial DOC decomposition occurs. The sequen- tial chemostats were a useful approach to study the complex process of bacterial HS decomposition.

Chromatographic Separation of Natural Compounds

THE isolation of more abundant natural compounds mostly uses very simple means—extraction, distiltion, crystallization—and combinations and refinements of these methods. The problem of isolatiing, less abundant natural substances is associated with two outstanding difficulties. The scale of the extraction process is much greater, and the desired material is often accompanied by a variety of very similar materials. Without doubt chromatography has done more than any other experimental advance to overcome these difficulties.