Chromosome-level genome of Thymus mandschuricus reveals molecular mechanism of aroma compounds biosynthesis.

Background: Thymus mandschuricus is an aromatic and medicinal plant with notable antibacterial and antioxidant properties. However, traditional breeding methods rely on phenotypic selection due to a lack of molecular resources. A high-quality reference genome is crucial for marker-assisted breeding, genome editing, and molecular genetics. Results: We utilized PacBio and Hi-C technologies to generate a high-quality chromosome-level reference genome for T. mandschuricus, with a size of 587.05 Mb and an N50 contig size of 8.41 Mb. The assembled genome contained 29,343 predicted protein-coding genes, and evidence of two distinct whole-genome duplications in T. mandschuricus was discovered. Comparative genomic analysis revealed rapid evolution of genes involved in phenylpropanoid biosynthesis and the CYP450 gene family in T. mandschuricus. Additionally, we reconstructed the gene families of terpenoid biosynthesis structural genes, such as TPS, BAHD, and CYP, and identified regulatory networks controlling the expression of aroma-synthesis genes by integrating transcriptome data from various organs and developmental stages. We discovered that hormones and transcription factors may collaborate in controlling aroma-synthesis gene expression. Conclusion: This study provides the first high-quality genome sequence and gene annotation for T. mandschuricus, an indigenous thyme species unique to China. The genome assembly and the comprehension of the genetic basis of fragrance synthesis acquired from this research could potentially serve as targets for future breeding programs and functional studies.

A comparative analysis of soil physicochemical properties and microbial community structure among four shelterbelt species in the northeast China plain.

Conducting studies that focus on the alterations occurring in the soil microbiome within protection forests in the northeast plain is of utmost importance in evaluating the ecological rehabilitation of agricultural lands in the Mollisols region. Nevertheless, the presence of geographic factors contributes to substantial disparities in the microbiomes, and thus, addressing this aspect of influence becomes pivotal in ensuring the credibility of the collected data. Consequently, the objective is to compare the variations in soil physicochemical properties and microbial community structure within the understory of diverse shelterbelt species. In this study, we analyzed the understory soils of Juglans mandshurica (Jm), Fraxinus mandschurica (Fm), Acer mono (Am), and Betula platyphylla (Bp) from the same locality. We employed high-throughput sequencing technology and soil physicochemical data to investigate the impact of these different tree species on soil microbial communities, chemical properties, and enzyme activities in Mollisols areas. Significant variations in soil nutrients and enzyme activities were observed among tree species, with soil organic matter content ranging from 49.1 to 67.7 g/kg and cellulase content ranging from 5.3 to 524.0 µg/d/g. The impact of tree species on microbial diversities was found to be more pronounced in the bacterial community (Adnoism: R = 0.605) compared to the fungal community (Adnoism: R = 0.433). The linear discriminant analysis effect size (LEfSe) analysis revealed a total of 5 (Jm), 3 (Bp), and 6 (Am) bacterial biomarkers, as well as 2 (Jm), 6 (Fm), 4 (Bp), and 1 (Am) fungal biomarker at the genus level (LDA3). The presence of various tree species was observed to significantly alter the relative abundance of specific microbial community structures, specifically in Gammaproteobacteria, Ascomycota, and Basidiomycota. Furthermore, environmental factors, such as pH, total potassium, and available phosphorus were important factors influencing changes in bacterial communities. We propose that Fm be utilized as the primary tree species for establishing farmland protection forests in the northeastern region, owing to its superior impact on enhancing soil quality. IMPORTANCE: The focal point of this study lies in the implementation of a controlled experiment conducted under field conditions. In this experiment, we deliberately selected four shelterbelts within the same field, characterized by identical planting density, and planting year. This deliberate selection effectively mitigated the potential impact of extraneous factors on the three microbiomes, thereby enhancing the reliability and validity of our findings.

Integrating metagenomics and metabolomics to study the response of microbiota in black soil degradation.

As the largest commercial food production base and ecological security barrier, land degradation in black soil areas seriously threatens the global food supply and natural ecosystems. Therefore, determining the response of soil microbiota is crucial to restoring degraded soils. This study combined metagenomics and metabolomics to investigate the effect of different degrees of soil degradation on microbial community composition and metabolic function in black soils. It was found that alpha diversity in degraded soils (Shannon: 22.3) was higher than in nondegraded soil (ND) (Shannon: 21.8), and the degree of degradation significantly altered the structure and composition of soil microbial communities. The results of LEfSe analysis obtained 9 (ND), 7 (lightly degraded, LD), 10 (moderately degraded, MD), and 1 (severely degraded, SD) biomarkers in four samples. Bradyrhizobium, Sphingomonas, and Ramlibacter were significantly affected by soil degradation and can be considered biomarkers of ND, MD, and SD, respectively. Soil nutrient and enzyme activities decreased significantly with increasing black soil degradation, soil organic matter (SOM) content decreased from 11.12 % to 1.97 %, and Sucrase decreased from 23.53 to 6.59 mg/g/d. In addition, C was the critical driver affecting microbial community structure, contributing 61.2 % to differences in microbial community distribution, and microbial altering relative abundance which participle in the carbon cycle to respond to soil degradation. Metabolomic analyses indicated that soil degradation significantly modified the soil metabolite spectrum, and the metabolic functions of most microorganisms responding to soil degradation were adversely affected. The combined multi-omics analysis further indicated that biomarkers dominate in accumulating metabolites. These findings confirmed that due to their role in the composition and functioning of these degraded soils, these biomarkers could be employed in strategies for managing and restoring degraded black soils.

An improved approach of salting-out solvent-free microwave mediated rotary distillation for essential oil preparation from fresh leaves of magnolia (Oyama sieboldii).

An improved method denoted as salting out-solvent-free microwave rotary distillation (SOSFMRD) was successfully developed for the extraction of essential oils from fresh magnolia (Oyama sieboldii) leaves, in which we achieved the rotation of the reaction material by means of a rotating motor to subject the material to uniform microwave irradiation. Magnesium chloride was selected as the salting-out agent through a comparative study on the salting-out effects of different anions and cations of metal salts. The variables of SOSFMRD were systematically optimized. Under the obtained optimization conditions, the essential oil yield was 21.68 ± 1.02 mL/kgDW. Gas chromatography-mass spectrometry analysis showed that the main chemical constituent of O. sieboldii essential oil was dehydrocostuslactone, the content of which reached 30.23 ± 1.27 %. Compared with the other conventional methods, this method has a high yield and low energy consumption, which can effectively reduce impact on the environment.

Tea saponin additive to extract eleutheroside B and E from Eleutherococcus senticosus by ultrasonic mediation and its application in a semi-pilot scale.

The safety of ethanol in operations and its effects on human health are gradually being questioned. Under this premise, we attempted to use the natural surfactant tea saponin, which originates from the processing residues of camellia oil, as the additive of the extraction solvent and to extract eleutheroside B and eleutheroside E in the roots and rhizomes of E. senticosus by ultrasonic mediation. After a single-factor experiment, extraction kinetics at different powers and reaction temperatures, and Box-Behnken design optimization, the optimal conditions obtained were 0.3% tea saponin solution as the extraction solvent, 20 mL/g liquid-solid ratio, 250 W ultrasonic irradiation power (43.4 mW/g ultrasonic power density) and 40 min ultrasonic irradiation time. Under optimal conditions, satisfactory yields of eleutheroside B (1.06 ± 0.04 mg/g) and eleutheroside E (2.65 ± 0.12 mg/g) were obtained with semi pilot scale ultrasonic extraction equipment. The experiments showed that compared with the traditional thermal extraction process, the extraction time is significantly reduced at lower operating temperatures.

Ultrasound-Assisted Extraction of Taxifolin, Diosmin, and Quercetin from Abies nephrolepis (Trautv.) Maxim: Kinetic and Thermodynamic Characteristics.

Extraction behaviors of the 3 flavonoids taxifolin, diosmin, and quercetin have been investigated in Abies nephrolepis leaves and bark. The following operation parameters-ethanol volume fraction, liquid-solid ratio, temperature, ultrasound irradiation power and time, and ultrasound frequency-were varied to study their effect on the yield of the 3 flavonoids during extraction. The results showed that a low extraction efficiency occurred at 293.15 K due to slow kinetics, while the situation was significantly improved at 333.15 K. The kinetic data for the extraction yields of the 3 flavonoids achieved good fits by the first-order kinetic model. From the thermodynamic analysis results, we realized that the ultrasound-assisted extraction of taxifolin, diosmin, and quercetin from the leaves and bark of A. nephrolepis was a spontaneous and endothermic process in which the disorder increased (ΔG0 < 0, ΔH0 > 0, and ΔS0 > 0). According to the response surface methodology (RSM) analysis, under the optimal operation conditions (ethanol concentration of 50%, liquid-solid ratio of 20 mL/g, frequency of 45 kHz, extraction time of 39.25 min, ultrasound irradiation power of 160 W and temperature of 332.19 K), the total yield of the 3 flavonoids were 100.93 ± 4.01 mg/g from the leaves of A. nephrolepis (with 31.03 ± 1.51 mg/g, 0.31 ± 0.01 mg/g, 69.59 ± 2.57 mg/g for taxifolin, diosmin, and quercetin, respectively), and under the optimal operation conditions (ethanol concentration of 50%, liquid-solid ratio of 20 mL/g, frequency of 45 kHz, extraction time of 36.80 min, ultrasound irradiation power of 150 W and temperature of 328.78 K), 16.05 mg/g ± 0.38 mg/g were obtained from the bark of A. nephrolepis (with 1.44 ± 0.05 mg/g, 0.47 ± 0.01 mg/g, 14.14 ± 0.38 mg/g for taxifolin, diosmin, and quercetin, respectively), which were close to the prediction values.

Efficient Homogenization-Ultrasound-Assisted Extraction of Anthocyanins and Flavonols from Bog Bilberry (Vaccinium uliginosum L.) Marc with Carnosic Acid as an Antioxidant Additive.

To explore the optimum conditions for the extraction of anthocyanins and flavonols from bog bilberry (Vaccinium uliginosum L.) marc on a single-factor experimental basis, a response surface methodology was adopted for this intensive study. The extraction procedure was carried out in a Waring blender and followed an ultrasonic bath, and the natural antioxidant carnosic acid was added to inhibit oxidation. The optimum extraction conditions were as follows: a volume fraction of ethanol of 70%, an antioxidant content of 0.02% (the mass of sample) carnosic acid, a liquid-solid ratio of 16 mL/g, a homogenization time of 3 min, a reaction temperature of 55 °C, an ultrasound irradiation frequency of 80 kHz, an ultrasound irradiation power of 200 W, and an ultrasound irradiation time of 40 min. Satisfactory yields of anthocyanins (13.95 ± 0.37 mg/g) and flavonols (3.51 ± 0.16 mg/g) were obtained. The experimental results showed that the carnosic acid played an effective antioxidant role in the extraction process of anthocyanins and flavonols with a green and safety guarantee.

A modified approach for isolation of essential oil from fruit of Amorpha fruticosa Linn using microwave-assisted hydrodistillation concatenated liquid-liquid extraction.

In this work, a modified technique was developed to separate essential oil from the fruit of Amorpha fruticosa using microwave-assisted hydrodistillation concatenated liquid-liquid extraction (MHD-LLE). The new apparatus consists of two series-wound separation columns for separating essential oil, one is the conventional oil-water separation column, and the other is the extraction column of components from hydrosol using an organic solvent. Therefore, the apparatus can simultaneously collect the essential oil separated on the top of hydrosol and the components extracted from hydrosol using an organic solvent. Based on the yield of essential oil in the first and second separation columns, the effects of parameters were investigated by single factor experiments and Box-Behnken design. Under the optimum conditions (2mL ethyl ether as the extraction solvent in the second separation column, 12mL/g liquid-solid ratio, 4.0min homogenate time, 35min microwave irradiation time and 540W microwave irradiation power), satisfactory yields for the essential oil in the first separation column (10.31±0.33g/kg) and second separation column (0.82±0.03g/kg) were obtained. Compared with traditional methods, the developed method gave a higher yield of essential oil in a shorter time. In addition, GC-MS analysis of the essential oil indicated significant differences of the relative contents of individual volatile components in the essential oils obtained in the two separation columns. Therefore, the MHD-LLE technique developed here is a good alternative for the isolation of essential oil from A. fruticosa fruit as well as other herbs.

An efficient approach for the extraction of orientin and vitexin from Trollius chinensis flowers using ultrasonic circulating technique.

Ultrasonic circulating extraction (UCE) approach was developed for effective extraction of orientin and vitexin from the flowers of Trollius chinensis successfully. In this study, some parameters potentially influencing the yields of orientin and vitexin were systematically investigated and optimized by Plackett-Burman and Box-Behnken design, and the optimum operational conditions obtained were 60% ethanol volume fraction, 1000r/min stirring speed, 30°C temperature, 28min ultrasonic irradiation time, 10mL/g liquid-solid ratio and 738W ultrasonic irradiation power. Satisfactory yields of orientin (6.05±0.19mg/g) and vitexin (0.96±0.03mg/g) were obtained in a relatively shorter extraction time under the derived optimum conditions, compared to other ultrasonic extraction methods and heat extraction methods. The mechanism of UCE procedure was discussed in detail, to illustrate the advantage of UCE in the extraction process. In addition, no degradation of orientin and vitexin and high reproducibility of the developed UCE method were observed under the optimum conditions. The proposed UCE technique with high-capacity and circulation function is a rapid and efficient sample extraction technique, and performs promising in large-scale sample preparation.

Optimization of ultrasonic circulating extraction of samara oil from Acer saccharum using combination of Plackett-Burman design and Box-Behnken design.

In this study, ultrasonic circulating extraction (UCE) technique was firstly and successfully applied for extraction of samara oil from Acer saccharum. The extraction kinetics were fitted and described, and the extraction mechanism was discussed. Through comparison, n-hexane was selected as the extraction solvent, the influence of solvent type on the responses was detailedly interpreted based on the influence of their properties on the occurrence and intensity of cavitation. Seven parameters potentially influencing the extraction yield of samara oil and content of nervonic acid, including ultrasound irradiation time, ultrasound irradiation power, ultrasound temperature, liquid-solid ratio, soaking time, particle size and stirring rate, were screened through Plackett-Burman design to determine the significant variables. Then, three parameters performed statistically significant, including liquid-solid ratio, ultrasound irradiation time and ultrasound irradiation power, were further optimized using Box-Behnken design to predict optimum extraction conditions. Satisfactory yield of samara oil (11.72±0.38%) and content of nervonic acid (5.28±0.18%) were achieved using the optimal conditions. 1% proportion of ethanol in extraction solvent, 120°C of drying temperature and 6.4% moisture were selected and applied for effective extraction. There were no distinct differences in the physicochemical properties of samara oil obtained by UCE and Soxhlet extraction, and the samara oil obtained by UCE exhibited better antioxidant activities. Therefore, UCE method has enormous potential for efficient extraction of edible oil with high quality from plant materials.

Brönsted acidic ionic liquid based ultrasound-microwave synergistic extraction of pectin from pomelo peels.

3-Methyl-1-(4-sulfonylbutyl) imidazolium hydrogensulfate, [HO3S(CH2)4mim]HSO4, was applied as an extractant in an ultrasound-microwave synergistic extraction approach to substitute conventional solvent for the extraction of pectin from the albedo part of pomelo peels. The analysis of variance (ANOVA) test and response surface method were employed for the optimization of the extraction conditions. A pectin yield of 328.64±4.19mg/g was achieved using the obtained optimal conditions, which was significantly higher than yields of conventional methods with reference solvents. Pectin samples extracted with [HO3S(CH2)4mim]HSO4 and hydrochloric acid solutions were tested by ANOVA and showed no significant differences in total carbohydrate content and degree of esterification; while galacturonic acid content was significantly different for the pectin from each extraction solvents. The differences revealed from images of atomic force microscopy and scanning electron microscope, Fourier transform infrared spectroscopy, and thermogravimetric analysis suggested the physiochemical properties of pectin could be affected by the extraction solvent. The [HO3S(CH2)4mim]HSO4 proved to be a promising alternative to conventional solvents and the proposed method is efficient for the extraction of pectin from the albedo of pomelo peels.

An approach for extraction of kernel oil from Pinus pumila using homogenate-circulating ultrasound in combination with an aqueous enzymatic process and evaluation of its antioxidant activity.

In this study, a novel approach involving homogenate-circulating ultrasound in combination with aqueous enzymatic extraction (H-CUAEE) was developed for extraction of kernel oil from Pinus pumila. Following comparison of enzyme types and concentrations, an enzyme mixture consisting of cellulase, pectinase and hemicellulase (1:1:1, w/w/w) at a concentration of 2.5% was selected and applied for effective oil extraction and release. Several variables potentially influencing extraction yields, namely, homogenization time, incubation temperature, incubation time, mark-space ratio of ultrasound irradiation, ultrasound irradiation power, liquid-solid ratio, pH and stirring rate, were optimized by Plackett-Burman design. Among the eight variables, incubation temperature, incubation time and liquid-solid ratio were statistically significant and were further optimized by Box-Behnken design to predict optimum extraction conditions and ascertain operability ranges for maximum extraction yield. Under optimum operating conditions, extraction yields of P. pumila kernel oil were 31.89±1.12% with a Δ5-unsaturated polymethylene interrupted fatty acid content of 20.07% and an unsaturated fatty acid content of 93.47%. Our study results indicate that the proposed H-CUAEE process has enormous potential for efficient and environmentally friendly extraction of edible oils.

Application of ionic liquids in vacuum microwave-assisted extraction followed by macroporous resin isolation of three flavonoids rutin, hyperoside and hesperidin from Sorbus tianschanica leaves.

Rutin, hyperoside and hesperidin were effectively extracted from Sorbus tianschanica leaves by an ionic liquid vacuum microwave-assisted method. A series of ionic liquids with various anions and alkyl chain length of the cations were studied and the extraction was performed in [C6mim][BF4] aqueous solution. After optimization by a factorial design and response surface methodology, total extraction yield of 2.37mg/g with an error of 0.12mg/g (0.71±0.04mg/g, 1.18±0.06mg/g and 0.48±0.02 for rutin, hyperoside and hesperidin, respectively) was achieved under -0.08MPa for vacuum, 19min and 420W for microwave irradiation time and power, and 15mL/g for liquid-solid ratio. The proposed method here is more efficient and needs a shorter extraction time for rutin, hyperoside and hesperidin from S. tianschanica leaves than reference extraction techniques. In stability studies performed with standard rutin, hyperoside and hesperidin, the target analytes were stable under the optimum conditions. The proposed method had a high reproducibility and precision. In addition, separation of rutin, hyperoside and hesperidin from [C6mim][BF4] extraction solution was completed effectively by AB-8 macroporous resin adsorption and desorption process. Ionic liquid vacuum microwave-assisted extraction is a simple, rapid and efficient sample extraction technique.

Application of alkyl polyglycoside surfactant in ultrasonic-assisted extraction followed by macroporous resin enrichment for the separation of vitexin-2″-O-rhamnoside and vitexin from Crataegus pinnatifida leaves.

An alkyl polyglycoside (APG) surfactant was used in ultrasonic-assisted extraction to effectively extract vitexin-2″-O-rhamnoside (VOR) and vitexin (VIT) from Crataegus pinnatifida leaves. APG0810 was selected as the surfactant. The extraction process was optimized for ultrasonic power, the APG concentration, ultrasonic time, soaking time, and liquid-solid ratio. The proposed approach showed good recovery (99.80-102.50% for VOR and 98.83-103.19% for VIT) and reproducibility (relative standard deviation, n=5; 3.7% for VOR and 4.2% for VIT) for both components. The proposed sample preparation method is both simple and effective. The use of APG for extraction of key herbal ingredients shows great potential. Ten widely used commercial macroporous resins were evaluated in a screening study to identify a suitable resin for the separation and purification of VOR and VIT. After comparing static and dynamic adsorption and desorption processes, HPD100B was selected as the most suitable resin. After column adsorption and desorption on this resin, the target compounds VOR and VIT can be effectively separated from the APG0810 extraction solution. Recoveries of VOR and VIT were 89.27%±0.42% and 85.29%±0.36%, respectively. The purity of VOR increased from 35.0% to 58.3% and the purity of VIT increased from 12.5% to 19.9%.

Enzymatic Hydrolysis and Simultaneous Extraction for Preparation of Genipin from Bark of Eucommia ulmoides after Ultrasound, Microwave Pretreatment.

A continuous process based on the combination of ultrasounds and/or microwaves pretreatments followed by enzymatic hydrolysis and simultaneous extraction (EHSE) has been proposed to recover genipin from Eucommia ulmoides bark. At first, in the pretreatment step, the mixture of 1.0 g dried bark powder and 10 mL deionized water were irradiated by microwave under 500 W for 10 min. Then, in hydrolysis step, the optimal conditions were as follows: 0.5 mg/mL of cellulase concentration, 4.0 pH of enzyme solution, 24 h of incubation time and 40 °C of incubation temperature. After incubation, 10 mL ethanol was added to extract genipin for 30 min by ultrasound. After EHSE treatment, the yield of genipin could reach 1.71 µmol/g. Moreover, scanning electron micrographs illustrated that severe structural disruption of plant was obtained by EHSE. The results indicated that the EHSE method provided a good alternative for the preparation of genipin from Eucommia ulmoides bark as well as other herbs.

An approach of ionic liquids/lithium salts based microwave irradiation pretreatment followed by ultrasound-microwave synergistic extraction for two coumarins preparation from Cortex fraxini.

Ionic liquids/lithium salts solvent system was successfully introduced into the separation technique for the preparation of two coumarins (aesculin and aesculetin) from Cortex fraxini. Ionic liquids/lithium salts based microwave irradiation pretreatment followed by ultrasound-microwave synergy extraction (ILSMP-UMSE) procedure was developed and optimized for the sufficient extraction of these two analytes. Several variables which can potentially influence the extraction yields, including pretreatment time and temperature, [C4mim]Br concentration, LiAc content, ultrasound-microwave synergy extraction (UMSE) time, liquid-solid ratio, and UMSE power were optimized by Plackett-Burman design. Among seven variables, UMSE time, liquid-solid ratio, and UMSE power were the statistically significant variables and these three factors were further optimized by Box-Behnken design to predict optimal extraction conditions and find out operability ranges with maximum extraction yields. Under optimum operating conditions, ILSMP-UMSE showed higher extraction yields of two target compounds than those obtained by reference extraction solvents. Method validation studies also evidenced that ILSMP-UMSE is credible for the preparation of two coumarins from Cortex fraxini. This study is indicative of the proposed procedure that has huge application prospects for the preparation of natural products from plant materials.

[Effects of human disturbance on coarse woody debris of Larix gmelinii forest on northern slope of Greater Hinggan Mountains].

An investigation was conducted on the living tree volume, coarse woody debris (CWD) loading, and composition of Larix gmelinii forest along a human disturbance gradient, i.e., no disturbance (natural larch forest), one-time disturbance (natural forest was disturbed once only), and two-time disturbance (natural forest was disturbed two times consecutively), on the northern slope of Greater Hinggan Mountains. The results showed that under no disturbance, one-time disturbance, and two-time disturbance, the average living tree volume of L. gmelinii forest was 161.6, 138.3, and 114. 8 m3 x hm(-2), and the average CWD loading was 69.77, 36.64 and 32.61 m3 x hm(-2), respectively. In natural L. gmelinii forest, most of CWD was of 20-40 cm diameter class, among which, fallen logs occupied 72%, and snags and stumps occupied 28% ; while in one-time and two-time disturbance L. gmelinii forests, most CWD was of 10-30 cm diameter class, with the fallen logs, snags, and stumps occupied 70%, 14% and 16%, and 57%, 15% and 28%, respectively. Human disturbance reduced the CWD loading of L. gmelinii forest and altered the composition of the CWD.

[Dynamics of soil nutrient contents in cutting forestlands of broad-leaved Korean pine forest on Changbai Mountains].

A comparative study was made to analyze the dynamics of soil nutrient contents in different cutting forestlands of broad-leaved Korean pine forest on Changbai Mountains. The results showed that soil nutrient contents in different forestlands with different cutting times had obvious differences. With the cutting time going on after logging, soil pH value had the trend of declining firstly and increasing afterwards. The acidity of the topsoil was the highest after cutting for five years, and that of 10-20 cm soil layer was the strongest after cutting for ten years. The organic matter content as well as the total and available contents of nutrients increased at the first 2-5 years, and then declined rapidly, especially for those in topsoils. The cation exchange capacity (CEC), exchangeable Ca and exchangeable Mg had the same trend. Therefore, after forest cutting, artificial planting and regeneration should be carried out in time to resume the vegetation to reduce and prevent soil nutrient loss.