This research was supported by the Institute of Cannabis Research at Colorado State University-Pueblo, the Korea Innovation Foundation grant funded by the Korean government (MSIT) (2021-DD-UP-0379), and Chuncheon city (Hemp R&#38;D and industrialization, 2020-2021).

1. Plant material preparation
<ol>
	<li>Obtain Cherry Wine inflorescences from plants grown in the field, planted in a south-to-north configuration, with plants 1 m apart in center and rows 1.2 m apart (cultivation located in Longmont, Colorado, USA).</li>
	<li>Air-dry the inflorescences at 35 &#176;C for 48 h. Grind the inflorescences using a grinding machine set a 177 &#181;m.</li>
	<li>Pass the pulverized material through the No. 80 mesh sieve. Store the resulting powder in a sealed bag at room temperature for future use.</li>
</ol>
2. Ultrasound extraction
<ol>
	<li>Weigh 0.5 g of the Cannabis inflorescence powder into a 50 mL conical tube. Add 40 mL of the solvent (e.g., 50% ethanol in deionized water) to the vessel.</li>
	<li>Place the extraction vessel in the ultrasonic bath set at 40 kHz and at room temperature (sonication power is 100 W).</li>
	<li>Perform the extraction in the ultrasonic bath for 30 min, increasing the temperature of the bath from 25 &#176;C to 30 &#176;C.</li>
	<li>Decant the extraction fluid into a centrifuge tube.</li>
	<li>Centrifuge the fluid at 3,000 x g at 15 &#176;C for 15 min. Filter the supernatant under vacuum through an 8 &#181;m filter paper.</li>
</ol>
3. High-performance liquid chromatography (HPLC) quantitative analysis
<ol>
	<li>Dilute seven cannabinoid standards: cannabichromene (CBC), CBD, CBDA, cannabinol (CBN), tetrahydrocannabinolic acid (THCA), &#916;8-THC, and &#916;9-THC&#160;to operating concentrations of 100, 50, 25, and 12.5 &#181;g/mL in 100% methanol. Mix and sonicate for 5 min in an ultrasonic bath set at 40 kHz and sonication power of 100 W</li>
	<li>Filter the standards through a 0.45 &#181;m polytetrafluoroethylene (PTFE) syringe filter. Filter the sample supernatant (from step 2.5) through a 0.45 &#181;m PTFE syringe filter.</li>
	<li>Put the sample to be analyzed in a 1.5 mL vial into the HPLC autosampler and load 10 &#181;L at a time.</li>
	<li>Run HPLC as per the conditions and parameters provided in Table 1. Derive cannabinoid concentrations in 50-200 &#181;g/mL from the generated standard curve.</li>
	<li>Multiply with the volume of the solvent (40 mL) used in the extraction process to obtain &#181;g of cannabinoid. Convert the &#181;g of cannabinoid to mg of cannabinoid by dividing it by 1000.</li>
	<li>Divide with the original weight of the plant material (0.5 g) used in the extraction to obtain mg/g dry weight.</li>
</ol>
4. Optimization using response surface methodology
<ol>
	<li>Establish the model, consisting of 15 experimental runs with 12 factorial points and three center points as shown in Table 4 using a data analysis tool.</li>
	<li>Optimize the extraction parameters, extraction time (T), solvent concentration (S), and sample/solvent ratio (R) using response surface methodology and central composite design. Set the range of variables T, S, and R as 5-30 min, 20%-100%, and 60-100 (1:X), respectively.</li>
	<li>Select the total yield of lipophilic extract and the yields of extracted CBD and CBDA as response factors (RF).</li>
</ol>

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The authors declare no competing interests.

The polarity of a solvent plays a critical role in the effective extraction of compounds. Since acidic cannabinoids are slightly polar in nature, due in large part to the carboxylic acid moiety, it was assumed that a polar solvent such as methanol or ethanol would be most effective. Garrett and Hunt19, in their study using THC, demonstrated that solubility in aqueous ethanol was based on percent ethanol in the solution and ionic strength of the solution. While ionic strength was not examined in th...

Industrial hemp (Cannabis spp.) produces acidic cannabinoids in various plant tissues (flowers, leaves, and stems), with the highest concentration found in the flower1. The Cannabis industry utilizes several methods to extract these compounds. One such method is solvent extraction that utilizes a non-polar and/or polar solvent, of which ethanol is the most commonly used. However, solvent extraction alone is limited in its ability; therefore, augmentative extraction techniques, such as microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE), are designed to increase the yield. In addition, high concentration cannabidiol (CBD) can be extracted using supercritical fluid technologies2.
Extraction is a dynamic process, and several factors influence its efficiency, namely moisture content, particle size, and solvent3. Specifically, for the UAE technique, efficiency is governed by temperature, pressure, frequency, and time4.
Ultrasonic-assisted extraction is the process where ultrasonic waves are passed through a liquid to agitate particles. During the agitation process, plant materials experience acoustic cavitation, cycles of compression and expansion which form bubbles that collapse in solution resulting in the generation of extreme temperature and pressure5. The pressure and temperature changes alter the physical properties of the solvents, which can result in increased efficacy of extraction6. Additionally, cavitation can disrupt molecular interactions leading to organic and inorganic compounds leaching from the plant matrix7. The process involves two main types of physical phenomena: (1) diffusion across the cell wall, and (2) rinsing of the cellular contents after breaking the wall8. However, the use of UAE is not without its pitfalls; there are several reports that UAE can degrade compounds9,10. Additionally, the temperatures generated at the cavitation sites are above those necessary for decarboxylation of cannabinoids. However, Mudge et al.11 used UAE and did not observe large decarboxylation of CBD or tetrahydrocannabinol (THC), thereby demonstrating that UAE is an efficient and green method for the extraction of cannabinoids since they can be extracted quickly using low energy.
De Vita et al.12 examined the use of MAE and UAE methods specifically and found that when applying the optimal conditions for each method, UAE extracted more of the acidic and neutral CBD and THC present in the plant material. Similarly, Ro&#382;anc et al.13 compared multiple methods of extraction (UAE, soxhlet, maceration, and supercritical fluid) and examined the extracts&#39; biological activity. Ro&#382;anc demonstrated that all the methods were effective at extracting cannabinoids; however supercritical fluid and UAE were most effective at extracting cannabidiolic acid (CBDA). Additionally, the UAE extraction had the highest biological activity when measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Ro&#382;anc&#39;s study also showed that while the extraction processes are effective at producing crude extracts, there remains a portion of non-cannabinoid compounds that influence the extracts&#39; biological activity. Additionally, these compounds can complicate the isolation and purification of individual cannabinoid compounds from the crude extracts13.
Supercritical fluid extraction (SFE) techniques have been used to extract neutral cannabinoids. Several studies demonstrated that SFE plus an organic solvent, such as ethanol, resulted in higher extraction efficiencies of neutral cannabinoids2,3. When the pressure was increased to levels capable of extracting the acidic cannabinoids, non-cannabinoid content also increased. As such, these high pressures are not practical for industrial processing as the selectivity of SFE for cannabinoids decreased and additional post-processing is required. Consequently, decarboxylation must be done prior to SFE, which can result in cannabinoid losses of up to 18%2. To increase efficiencies in SFE, it has been combined with techniques such as solid-phase extraction to increase the purity of the final extract14. However, despite having high purity as the final product, only neutral cannabinoids are obtained.
Traditionally, in the analytical laboratory, cannabinoids were extracted in a 9:1 methanol:chloroform mixture. However, Mudge et al.11 demonstrated that effective extraction can be carried out with single solvents when employing UAE. The study showed that 80% methanol was as effective as the traditional 9:1 methanol:chloroform extraction, thereby indicating that greener solvents can be as effective. As such, UAE was examined for its potential use due to having several benefits, including low capital cost, reduced extraction time, and lower energy use and solvent volumes. However, in the case of UAE, when polar solvents are used, chlorophyll and other non-cannabinoids can be extracted, which may cause a problem in color7. Consequently, to examine the potential for obtaining acidic cannabinoids at a commercial scale, UAE was employed using the industrial hemp variety Cherry Wine. Cherry Wine is a hybrid of C. sativa and C. indica, a cross between the varieties of The Wife and Charlotte&#39;s Cherries. The Cherry Wine varietal is a high CBDA producing strain (15% to 25% CBD) with low levels of tetrahydrocannabinolic acid (THCA). The varietal is a C. indica-dominate strain that has 7 to 9 weeks of flowering.
In order to establish the optimal UAE extraction protocol, two approaches were taken: the traditional one factor at a time (OFT) optimization and a Design of Experiment (DoE) approach using a Central Composite Design (CCD)15. For the DoE, CBDA/CBD extraction was optimized based on the sample/solvent ratio, extraction time, and solvent concentration as factors, and the resulting data was analyzed by Response Surface Methodology (RSM). In conclusion, the protocol described outlines the optimal method for extracting the highest amount of CBDA/CBD.

<table><tbody><tr><td>Acetonitrile</td><td>J.K.Baker</td><td>9017-88</td><td>solvent</td></tr><tr><td>Cannabichromene</td><td>Cerilliant</td><td>C-143</td><td>Cannabinoids standard</td></tr><tr><td>Cannabidiol</td><td>Cerilliant</td><td>C-045</td><td>Cannabinoids standard</td></tr><tr><td>Cannabidiolic acid</td><td>Cerilliant</td><td>C-144</td><td>Cannabinoids standard</td></tr><tr><td>Cannabidivarin</td><td>Cerilliant</td><td>C-140</td><td>Cannabinoids standard</td></tr><tr><td>Cannabigerol</td><td>Cerilliant</td><td>C-141</td><td>Cannabinoids standard</td></tr><tr><td>Cannabinol</td><td>Cerilliant</td><td>C-046</td><td>Cannabinoids standard</td></tr><tr><td>Centrifuge</td><td>Hanil Scientific Inc</td><td>Supra 22K</td><td>Centrifuge</td></tr><tr><td>Cherry Wine hemp</td><td>CFH, Ltd.</td><td>-</td><td>Flower extraction material</td></tr><tr><td>Distilled water</td><td>TEDIA</td><td>WS2211-001</td><td>solvent</td></tr><tr><td>Ethanol</td><td>TEDIA</td><td>ES1431-001</td><td>solvent</td></tr><tr><td>Filter paper</td><td>Whatman</td><td>#2</td><td>Filtering</td></tr><tr><td>Grinder</td><td>Daesung Artlon</td><td>DA280-S</td><td>Milling</td></tr><tr><td>HPLC</td><td>Shimadzu</td><td>LC-10 system</td><td>Analysis of Cannabinoid</td></tr><tr><td>Methanol</td><td>TEDIA</td><td>MS1922-001</td><td>solvent</td></tr><tr><td>Minitab 16.2.0</td><td>Minitab Inc.</td><td></td><td></td></tr><tr><td>Syringe filters</td><td>Whatman</td><td>6779-1304</td><td>Filtering</td></tr><tr><td>Tetrahydrocannabivarin</td><td>Cerilliant</td><td>T-094</td><td>Cannabinoids standard</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-aldrich</td><td>302031-1L</td><td>HPLC flow solvent</td></tr><tr><td>Untrasonic bath</td><td>Jinwoo</td><td>4020P</td><td>Ultrasonic extraction</td></tr><tr><td>Zorbax Eclipse plus C18 HPLC column</td><td>Agilent</td><td>9599990-902</td><td>HPLC column</td></tr><tr><td>&amp;Delta;&lt;sup&gt;8&lt;/sup&gt; - Tetrahydrocannabinol</td><td>Cerilliant</td><td>T-032</td><td>Cannabinoids standard</td></tr><tr><td>&amp;Delta;&lt;sup&gt;9&lt;/sup&gt; - Tetrahydrocannabinol</td><td>Cerilliant</td><td>T-005</td><td>Cannabinoids standard</td></tr><tr><td>&amp;Delta;&lt;sup&gt;9 &lt;/sup&gt;- Tetrahydrocannabinolic acid</td><td>Cerilliant</td><td>T-093</td><td>Cannabinoids standard</td></tr></tbody></table>

ultrasonic-assisted extraction of cannabidiolic acid from cannabis biomass

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

The solvents utilized range from the middle of the polarity index (0.460 - ACN) to polar (1.000 - water). From Table 2, it can be seen that water did not make an effective extractant for cannabinoids, which is not unexpected, as cannabinoids have limited solubility in water due to their hydrophobicity13. In contrast to water, the other solvents had similar extracted values of CBD and CBDA, with the least polar solvent acetonitrile (ACN) having higher extraction when compared to th...

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Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Ultrasonic-assisted extraction (UAE) increases extraction efficiency of solvents and when applied to Cannabis spp. biomass it reduces the time required for extraction. This decreases the cost and potential cannabinoid loss due to degradation. Additionally, UAE is considered a green method due to low solvent use.

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center ...

ultrasonic-assisted-extraction-cannabidiolic-acid-from-cannabis

Research

JoVE Journal

Biochemistry

6.0K Views.  Colorado State University-Pueblo. Dit protocol demonstreert het hele proces van CBD-extractie, te beginnen met het voorbereiden van het materiaal, oplosmiddelextractie en het meten van het CBD-gehalte. Door de geoptimaliseerde oplosmiddel- en oplosmiddelconcentratie voor te stellen, elimineert deze methode onnodige processen en kan deze helpen bij efficiënte oplosmiddelextractie voor industriële doeleinden. Begin met het verkrijgen van kersenwijn bloeiwijzen van planten die in het veld worden gekweekt.Droog de bloeiw...

Video: Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Environment

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Chemistry

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The biosynthetic pathways for cannabinoid and volatile terpene metabolism are localized primarily in the Cannabis trichomes, and isolated trichomes are beneficial for transcriptome analysis. The existing protocols for isolating glandular trichomes for transcriptomic characterization are inconvenient and deliver compromised trichome heads and a relatively low amount of isolated trichomes. Furthermore, they rely on expensive apparatus and isolation media containing protein inhibitors to avoid RNA degradation. The present protocol suggests combining three individual modifications to obtain a large amount of isolated glandular capitate stalked and sessile trichomes from C. sativa mature female inflorescences and fan leaves, respectively. The first modification involves substituting liquid nitrogen for the conventional isolation medium to facilitate the passage of trichomes through the micro-sieves. The second modification involves using dry ice to detach the trichomes from the plant source. The third modification involves passing the plant material consecutively through five micro-sieves of diminishing pore sizes. Microscopic imaging demonstrated the effectiveness of the isolation technique for both trichome types. In addition, the quality of RNA extracted from the isolated trichomes was appropriate for downstream transcriptomic analysis.

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

A protocol is presented for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The protocol is based on a dry, non-buffer extraction of trichomes using only liquid nitrogen,&#160;dry ice, and nylon sieves and is suitable for RNA extraction and transcriptomic analysis.

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from ...

Genetics

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized.
Samples were collected to represent the entire plant, frozen at -80 &#176;C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

We present a fast and reliable method to quantify vitamin C in Lactuca spp. using UPLC-UV, potentially transferable to other plants. The key steps are the sample preparation and vitamin C extraction under stable conditions, the reduction of dehydroascorbic acid to ascorbic acid and the optimization of the chromatographic procedure.

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant ...

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. &#946;-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of ...

Biodiesel Synthesis by Ultrasonic-Assisted Transesterification of Vegetable Oils

Ultrasonic-Assisted Preparation of Biodiesel Products from Vegetable Oils

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel synthesis. This alkaline-catalyzed procedure harnesses ultrasound as a potent energy input, facilitating the rapid conversion of extra virgin olive oil into biodiesel. In this demonstration, the reaction is run in an ultrasonic bath under ambient conditions for 15 min, requiring a 1:6 molar ratio of extra virgin olive oil to methanol and a minimum amount of KOH as the catalyst. The physiochemical properties of biodiesel are also reported. Emphasizing the remarkable advantages of ultrasonic-assisted transesterification, this method demonstrates notable reductions in reaction and separation times, achieving near-perfect purity (~100%), high yields, and negligible waste generation. Importantly, these benefits are achieved within a framework that prioritizes safety and environmental sustainability. These compelling findings underscore the effectiveness of this approach in converting vegetable oil into biodiesel, positioning it as a viable option for both research and practical applications.

Author Spotlight: Employing Green-Chemistry Principles for Safe and Sustainable Synthesis of Biodiesels

A safe ultrasonic-assisted transesterification method for vegetable oils using an alkaline catalyst is presented here. The method is rapid and efficient for preparing pure biodiesel products.

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

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