High-Throughput Quantitation of Cannabinoids by Liquid Chromatography Triple-Quadrupole Mass Spectrometry

The high-throughput quantitation of cannabinoids is important for the cannabis industry. As medicinal products increase, and research into compounds that have pharmacological benefits increase, and the need to quantitate more than just the main cannabinoids becomes more important. This study aims to provide a rapid, high-throughput method for cannabinoid quantitation using a liquid chromatography triple-quadrupole mass spectrometer (LC-QQQ-MS) with an ultraviolet diode array detector (UV-DAD) for 16 cannabinoids: CBDVA, CBDV, CBDA, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, THC, Δ8-THC, CBL, CBC, THCA-A and CBCA. Linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, recovery and matrix effect were all evaluated. The validated method was used to determine the cannabinoid concentration of four different Cannabis sativa strains and a low THC strain, all of which have different cannabinoid profiles. All cannabinoids eluted within five minutes with a total analysis time of eight minutes, including column re-equilibration. This was twice as fast as published LC-QQQ-MS methods mentioned in the literature, whilst also covering a wide range of cannabinoid compounds.     

Direct quantification of cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry

The first method for quantifying cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated. Solid-phase extraction followed protein precipitation with acetonitrile. High-performance liquid chromatography separation was achieved in 16 min via gradient elution. Electrospray ionization was utilized for cannabinoid detection; both positive (Δ⁹-tetrahydrocannabinol [THC] and cannabinol [CBN]) and negative (11-hydroxy-THC [11-OH-THC], 11-nor-9-carboxy-THC [THCCOOH], cannabidiol [CBD], THC-glucuronide, and THCCOOH-glucuronide) polarity were employed with multiple reaction monitoring. Calibration by linear regression analysis utilized deuterium-labeled internal standards and a 1/x ² weighting factor, yielding R ² values >0.997 for all analytes. Linearity ranged from 0.5 to 50 μg/L (THC-glucuronide), 1.0–100 μg/L (THC, 11-OH-THC, THCCOOH, CBD, and CBN), and 5.0–250 μg/L (THCCOOH-glucuronide). Imprecision was <10.5% CV, recovery was >50.5%, and bias within ±13.1% of target for all analytes at three concentrations across the linear range. No carryover and endogenous or exogenous interferences were observed. This new analytical method should be useful for quantifying cannabinoids in whole blood and further investigating cannabinoid glucuronides as markers of recent cannabis intake.     

Simultaneous quantification of cannabinoids and metabolites in oral fluid by two-dimensional gas chromatography mass spectrometry

Development and validation of a method for simultaneous identification and quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), and metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in oral fluid. Simultaneous analysis was problematic due to different physicochemical characteristics and concentration ranges. Neutral analytes, such as THC and CBD, are present in ng/mL, rather than pg/mL concentrations, as observed for the acidic THCCOOH biomarker in oral fluid. THCCOOH is not present in cannabis smoke, definitively differentiating cannabis use from passive smoke exposure. THC, 11-OH-THC, THCCOOH, CBD, and CBN quantification was achieved in a single oral fluid specimen collected with the Quantisal™ device. One mL oral fluid/buffer solution (0.25 mL oral fluid and 0.75 mL buffer) was applied to conditioned CEREX^® Polycrom™ THC solid-phase extraction (SPE) columns. After washing, THC, 11-OH-THC, CBD, and CBN were eluted with hexane/acetone/ethyl acetate (60:30:20, v/v/v), derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide and quantified by two-dimensional gas chromatography electron ionization mass spectrometry (2D-GCMS) with cold trapping. Acidic THCCOOH was separately eluted with hexane/ethyl acetate/acetic acid (75:25:2.5, v/v/v), derivatized with trifluoroacetic anhydride and hexafluoroisopropanol, and quantified by the more sensitive 2D-GCMS–electron capture negative chemical ionization (NCI-MS). Linearity was 0.5–50 ng/mL for THC, 11-OH-THC, CBD and 1–50 ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5–500 pg/mL. Intra- and inter-assay imprecision as percent RSD at three concentrations across the linear dynamic range were 0.3–6.6%. Analytical recovery was within 13.8% of target. This new SPE 2D-GCMS assay achieved efficient quantification of five cannabinoids in oral fluid, including pg/mL concentrations of THCCOOH by combining differential elution, 2D-GCMS with electron ionization and negative chemical ionization. This method will be applied to quantification of cannabinoids in oral fluid specimens from individuals participating in controlled cannabis and Sativex^® (50% THC and 50% CBD) administration studies, and during cannabis withdrawal.     

Extraction of Phenolic and Flavonoid Compounds from Mung Bean (Vigna radiata L.) Seed Coat by Pressurized Liquid Extraction

Mung bean seed coat (MBC) is a by-product of the mung bean processing industry. It contains a large number of phenolic compounds with therapeutic anti-inflammatory, anti-diabetic and antioxidant properties. This research aimed to investigate the optimum conditions for phenolic and flavonoid extraction from MBC by pressurized liquid extraction (PLE). Response surface methodology (RSM) was used to study the effects of temperature (80–160 °C), pressure (1200–1800 psi) and ethanol concentration (5–95%) on total phenolic content (TPC), total flavonoid content (TFC) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (ABTS). Scale-up extraction was also performed. The optimum conditions for extraction were 160 °C, 1300 psi and 50% ethanol. Under optimum conditions, the TPC was 55.27 ± 1.14 mg gallic acid equivalent (GAE)/g MBC, TFC was 34.04 ± 0.72 mg catechin equivalent (CE)/g MBC and ABTS scavenging activity was 195.05 ± 2.29 mg trolox equivalent (TE)/g MBC. The TFC and ABTS scavenging activity of the extracts obtained at the pilot scale (10 L) was not significantly different from the laboratory scale, while TPC was significantly increased. The freeze-dried MBC extract contained vitexin and isovitexin 130.53 ± 17.89, 21.21 ± 3.22 mg/g extract, respectively. In conclusion, PLE was able to extract phenolics, flavonoids with ABTS scavenging activity from MBC with the prospect for future scale-up for food industry.     

Response Surface Methodology (RSM)-Based Optimization of Ultrasound-Assisted Extraction of Sennoside A,Sennoside B,Aloe-Emodin,Emodin, and Chrysophanol from Senna alexandrina (Aerial Parts): HPLC-UV and Antioxidant Analysis

In this study, ultrasound-assisted extraction conditions were optimized to maximize the yields of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol from S. alexandrina (aerial parts). The three UAE factors, extraction temperature (S₁), extraction time (S₂), and liquid to solid ratio (S₃), were optimized using response surface methodology (RSM). A Box–Behnken design was used for experimental design and phytoconstituent analysis was performed using high-performance liquid chromatography-UV. The optimal extraction conditions were found to be a 64.2 °C extraction temperature, 52.1 min extraction time, and 25.2 mL/g liquid to solid ratio. The experimental values of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol (2.237, 12.792, 2.457, 0.261, and 1.529%, respectively) agreed with those predicted (2.152, 12.031, 2.331, 0.214, and 1.411%, respectively) by RSM models, thus demonstrating the appropriateness of the model used and the accomplishment of RSM in optimizing the extraction conditions. Excellent antioxidant properties were exhibited by S. alexandrina methanol extract obtained using the optimized extraction conditions with a DPPH assay (IC50 = 59.7 ± 1.93, µg/mL) and ABTS method (47.2 ± 1.40, µg/mL) compared to standard ascorbic acid.     

Supercritical carbon dioxide extraction of Usnea longissima (L.) Ach.: Optimization by Box-Behnken design (BBD)

Usnic acid (UA) was extracted from Usnea longissima (L.) Ach. in supercritical carbon dioxide (SC-CO₂) medium. The selected process parameters were extraction temperature (35–45 °C), amount of co-solvent (0%–5%) and extraction time (5–9 h). These parameters were applied to Box-Behnken design (BBD) belonging to response surface methodology (RSM) to determine optimum process parameters for the highest amount of UA in the extract. g UA/100g lichen, extraction yield % and UA content values were calculated in the range of 0.045–0.317, 2.77–5.4 and 71%–82% in different experimental conditions, respectively. The optimum conditions were predicted as 42 °C, 4.3% (ethanol) and 7.48 h. It was determined that the predicted and experimental values of g UA/100g lichen were compatible, and the suggested model was valid.     

Identification and quantification of cannabinoids in Cannabis sativa L. plants by high performance liquid chromatography-mass spectrometry

High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has been successfully applied to cannabis plant extracts in order to identify cannabinoid compounds after their quantitative isolation by means of supercritical fluid extraction (SFE). MS conditions were optimized by means of a central composite design (CCD) approach, and the analysis method was fully validated. Six major cannabinoids [tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabigerol (CBG), and cannabinol (CBN)] were quantified (RSD < 10%), and seven more cannabinoids were identified and verified by means of a liquid chromatograph coupled to a quadrupole-time-of-flight (Q-ToF) detector. Finally, based on the distribution of the analyzed cannabinoids in 30 Cannabis sativa L. plant varieties and the principal component analysis (PCA) of the resulting data, a clear difference was observed between outdoor and indoor grown plants, which was attributed to a higher concentration of THC, CBN, and CBD in outdoor grown plants. Graphical Abstract

Representative figure of the identification and quantification process of cannabinoids     

Supercritical Fluid Extraction of Phenolic Compounds from Mango (Mangifera indica L.) Seed Kernels and Their Application as an Antioxidant in an Edible Oil

Phenolic compounds from mango (M. indica) seed kernels (MSK) var. Sugar were obtained using supercritical CO₂ and EtOH as an extraction solvent. For this purpose, a central composite design was carried out to evaluate the effect of extraction pressure (11–21 MPa), temperature (40–60 °C), and co-solvent contribution (5–15% w/w EtOH) on (i) extraction yield, (ii) oxidative stability (OS) of sunflower edible oil (SEO) with added extract using the Rancimat method, (iii) total phenolics content, (iv) total flavonoids content, and (v) DPPH radical assay. The most influential variable of the supercritical fluid extraction (SFE) process was the concentration of the co-solvent. The best OS of SEO was reached with the extract obtained at 21.0 MPa, 60 °C and 15% EtOH. Under these conditions, the extract increased the OS of SEO by up to 6.1 ± 0.2 h (OS of SEO without antioxidant, Control, was 3.5 h). The composition of the extract influenced the oxidative stability of the sunflower edible oil. By SFE it was possible to obtain extracts from mango seed kernels (MSK) var. Sugar that transfer OS to the SEO. These promissory extracts could be applied to foods and other products.     

Oral fluid cannabinoid concentrations following controlled smoked cannabis in chronic frequent and occasional smokers

Oral fluid (OF) is an alternative biological matrix for monitoring cannabis intake in drug testing, and drugged driving (DUID) programs, but OF cannabinoid test interpretation is challenging. Controlled cannabinoid administration studies provide a scientific database for interpreting cannabinoid OF tests. We compared differences in OF cannabinoid concentrations from 19 h before to 30 h after smoking a 6.8 % THC cigarette in chronic frequent and occasional cannabis smokers. OF was collected with the Statsure Saliva Sampler? OF device. 2D-GC-MS was used to quantify cannabinoids in 357 OF specimens; 65 had inadequate OF volume within 3 h after smoking. All OF specimens were THC-positive for up to 13.5 h after smoking, without significant differences between frequent and occasional smokers over 30 h. Cannabidiol (CBD) and cannabinol (CBN) had short median last detection times (2.5–4 h for CBD and 6–8 h for CBN) in both groups. THCCOOH was detected in 25 and 212 occasional and frequent smokers’ OF samples, respectively. THCCOOH provided longer detection windows than THC in all frequent smokers. As THCCOOH is not present in cannabis smoke, its presence in OF minimizes the potential for false positive results from passive environmental smoke exposure, and can identify oral THC ingestion, while OF THC cannot. THC?≥?1 μg/L, in addition to CBD?≥?1 μg/L or CBN?≥?1 μg/L suggested recent cannabis intake (≤13.5 h), important for DUID cases, whereas THC?≥?1 μg/L or THC?≥?2 μg/L cutoffs had longer detection windows (≥30 h), important for workplace testing. THCCOOH windows of detection for chronic, frequent cannabis smokers extended beyond 30 h, while they were shorter (0–24 h) for occasional cannabis smokers.     

Pilot-Scale Optimization of Supercritical CO2 Extraction of Dry Paprika Capsicum annuum: Influence of Operational Conditions and Storage on Extract Composition

Supercritical carbon dioxide extraction was used to extract carotenoids from dry paprika Capsicum annuum. Studies regarding the effect of process parameters, including pressure (25–45 MPa), temperature (40–60 °C), and time (10–110 min), were carried out using response surface methodology. It was found that under optimal conditions (pressure of 45 MPa, temperature of 50 °C, and time of 74 min), the extract yield was 10.05%, and the total content of carotenoids in the extract was 4.21%, in good agreement with the predicted values (10.24% and 4.24%, respectively). Composition analysis showed that paprika extract mainly consisted of linoleic acid. There was no significant difference between the fatty acid content of the extracts obtained by SC-CO₂ extraction and n-hexane Soxhlet extraction. For functional purposes, the effect of storage conditions and time on the quality of paprika extract was also specified.     

Botanically-Derived Δ9-Tetrahydrocannabinol and Cannabidiol,and Their 1:1 Combination,Modulate Toll-like Receptor 3 and 4 Signalling in Immune Cells from People with Multiple Sclerosis

The innate immune response to bacterial and viral molecules involves the coordinated production of cytokines, chemokines, and type I interferons (IFNs), which is orchestrated by toll-like receptors (TLRs). TLRs, and their intracellular signalling intermediates, are closely associated with multiple sclerosis (MS) pathogenesis. Recent data from our laboratory reported that the plant-derived cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD), regulate viral and bacterial inflammatory signalling pathways controlled by TLR3 and TLR4 in macrophages. The aim of this study was to assess the impact of THC and CBD, when delivered in isolation and in combination (1:1), on TLR3- and TLR4-dependent signalling in peripheral blood mononuclear cells (PBMCs) from people with MS (pwMS; n = 21) and healthy controls (HCs; n = 26). We employed the use of poly(I:C) and lipopolysaccharide (LPS) to induce viral TLR3 and bacterial TLR4 signalling, and PBMCs were pre-exposed to plant-derived highly purified THC (10 μM), CBD (10 μM), or a combination of both phytocannabinoids (1:1 ratio, 10:10 μM), prior to LPS/poly(I:C) exposure. TLR3 stimulation promoted the protein expression of the chemokine CXCL10 and the type I IFN-β in PBMCs from both cohorts. THC and CBD (delivered in 1:1 combination at 10 μM) attenuated TLR3-induced CXCL10 and IFN-β protein expression in PBMCs from pwMS and HCs, and this effect was not seen consistently when THC and CBD were delivered alone. In terms of LPS, TLR4 activation promoted TNF-α expression in PBMCs from both cohorts, and, interestingly, CBD when delivered alone at 10 μM, and in combination with THC (in 1:1 combination at 10 μM), exacerbated TLR4-induced TNF-α protein expression in PBMCs from pwMS and HCs. THC and CBD displayed no evidence of toxicity in primary PBMCs. No significant alteration in the relative expression of TLR3 and TLR4 mRNA, or components of the endocannabinoid system, including the cannabinoid receptor CB₁ (encoded by CNR1 gene) and CB₂ (encoded by CNR2 gene), and endocannabinoid metabolising enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGLL), was determined in PBMCs from pwMS versus HCs. Given their role in inflammation, TLRs are clinical targets, and data herein identify CBD and THC as TLR3 and TLR4 modulating drugs in primary immune cells in vitro. This offers insight on the cellular target(s) of phytocannabinoids in targeting inflammation in the context of MS.     

Cannabis-Derived Compounds Cannabichromene and Δ9-Tetrahydrocannabinol Interact and Exhibit Cytotoxic Activity against Urothelial Cell Carcinoma Correlated with Inhibition of Cell Migration and Cytoskeleton Organization

Cannabis sativa contains more than 500 constituents, yet the anticancer properties of the vast majority of cannabis compounds remains unknown. We aimed to identify cannabis compounds and their combinations presenting cytotoxicity against bladder urothelial carcinoma (UC), the most common urinary system cancer. An XTT assay was used to determine cytotoxic activity of C. sativa extracts on T24 and HBT-9 cell lines. Extract chemical content was identified by high-performance liquid chromatography (HPLC). Fluorescence-activated cell sorting (FACS) was used to determine apoptosis and cell cycle, using stained F-actin and nuclei. Scratch and transwell assays were used to determine cell migration and invasion, respectively. Gene expression was determined by quantitative Polymerase chain reaction (PCR). The most active decarboxylated extract fraction (F7) of high-cannabidiol (CBD) C. sativa was found to contain cannabichromene (CBC) and Δ9-tetrahydrocannabinol (THC). Synergistic interaction was demonstrated between CBC + THC whereas cannabinoid receptor (CB) type 1 and type 2 inverse agonists reduced cytotoxic activity. Treatments with CBC + THC or CBD led to cell cycle arrest and cell apoptosis. CBC + THC or CBD treatments inhibited cell migration and affected F-actin integrity. Identification of active plant ingredients (API) from cannabis that induce apoptosis and affect cell migration in UC cell lines forms a basis for pre-clinical trials for UC treatment.     

A Rapid Nano-Liquid Chromatographic Method for the Analysis of Cannabinoids in Cannabis sativa L. Extracts

Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H₂O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization–mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia’s protocol on medicinal plants, were carried out, and the results achieved were compared.     

Optimising the Polyphenolic Content and Antioxidant Activity of Green Rooibos (Aspalathus linearis) Using Beta-Cyclodextrin Assisted Extraction

Antioxidant activity associated with green rooibos infusions is attributed to the activity of polyphenols, particularly aspalathin and nothofagin. This study aimed to optimise β-cyclodextrin (β-CD)-assisted extraction of crude green rooibos (CGRE) via total polyphenolic content (TPC) and antioxidant activity assays. Response surface methodology (RSM) permitted optimisation of β-CD concentration (0–15 mM), temperature (40–90 °C) and time (15–60 min). Optimal extraction conditions were: 15 mM β-CD: 40 °C: 60 min with a desirability of 0.985 yielding TPC of 398.25 mg GAE·g⁻¹, metal chelation (MTC) of 93%, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging of 1689.7 µmol TE·g⁻¹, ferric reducing antioxidant power (FRAP) of 2097.53 µmol AAE·g⁻¹ and oxygen radical absorbance capacity (ORAC) of 11,162.82 TE·g⁻¹. Aspalathin, hyperoside and orientin were the major flavonoids, with quercetin, luteolin and chrysoeriol detected in trace quantities. Differences (p < 0.05) between aqueous and β-CD assisted CGRE was only observed for aspalathin reporting the highest content of 172.25 mg·g⁻¹ of dry matter for extracts produced at optimal extraction conditions. Positive, strong correlations between TPC and antioxidant assays were observed and exhibited regression coefficient (R²) between 0.929–0.978 at p < 0.001. These results demonstrated the capacity of β-CD in increasing polyphenol content of green rooibos.     

A Pilot-Scale Supercritical Carbon Dioxide Extraction to Valorize Colombian Mango Seed Kernel

Colombian mango production, which exceeded 261,000 t in 2020, generates about 40% of the whole fruit as solid waste, of which more than 50% are seed kernels (over 52,000 t solid by-product); though none is currently used for commercial purposes. This study reports the results of the supercritical carbon dioxide (scCO₂) extraction of an oil rich in essential fatty acids (EFAs) from revalorized mango seed kernels and the optimization of the process by the Response Surface Methodology (RSM). In pilot-scale scCO₂ experiments, pressure (23–37 MPa) and temperature (52–73 °C) were varied, using 4.5 kg of CO₂. The highest experimental oil extraction yield was 83 g/kg (37 MPa and 63 °C); while RSM predicted that 84 g/kg would be extracted at 35 MPa and 65 °C. Moreover, by fine-tuning pressure and temperature it was possible to obtain an EFA-rich lipid fraction in linoleic (37 g/kg) and α-linolenic (4 g/kg) acids, along with a high oleic acid content (155 g/kg), by using a relatively low extraction pressure (23 MPa), which makes the process a promising approach for the extraction of oil from mango waste on an industrial scale, based on a circular economy model.     

Microwave-Assisted Extraction of Anticancer Flavonoid, 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethyl Chalcone (DMC), Rich Extract from Syzygium nervosum Fruits

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethyl chalcone (DMC) is a biological flavonoid that is present in the fruits of Syzygium nervosum (Ma-Kiang in Thai). Microwave-assisted extraction (MAE), which utilizes microwave radiation to heat the extraction solvent quickly and effectively, was used to recover DMC-rich extract from Syzygium nervosum fruit. To determine the DMC content, a highly accurate and precise HPLC technique was developed. The influences of MAE conditions, including the solid–liquid ratio, microwave power, and microwave duration on the content of DMC, were sequentially employed by a single factor investigation and response surface methodology (RSM) exploratory design. The predicted quadratic models were fitted due to their highly significant (p < 0.0001) and excellent determination coefficient (R² = 0.9944). The optimal conditions for producing DMC-rich extract were a ratio of sample to solvent of 1:35 g/mL, a microwave power of 350 W, and a microwave time of 38 min. Under the optimal MAE setting, the DMC content reached 1409 ± 24 µg/g dry sample, which was greater than that of the conventional heat reflux extraction (HRE) (1337 ± 37 µg/g dry sample) and maceration (1225 ± 81 µg/g dry sample). The DMC-rich extract obtained from MAE showed stronger anticancer activities against A549 (human lung cancer cells) and HepG2 (human liver cancer cells) than the individual DMC substance, which makes MAE an effective method for extracting essential phytochemicals from plants in the nature.     

Ultrasound-Assisted Extraction of Carotenoids from Carrot Pomace and Their Optimization through Response Surface Methodology

Ultrasound-assisted extraction (UAE) was used to extract carotenoids from the carrot pomace. To investigate the effect of independent variables on the UAE, the response surface methodology (RSM) with central-composite design (CCD) was employed. The study was conducted with three independent variables including extraction time (min), temperature (°C), and ethanol concentration (%). The results showed that the optimal conditions for UAE were achieved with an extraction time of 17 min, temperature of 32 °C, and ethanol concentration of 51% of total carotenoids (31.82 ± 0.55); extraction time of 16 min, temperature of 29 °C, and ethanol concentration of 59% for a combination of β-carotene (14.89 ± 0.40), lutein (5.77 ± 0.19), and lycopene (2.65 ± 0.12). The non-significant (p > 0.05) correlation under optimal extraction conditions between predicted and experimental values suggested that UAE is the more productive process than conventional techniques for the extraction of carotenoids from the carrot pomace.     

Extraction and Study of Hypoglycemic Constituents from Myrica rubra Pomace

Myrica rubra pomace accounts for 20% of the fruit’s weight that is not utilized when it is juiced. The pomace contains bioactive phenolic substances such as anthocyanins and flavonoids. To improve the utilization value of Myrica rubra pomace, an optimized extraction method for the residual polyphenols was developed using response surface methodology (RSM). The resulting extract was analyzed by high performance liquid chromatography (HPLC), and the in vitro hypoglycemic activity and antioxidant activity of the polyphenolic compounds obtained were also investigated. The optimum extraction conditions (yielding 24.37 mg·g⁻¹ total polyphenols content) were: extraction temperature 60 °C, ultrasonic power 270 W, ethanol concentration 53%, extraction time 57 min, and solid to liquid ratio 1:34. Four polyphenolic compounds were identified in the pomace extract by HPLC: myricitrin, cyanidin-O-glucoside, hyperoside, and quercitrin. DPPH and hydroxyl radical scavenging tests showed that the Myrica rubra polyphenols extract had strong antioxidant abilities. It is evident that the residual polyphenols present in Myrica rubra pomace have strong hypoglycemic activity and the juiced fruits can be further exploited for medicinal purposes.     

Study on Extraction and Antioxidant Activity of Flavonoids from Hemerocallis fulva (Daylily) Leaves

Hemerocallis fulva is a medical and edible plant. In this study, we optimized the ultrasound-assisted extraction (UAE) process of extracting flavonoids from Hemerocallis fulva leaves by single-factor experiments and response surface methodology (RSM). The optimum extraction conditions generating the maximal total flavonoids content was as follows: 70.6% ethanol concentration; 43.9:1 mL/g solvent to sample ratio; 61.7 °C extraction temperature. Under the optimized extraction conditions, the total flavonoid content (TFC) in eight Hemerocallis fulva varieties were determined, and H. fulva (L.) L. var. kwanso Regel had the highest TFC. The cytotoxicity of the extract was studied using the Cell Counting Kit-8 (CCK-8 assay). When the concentration was less than 1.25 mg/mL, the extract had no significant cytotoxicity to HaCaT cells. The antioxidant activity was measured via chemical antioxidant activity methods in vitro and via cellular antioxidant activity methods. The results indicated that the extract had a strong ABTS and •OH radical scavenging activity. Additionally, the extract had an excellent protective effect against H₂O₂-induced oxidative damage at a concentration of 1.25 mg/mL, which could effectively reduce the level of ROS to 106.681 ± 9.733% (p < 0.001), compared with the 163.995 ± 6.308% of the H₂O₂ group. We identified five flavonoids in the extracts using high-performance liquid chromatography (HPLC). Infrared spectroscopy indicated that the extract contained the structure of flavonoids. The results showed that the extract of Hemerocallis fulva leaves had excellent biocompatibility and antioxidant activity, and could be used as a cheap and potential source of antioxidants in the food, cosmetics, and medicine industries.     

Optimization of Ultrasound-Assisted Extraction of Polyphenols from Ilex latifolia Using Response Surface Methodology and Evaluation of Their Antioxidant Activity

The polyphenolic extract of Ilex latifolia (PEIL) exhibits a variety of biological activities. An evaluation of the parameters influencing the ultrasonic extraction process and the assessment of PEIL antioxidant activity are presented herein. Response surface methodology (RSM) was used to optimize the experimental conditions for the polyphenols ultrasonic-assisted extraction (UAE) from the leaves of Ilex latifolia. We identified the following optimal conditions of PEIL: ethanol concentration of 53%, extraction temperature of 60 °C, extraction time of 26 min and liquid–solid ratio of 60 mL/g. Using these parameters, the UAE had a yield of 35.77 ± 0.26 mg GAE/g, similar to the value we predicted using RSM (35.864 mg GAE/g). The antioxidant activity of PEIL was assessed in vitro, using various assays, as well as in vivo. We tested the effects of various doses of PEIL on D-galactose induced aging. Vitamin C (Vc) was used as positive control. After 21 days of administration, we measured superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, malondialdehyde (MDA) levels in mouse serum and liver tissue. The results demonstrated that the PEIL exhibits potent radical scavenging activity against 1,1-diphenyl-2-picrythydrazyl (DPPH∙), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS⁺), and hydroxyl (∙OH) radicals. The serum concentrations of SOD and GSH-Px were higher, and MDA levels were lower, in the medium- and high-dose PEIL-treated groups than those in the aging group (p < 0.01), and the activity of MDA was lower than those of the model group (p < 0.01). The liver concentrations of SOD and GSH-Px were higher (p < 0.05), and MDA levels were lower, in the medium- and high-dose PEIL-treated groups than those in the aging control group (p < 0.01). These results suggest that optimizing the conditions of UAE using RSM could significantly increase the yield of PEIL extraction. PEIL possesses strong antioxidant activity and use as a medicine or functional food could be further investigated.