Enhancement of Sesquiterpenoid Production by Methyl Jasmonate in Atractylodes chinensis Adventitious Root Culture and its Transcriptional Regulation.

The adventitious root (AR) culture of Atractylodes chinensis is an efficient platform for sustainable production of its sesquiterpenoid compounds (atractylon and ß-eudesmol). However, their limited accumulation levels need an effective elicitation approach, and the present study solved this problem using methyl jasmonate (MeJA) as an elicitor. The effects of its treatment concentration and duration on metabolite production were investigated. The ARs treated with 100 µM MeJA for seven days increased atractylon and ß-eudesmol by 3.64- and 1.90-fold, respectively, compared with the control. This study further performed transcriptome analysis to explore the transcriptional regulation mechanism of the MeJA elicitation. A total of 124,464 unigenes were identified in A. chinensis ARs, of which 3,568 genes were upregulated and 3,864 genes were downregulated under the MeJA treatment. The MeJA treatment activated the endogenous JA biosynthesis and signaling pathways and sesquiterpenoid biosynthesis. The MeJA treatment more significantly activated the MEP pathway than the MVA pathway. In addition, 14 genes encoding terpene synthase were identified to be significantly upregulated. A total of 2,700 transcription factors (TFs) were identified in A. chinensis ARs, of which Tify, MYB, and MADS were significantly enriched under the MeJA treatment. We predicted a new antagonistic interaction between MYC2 and CPP TFs, which was significantly regulated by the MeJA treatment. The results of real-time quantitative PCR and enzyme activity assays proved the reliability of the transcriptome data. This study will help improve the in vitro production system of A. chinensis sesquiterpenoids and understand the transcriptional regulation mechanism of MeJA elicitation.

Melatonin alleviates UV-B stress and enhances phenolic biosynthesis in rosemary (Rosmarinus officinalis) callus.

Although used in in vitro culture to boost secondary metabolite production, UV-B radiation can seriously affect plant growth if not properly dosed. Rosemary callus can be used as an important source of effective ingredients in the food and medicine industry. To balance the positive and negative effects of UV-B on rosmary callus, this study investigated the effects of melatonin on rosemary callus under UV-B radiation. The results showed that melatonin improved rosemary callus growth, with fresh weight and dry weight increased by 15.81% and 8.30%, respectively. The addition of 100 µM melatonin increased antioxidant enzyme activity and NO content in rosemary callus. At the same time, melatonin also significantly reduced membrane lipid damage and H2O2 accumulation in rosemary callus under UV-B stress, with malondialdehyde (MDA) and H2O2 contents reduced by 13.03% and 14.55%, respectively. In addition, melatonin increased the total phenol and rosmarinic acid contents in rosemary callus by 19% and 54%, respectively. Melatonin significantly improved the antioxidant activity of the extracts from rosemary callus. These results suggest that exogenous melatonin can alleviate the adverse effects of UV-B stress on rosemary callus by promoting NO accumulation while further enhancing phenolic accumulation and biological activity.

Melatonin enhances biomass, phenolic accumulation, and bioactivities of rosemary (Rosmarinus officinalis) in vitro shoots under UV-B stress.

Melatonin is a stress-related hormone that plays a critical role in triggering the plant defence system and regulating secondary metabolism when plants are exposed to stress. To explore the potential roles of melatonin in response to Ultraviolet-B (UV-B) radiation, we examined the effects of exogenous melatonin on rosemary in vitro shoots under UV-B stress. The application of melatonin (50 µM) alleviated the adverse effects of UV-B stress on the biomass, photosynthetic pigment contents, and membrane lipids of the rosemary in vitro shoots. Melatonin significantly increased superoxide dismutase (1.15.1.1, SOD), peroxidase (1.11.1.7, POD), and catalase (1.11.1.6, CAT) activities by 62%, 99%, and 53%, respectively. The contents of total phenols, rosmarinic acid, and carnosic acid increased under UV-B stress, and they further increased by the melatonin treatment by 41%, 68%, and 67%, respectively, compared with the control group. Under UV-B stress, the increased total phenol content in melatonin-pretreated plants could be attributed to the activation of phenylalanine ammonia-lyase (4.3.1.5, PAL) and tyrosine aminotransferase (2.6.1.5, TAT). In addition, melatonin enhanced the antioxidant and antibacterial activities of the rosemary in vitro shoots under UV-B stress. These results suggest that melatonin can alleviate the damage caused by UV-B stress and also enhance the secondary metabolism and bioactivity of rosemary in vitro shoots.

How does UV-B stress affect secondary metabolites of Scutellaria baicalensis in vitro shoots grown at different 6-benzyl aminopurine concentrations?

Ultraviolet-B (UV-B) radiation is one of the abiotic stresses that can significantly affect the secondary metabolite accumulation in in vitro tissue cultures of medicinal plants. The present study investigated the effects of UV-B radiation on the secondary metabolites and antioxidant activities of Scutellaria baicalensis in vitro shoots grown at different concentrations of 6-benzyl aminopurine (6-BA), which is the cytokinin most widely used in plant tissue culture. The UV-B radiation caused significant increases in lipid peroxidation, total phenolic, and flavonoid contents, and antioxidant activities in the in vitro shoots grown at lower 6-BA concentrations (0 and 1 mg L-1 ), while it did not cause any significant changes in those grown at higher 6-BA concentrations (2 and 3 mg L-1 ). However, the UV-B radiation significantly altered the contents of main individual flavonoids at both lower and higher 6-BA concentrations. Upon UV-B radiation, aglycones (including baicalein, wogonin, and scutellarein) increased, while glucuronides such as baicalin and wogonoside decreased; this was more evident at higher 6-BA concentrations. This study demonstrated that the effects of UV-B radiation on the secondary metabolites of S. baicalensis in vitro shoots highly depended on the 6-BA concentration in the culture medium.

In vitro production of atractylon and ß-eudesmol from Atractylodes chinensis by adventitious root culture.

Atractylodes chinensis is a medicinal plant widely used for the treatment of gastric disorders, and its main bioactive compounds are atractylon and ß-eudesmol. This study was purposed to establish the adventitious root culture system of A. chinensis for in vitro production of atractylon and ß-eudesmol. The main parameters in the adventitious root induction and suspension cultures were optimized to maximize the culture efficiency. Adventitious roots were induced most efficiently from leaf explants on Murashige and Skoog (MS) solid medium containing 1.5 mg/L naphthaleneacetic acid (NAA) and 30 g/L sucrose with the highest root induction rate of approximately 92% and 12.9 roots per explant. During the adventitious root suspension culture, the root biomass and the accumulated content of the target compounds simultaneously increased to reach the maximum values after 8 weeks of culture. The maximum yield of the target compounds (total concentration 3.38 mg/g DW, total yield 2.66 mg) was achieved in the roots cultured in ½ MS liquid medium supplemented with 2.0 mg/L IBA, 3.2 mg/L NAA, and 40 g/L sucrose with the inoculum density of 8 g/L. Through the central composite design experiment, it was found that the combined use of different types of auxins in the suspension culture could further improve root growth and metabolite accumulation than the application of only one type of auxin. This work provides a new possibility to have a promising candidate for the industrial production of A. chinensis pharmaceuticals without relying on wild resources or field cultivation. KEY POINTS: • The induction culture was optimized for efficient root induction. • Suspension culture was optimized for the atractylon and ß-eudesmol production. • Combined use of different auxins improves root growth and metabolite accumulation.

Ultrasound-assisted enzymatic extraction of Scutellaria baicalensis root polysaccharide and its hypoglycemic and immunomodulatory activities.

Scutellaria baicalensis is a medicinal plant possessing abundant bioactive polysaccharides. This study aimed to optimize the ultrasound-assisted enzymatic extraction of S. baicalensis root polysaccharide (SRP) and investigate its hypoglycemic and immunomodulatory activities. The optimal extraction conditions found in this study were as follows: cellulase concentration 165.6 U/mL, temperature 57.3 °C, liquid-solid ratio 44.8 mL/g, time 50 min, and ultrasonic power 225 W; with the yield reached up to 12.27 %. The ion exchange and gel filtration chromatographies were used to obtain a purified SRP. The carbohydrate content of SRP was 85.09 %, with a relatively high content of uronic acids (11.27 %). The SRP had a molecular weight of 89.7 kDa and was composed of eight monosaccharides. The inhibitory activity of SRP against α-amylase and α-glucosidase was determined. It was revealed that SRP could effectively inhibit these two enzymes with IC50 values of 1.23 and 0.63 mg/mL, respectively. Finally, the immunomodulatory effect of SRP on the dendritic cell activation was investigated, and the expressions of MHC II, CD80, CD86, and CD40 increased by 1.56, 1.96, 1.75, and 1.70 times, respectively, by the SRP treatment. This work will provide a foundation for SRP's efficient extraction and utilization for diabetes and immune therapy.

Optimization of ultrasound-assisted enzymatic pretreatment for enhanced extraction of baicalein and wogonin from Scutellaria baicalensis roots.

It is of great theoretical interest and industrial significance to improve the extraction efficiency of baicalein and wogonin from Scutellaria baicalensis roots because of their high pharmacological activities. The present study was aimed to establish the optimized ultrasound-assisted enzymatic pretreatment (UAEP) process by which ultrasound irradiation and the exogenous enzyme were simultaneously applied to efficiently transform baicalin and wogonoside into baicalein and wogonin, enhancing their extraction efficiency. Single-factor experiment and Box-Behnken design were used to optimize the main UAEP conditions to maximize the total extraction yield of baicalein and wogonin. The optimized UAEP conditions were cellulase concentration of 1.1%, pH of 5.5, UAEP temperature of 56.5 °C, UAEP time of 39.4 min, and ultrasonic power of 200 W with the total extraction yield of 82.51 ± 0.85 mg/g DW. The comparison of the established technique with the reference method based on the enzymatic pretreatment revealed that the productive efficiency was significantly improved with the transformation rates nearly doubled. These results suggest that the optimized UAEP process has the potential to be applied for the green, simple, and efficient extraction of baicalein and wogonin in the pharmaceutical and food industry.

Optimized ultrasound-assisted extraction of total polyphenols from Empetrum nigrum and its bioactivities.

Optimization of ultrasound-assisted extraction (UAE) of total polyphenols (TPP) from Empetrum nigrum aerial parts was carried out by response surface methodology (RSM). The optimum UAE conditions of extraction time, extraction temperature, ethanol concentration, and solvent-to-material ratio were 21.38 min, 42.32 °C, 61.93% and 53.29:1 mL/g, respectively. Under the optimum conditions, the extraction yield of TPP was 32.17 ± 0.46 mg/g, which was 1.29-1.44 folds to those by the conventional extraction methods. In addition, the bioactivities of the extracts were investigated. Antioxidant activity test by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay revealed that the TPP extracts had a high potential for free radical scavenging activity. The TPP extracts showed remarkable antibacterial activity against both Gram-positive and Gram-negative strains, especially against Gram-positive strains. The evaluation of antitumor activity by the MTT assay and flow cytometric analysis indicated that the TPP extracts significantly inhibited B 16F 10 melanoma cell proliferation and effectively induced apoptosis of melanoma cells. These results demonstrate that E. nigrum aerial parts are rich in TPP and show great application potential in the pharmaceutical industry.