Role of TDZ in the Quick Regeneration of Multiple Shoots from Nodal Explant of Vitex trifolia L.—an Important Medicinal Plant

The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0???M). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with ??-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0???M BA and 0.5???M NAA. The highest shoot regeneration frequency (90?%) and maximum number (22.3?±?0.2) of shoots per explant with shoot length of (5.2?±?0.2?cm) was recorded on MS medium fortified with 5.0???M TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5???M NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.     

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0–4.0 μM) alone or in combination with IBA (0.2–0.6 μM) or 2, 4-D (0.5–1.5 μM). The optimum frequency (94 %) of callus induction was recorded on MS medium supplemented with 3.0 μM Kn and 0.4 μM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0–7.0 μM) of BA or TDZ alone or in combination with NAA (0.2–1.0 μm) was employed. The highest frequency of shoot regeneration (91 %) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 μM BA and 0.7 μM NAA. The shoots were excised and cultured on MS medium with 4.0 μM IBA produced 3.4 roots per shoot in 88 % cultures. Of the 65 plants transferred to soil 54 survived (83 %). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.     

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg?l^?1) in combination with 6-benzylaminopurine (BA; 0.8 mg?l^?1). For callus regeneration, various concentrations of BA (1.0–5.0 mg?l^?1) or thidiazuron (TDZ; 1.0–5.0 mg?l^?1) alone or in combination with indole-3-acetic acid (IAA; 0.2–1.0 mg?l^?1) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg?l^?1) and IAA (0.5 mg?l^?1) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.     

Synergistic Effects of Plant Growth Regulators and Elicitors on α-Humulene and Zerumbone Production in Zingiber zerumbet Smith Adventitious Root Cultures

Zingiber zerumbet, also known as ‘Lempoyang’, possesses various phytomedicinal properties, such as anticancer, antimicrobial, anti-inflammatory, antiulcer, and antioxidant properties. Secondary metabolites possessing such properties i.e., zerumbone and α-humulene, are found dominantly in the plant rhizome. Synergistic effects of plant growth hormones and elicitors on in vitro α-humulene and zerumbone production, and biomass growth, in adventitious root culture (AdRC) of Z. zerumbet cultivated in a two-stage culture are reported. The culture was induced by supplementation of 1.0 mg/L NAA and 2.0 mg/L IBA (dark), and subsequently maintained in medium supplemented with 1 mg/L NAA and 3 mg/L BAP (16:08 light-dark cycle), yielded the production of zerumbone at 3440 ± 168 µg/g and α-humulene at 3759 ± 798 µg/g. Synergistic elicitation by 400 μM methyl jasmonate (MeJa) and 400 μM salicylic acid (SA) resulted in a 13-fold increase in zerumbone (43,000 ± 200 µg/g), while 400 μM MeJa and 600 μM SA produced a 4.3-fold increase in α-humulene (15,800 ± 5100 µg/g) compared to control.     

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Petiole Explants of Cassia angustifolia Vahl.—a Potential Medicinal Plant

An effective protocol was developed for in vitro regeneration of the Cassia angustifolia via indirect organogenesis from petiole explants excised from 21-day-old axenic seedlings. Organogenic callus were induced on Murashige and Skoog (MS) medium supplemented with 5.0 μM 2,4-dichlorophenoxy acetic acid and 2.5 μM thidiazuron (TDZ). Adventitious shoot regeneration was achieved on MS medium supplemented with 5.0 μM TDZ as it induced 8.5 ± 0.98 shoots in 85% cultures. The number of shoots and shoot length was significantly enhanced when cultures were subcultured on auxin–cytokinin-containing medium. The highest number of shoots (12.5 ± 1.10) and shoot length (4.3 ± 0.20 cm) was recorded on MS medium supplemented with 5.0 μM TDZ and 1.5 μM indole-3-acetic acid. Regenerated shoots were rooted best on MS medium supplemented with 10.0 μM indole-3-butyric acid followed by their transfer to liquid MS filter paper bridge medium. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 70% survival rate. The plants showed normal morphological characteristics similar to the field grown plants.     

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.     

High Frequency Shoots Regeneration for Mass Multiplication of Phyllanthus fraternus Webster—An Important Antiviral and Hepatoprotective Plant

An efficient, rapid, and highly reproducible regeneration protocol was successfully developed for Phyllanthus fraternus from the field-derived mature nodal segments. The explants induced multiple shoots on cytokinin containing medium. The highest frequency (99 %) and maximum number of shoots (19.75) were induced on Murashige and Skoog’s (MS) medium supplemented with 2.22 μM 6-benzylaminopurine after 3–4 weeks of culture initiation. The elongated shoots were rooted on MS medium supplemented with indol-3-butyric acid (IBA) or α-naphthalene acetic acid. Pulse treatment of microshoots promoted significant increase in the percentage of rooting and number of root regeneration per shoot. The highest rooting (100 %) and maximum number of roots (8.75) per shoot was obtained when shoots were dipped in IBA solution (0.98 mM) for 5 min and further subcultured on MS basal medium. Plantlets were successfully acclimatized and established in soil. Regenerated plants were grown normally in the field without showing any morphological variations. This cost-effective protocol will help the mass multiplication of P. fraternus for commercial propagation and high biomass production of this valuable medicinal plant.     

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Inflorescence Explants and Peroxidase Involvement in Morphogenesis of Populus euphratica Olivier

The inflorescences as explants for rapid propagation in vitro remained unknown in Populus euphratica Olivier. Here, we reported that multiple shoots were initiation from calli of both male and female inflorescences. The optimum medium for shoot induction from male inflorescences was lactose sulfite medium containing 1.0?mg?L^?1 6-benzylaminopurine (BA) and 0.5?mg?L^?1 ??-naphthalene acetic acid (NAA) or Murashige and Skoog (MS) medium containing 0.5?mg?L^?1 BA and 0.2?mg?L^?1 NAA. The optimum medium of shoot induction from female inflorescence calli was the MS medium containing 0.5?mg?L^?1 BA and 0.2?mg?L^?1 NAA. Rooting of regenerated shoots was obtained on 1/2 MS medium supplemented with 0.5??1.0?mg?L^?1 indole-3-butyric acid (IBA) and the highest frequency rooting was on medium containing 0.5?mg?L^?1 IBA. No shoots were obtained on medium without BA and NAA. Peroxidase (POD) activity was measured by polyacrylamide gel electrophoresis during shoot induction and differentiation stages. The results showed that two bands of POD (2a and 2b) activity appeared lowest during the early 8?days at the dedifferentiation phase of leaves inducing calli, whereas POD 2a, 2b activity appeared to be increasing at the homeochronous dedifferentiation phase of inflorescence. Five most intensive bands, POD 1a, 1b, 1c, 2a, and ab, appeared in 8th and 28th days at the redifferentiation phase during shoot morphogenesis. These results demonstrated that the POD was involved in shoot morphogenesis from both leaf and inflorescence explants of Populus euphratica.     

Preconditioning of Axillary Buds in Thidiazuron-Supplemented Liquid Media Improves In Vitro Shoot Multiplication in <Emphasis Type="Italic">Nyctanthes arbor-tristis</Emphasis> L.

An efficient tissue culture technology has been designed for mass multiplication of Nyctanthes arbor-tristis L. by preculturing nodal explants in thidiazuron (TDZ)-supplemented liquid Murashige and Skoog (MS) media. Direct inoculation of nodal segments on semi-solid MS medium augmented with various concentrations of TDZ (0.1 to 0.9 μM) produced shoots but with low regeneration response and few shoots per explant. Hence, nodal explants were pretreated with greater concentrations of TDZ (5 to 100 μM) in liquid MS media for different durations (4, 8, 12, and 16 days) with the aim of improving shoot regeneration response from cultured explants. After pretreatment, explants were transferred to agar-solidified hormone-free MS medium. Best response in terms of percent regeneration (94%), number of shoots per explant (20.00 ± 1.15), and greatest shoot length (7.23 ± 0.83 cm) were obtained with nodal segments pretreated in75 μM TDZ for 8 days. Similarly, root induction was obtained from pulse-treated microshoots for 24 h with 200 μM indole-3-butyric acid (IBA) followed by their transfer to 1/2 MS medium which produced an average of 5.50 ± 0.92 roots per microshoot. The rooted plantlets were transplanted to soil with 80% success rate.     

Effect of Adenine Sulphate Interaction on Growth and Development of Shoot Regeneration and Inhibition of Shoot Tip Necrosis Under In Vitro Condition in Adult Syzygium cumini L.—a Multipurpose Tree

An efficient method for cloning Syzygium cumini (above 40 years old) through mature nodal segments has been successfully developed and that could be exploited for large-scale production of this valuable multipurpose tree. Nodal segments from mature tree were taken as explants and cultured on MS basal medium with different cytokinins (BA, Kin, AdS). The application of BA proved to be the best responsive cytokinin for the induction of shoot buds and shoots, but the proliferated shoots exhibited slower and stunted growth accompanied with abscission of leaves and shoot tip necrosis (STN). The problem of leaf abscission and STN was considerably reduced by the application of an adjuvant, adenine sulphate (AdS) in the optimal medium which led to the production of a maximum of 14 shoots. Further improvement in shoot bud regeneration and improved growth pattern of the regenerating tissue was obtained on the media comprised of MS?+?BA (10 μM)?+?GA₃ (2.5 μM). A total number of 15 shoots with mean shoot length of 5.9 cm was obtained. The healthy elongated shoots were then rooted on MS basal augmented with NAA (5 μM). The plantlets obtained were healthy and were successfully acclimatized and transferred under field condition with 70 % survival rate.     

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC₅₀ values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.     

An Efficient and Reproducible Method for in vitro Clonal Multiplication of Rauvolfia tetraphylla L. and Evaluation of Genetic Stability using DNA-Based Markers

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or ??-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90?%) and maximum number of shoot (35.4?±?2.3) per explant were observed on WPM medium supplemented with 7.5???M BA, 2.5???M NAA, and 30?g/l sucrose at pH?5.8. Well-developed shoots, 4?C5?cm in length, were successfully rooted ex vitro at 90?% by a 30-min pulse treatment with 150???M IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90?% when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.     

Effect of auxins and cytokinins on efficient plant regeneration and multiple-shoot formation from cotyledons and cotyledonary-node expiants of groundnut (Arachis hypogaea L.) by in vitro culture technology

Tissue cultures were established from cotyledon and cotyledonarynode segments ofArachis hypogaea L. on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins (IAA, NAA, IBA, and 2, 4-D) and cytokinins (KIN and BAP). For callus initiation, high concentration of auxins and low concentration of cytokinins were used, whereas high concentration of cytokinins and low concentration of auxins were used for shoot-bud differentiation. Callus induction and shoot-bud regeneration frequency, however, varied with genotype, expiant, and the different plant-growth regulators combination in the medium. The shoot-bud multiplication was also influenced by genotype, explant type, and growth regulators. The combination of BAP and NAA produced more shoots than other combinations. The maximum number of shoots was obtained from cotyledonary-node segments on a medium containing BAP (5.0 mg/L) and IBA (1.0 mg/L). Rooting of regenerated shoots was achieved on a medium augmented with NAA or IBA (2.0 mg/L) in combination with KIN (0.5 mg/L). Rooted plantlets were successfully established in the soil, where 95% of them survived. Tissue-culture studies of these expiants suggests the shoots to be ofde nova origin, which would make the system suitable for gene-transfer technology.     

In Vitro and Clinical Evaluation of Cannabigerol (CBG) Produced via Yeast Biosynthesis: A Cannabinoid with a Broad Range of Anti-Inflammatory and Skin Health-Boosting Properties

Cannabigerol (CBG) is a minor non-psychoactive cannabinoid present in Cannabis sativa L. (C. sativa) at low levels (<1% per dry weight) that serves as the direct precursor to both cannabidiol (CBD) and tetrahydrocannabinol (THC). Consequently, efforts to extract and purify CBG from C. sativa is both challenging and expensive. However, utilizing a novel yeast fermentation technology platform, minor cannabinoids such as CBG can be produced in a more sustainable, cost-effective, and timely process as compared to plant-based production. While CBD has been studied extensively, demonstrating several beneficial skin properties, there are a paucity of studies characterizing the activity of CBG in human skin. Therefore, our aim was to characterize and compare the in vitro activity profile of non-psychoactive CBG and CBD in skin and be the first group to test CBG clinically on human skin. Gene microarray analysis conducted using 3D human skin equivalents demonstrates that CBG regulates more genes than CBD, including several key skin targets. Human dermal fibroblasts (HDFs) and normal human epidermal keratinocytes (NHEKs) were exposed in culture to pro-inflammatory inducers to trigger cytokine production and oxidative stress. Results demonstrate that CBG and CBD reduce reactive oxygen species levels in HDFs better than vitamin C. Moreover, CBG inhibits pro-inflammatory cytokine (Interleukin-1β, -6, -8, tumor necrosis factor α) release from several inflammatory inducers, such as ultraviolet A (UVA), ultraviolet B (UVB), chemical, C. acnes, and in several instances does so more potently than CBD. A 20-subject vehicle-controlled clinical study was performed with 0.1% CBG serum and placebo applied topically for 2 weeks after sodium lauryl sulfate (SLS)-induced irritation. CBG serum showed statistically significant improvement above placebo for transepidermal water loss (TEWL) and reduction in the appearance of redness. Altogether, CBG’s broad range of in vitro and clinical skin health-promoting activities demonstrates its strong potential as a safe, effective ingredient for topical use and suggests there are areas where it may be more effective than CBD.     

Facial Synthesis and Bioevaluation of Well-Defined OEGylated Betulinic Acid-Cyclodextrin Conjugates for Inhibition of Influenza Infection

Betulinic acid (BA) and its derivatives exhibit a variety of biological activities, especially their anti-HIV-1 activity, but generally have only modest inhibitory potency against influenza virus. The entry of influenza virus into host cells can be competitively inhibited by multivalent derivatives targeting hemagglutinin. In this study, a series of hexa-, hepta- and octavalent BA derivatives based on α-, β- and γ-cyclodextrin scaffolds, respectively, with varying lengths of flexible oligo(ethylene glycol) linkers was designed and synthesized using a microwave-assisted copper-catalyzed 1,3-dipolar cycloaddition reaction. The generated BA-cyclodextrin conjugates were tested for their in vitro activity against influenza A/WSN/33 (H1N1) virus and cytotoxicity. Among the tested compounds, 58, 80 and 82 showed slight cytotoxicity to Madin-Darby canine kidney cells with viabilities ranging from 64 to 68% at a high concentration of 100 μM. Four conjugates 51 and 69–71 showed significant inhibitory effects on influenza infection with half maximal inhibitory concentration values of 5.20, 9.82, 7.48 and 7.59 μM, respectively. The structure-activity relationships of multivalent BA-cyclodextrin conjugates were discussed, highlighting that multivalent BA derivatives may be potential antiviral agents against influenza infection.     

Binding Interaction of Betulinic Acid to α-Glucosidase and Its Alleviation on Postprandial Hyperglycemia

Inhibiting the intestinal α-glucosidase can effectively control postprandial hyperglycemia for type 2 diabetes mellitus (T2DM) treatment. In the present study, we reported the binding interaction of betulinic acid (BA), a pentacyclic triterpene widely distributed in nature, on α-glucosidase and its alleviation on postprandial hyperglycemia. BA was verified to exhibit a strong inhibitory effect against α-glucosidase with an IC₅₀ value of 16.83 ± 1.16 μM. More importantly, it showed a synergistically inhibitory effect with acarbose. The underlying inhibitory mechanism was investigated by kinetics analysis, surface plasmon resonance (SPR) detection, molecular docking, molecular dynamics (MD) simulation and binding free energy calculation. BA showed a non-competitive inhibition on α-glucosidase. SPR revealed that it had a strong and fast affinity to α-glucosidase with an equilibrium dissociation constant (K_D) value of 5.529 × 10⁻⁵ M and a slow dissociation. Molecular docking and MD simulation revealed that BA bound to the active site of α-glucosidase mainly due to the van der Waals force and hydrogen bond, and then changed the micro-environment and secondary structure of α-glucosidase. Free energy decomposition indicated amino acid residues such as PHE155, PHE175, HIE277, PHE298, GLU302, TRY311 and ASP347 of α-glucosidase at the binding pocket had strong interactions with BA, while LYS153, ARG210, ARG310, ARG354 and ARG437 showed a negative contribution to binding affinity between BA and α-glucosidase. Significantly, oral administration of BA alleviated the postprandial blood glucose fluctuations in mice. This work may provide new insights into the utilization of BA as a functional food and natural medicine for the control of postprandial hyperglycemia.     

Synthesis,Antitumor and Antibacterial Studies of New Shortened Analogues of (KLAKLAK)2-NH2 and Their Conjugates Containing Unnatural Amino Acids

(1) Background: (KLAKLAK)₂ is a representative of the antimicrobial peptide group which also shows good anticancer properties. (2) Methods: Herein, we report synthesis using SPPS and characterization by HPLC/MS of a series of shortened analogues of (KLAKLAK)₂. They contain single sequence KLAKLAK as C-terminal amides. In addition, substitution of some natural amino acids with unnatural β-Ala and nor-Leu is realized. In addition, these structures are conjugated with second pharmacophore with well proven anticancer properties 1,8-naphthalimide or caffeic acid. Cytotoxicity, antiproliferative effect and antimicrobial activity of newly synthesized structures were studied. (3) Results: The obtained experimental results reveal significant selective index for substances with common chemical structure KLβAKLβAK-NH₂. The antibacterial properties of newly synthesized analogues at two different concentrations 10 μM and 20 μM, were tested against Gram-negative microorganisms Escherichia coli K12 407. Only two of the studied compounds KLAKLAK-NH₂ and the one conjugated with second pharmacophore 1,8-naphthalimide and unnatural amino acid nor-Leu showed moderate activity against tested strains at concentration of 20 μM. (4) Conclusions: The obtained results reveal that the introducing of 1,8-naphthalimideGly- and Caf- increase the cytotoxicity and antiproliferative activity of the peptides but not their selectivity. Only two compounds KLAKLAK-NH₂ and 1,8-naphthalimideGKnLAKnLAK-NH₂ show moderate activity against Escherichia coli K12 at low concentration of 20 μM.     

Phenolic compounds,essential oil composition,and antioxidant activity of Angelica purpurascens (Avé-Lall.) Gill

In this study, methanol extracts (MEs) and essential oil (EO) of Angelica purpurascens (Avé-Lall.) Gill obtained from different parts (root, stem, leaf, and seed) were evaluated in terms of antioxidant activity, total phenolics, compositions of phenolic compound, and essential oil with the methods of 2,2-azino-bis(3ethylbenzo-thiazoline-6-sulfonic acid (ABTS•⁺), 2,2-diphenyl-1-picrylhydrazil (DPPH•) radical scavenging activities, and ferric reducing/antioxidant power (FRAP), the Folin–Ciocalteu, liquid chromatography−tandem mass spectrometry (LC−MS/MS), and gas chromatography-mass spectrometry (GC−MS), respectively. The root extract of A. purpurascens exhibited the highest ABTS•⁺, DPPH•, and FRAP activities (IC₅₀: 0.05 ± 0.0001 mg/mL, IC₅₀: 0.06 ± 0.002 mg/mL, 821.04 ± 15.96 µM TEAC (Trolox equivalent antioxidant capacity), respectively). Moreover, EO of A. purpurascens root displayed DPPH• scavenging activity (IC₅₀: 2.95 ± 0.084 mg/mL). The root extract had the highest total phenolic content (438.75 ± 16.39 GAE (gallic acid equivalent), µg/mL)). Twenty compounds were identified by LC−MS/MS. The most abundant phenolics were ferulic acid (244.39 ± 15.64 μg/g extract), benzoic acid (138.18 ± 8.84 μg/g extract), oleuropein (78.04 ± 4.99 μg/g extract), and rutin (31.21 ± 2.00 μg/g extract) in seed, stem, root, and leaf extracts, respectively. According to the GC−MS analysis, the major components were determined as α-bisabolol (22.93%), cubebol (14.39%), α-pinene (11.63%), and α-limonene (9.41%) among 29 compounds. Consequently, the MEs and EO of A. purpurascens can be used as a natural antioxidant source.     

Secondary Metabolites from Hericium erinaceus and Their Anti-Inflammatory Activities

Hericium erinaceus, a culinary and medicinal mushroom, is widely consumed in Asian countries. Chemical investigation on the fruiting bodies of Hericium erinaceus led to the isolation of one new ergostane-type sterol fatty acid ester, erinarol K (1); and eleven known compounds: 5α,8α -epidioxyergosta-6,22-dien-3β-yl linoleate (2); ethyl linoleate (3); linoleic acid (4); hericene A (5); hericene D (6); hericene E (7); ergosta-4,6,8(14),22-tetraen-3-one (8); hericenone F (9); ergosterol (10); ergosterol peroxide (11); 3β,5α,6α,22E-ergosta-7,22-diene-3,5,6-triol 6-oleate (12). The chemical structures of the compounds were determined by 1D and 2D NMR (nuclear magnetic resonance) spectroscopy, mass spectra, etc. Anti-inflammatory effects of the isolated aromatic compounds (5–7, 9) were evaluated in terms of inhibition of pro-inflammatory mediator (TNF-α, IL-6 and NO) production in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophage cells. The results showed that compounds 5 and 9 exhibited moderate activity against TNF-α (IC₅₀: 78.50 μM and 62.46 μM), IL-6 (IC₅₀: 56.33 μM and 48.50 μM) and NO (IC₅₀: 87.31 μM and 76.16 μM) secretion. These results supply new information about the secondary metabolites of Hericium erinaceus and their anti-inflammatory effects.     

Effects of Plant Elicitors on Growth and Gypenosides Biosynthesis in Cell Culture of Giao co lam (Gynostemma pentaphyllum)

Giao co lam (Gynostemma pentaphyllum (Thunb.) Makino) is used in Northeast and Southeast Asia countries for the treatment of various diseases, including hepatitis, diabetes, and cardiovascular disease. G. pentaphyllum saponins (gypenosides) are the major components responsible for the pharmacological activities. In this study, different concentrations of abiotic (25–200 μM methyl jasmonate-MeJA and salicylic acid-SA) or biotic elicitors (1–5 g/L yeast extract-YE and Fusarium biomass) were used as plant elicitors, in order to investigate their influences on cell growth and gypenosides accumulation in G. pentaphyllum suspension cells. Suspension cells were grown on a MS medium containing 2.0 mg/L KIN and 0.5 mg/L IBA, with initial inoculum sizes of 3 g and shaking speeds of 120 rpm for 18 days. Gypenoside and Rb1 contents were measured by colorimetric and HPLC methods. Among three elicitors, SA was suitable for gypenosides accumulation in individual treatment. The cell biomass had the same values in elicitated and control suspension cells. Gypenosides content in cells treated with 100 μM salicylic acid after 6 days of culture reached a maximum value of 79.721 mg gypenoside/g dry biomass (including 0.093 mg ginsenoside Rb1/mg dry weight), which was 2.18-folds higher than that of the natural product. The elicitation promises an efficiency strategy for the production gypenosides in Gynostemma pentaphyllum suspension cells.