Chimaphila umbellata; a biotechnological perspective on the coming-of-age prince's pine.

Chimaphila umbellata has been studied for almost two centuries now, with the first paper exploring the phytochemistry of the plant published in 1860. Almost all contemporary studies focus on the biotechnological advances of C. umbellata including its utilization as a natural alternative in the cosmetic, food, biofuel, and healthcare industry, with a special focus on its therapeutic uses. This literature review critically investigates the significance and applications of secondary metabolites extracted from the plant and presses on the biotechnological approaches to improve its utilization. C. umbellata is home to many industrially and medicinally important phytochemicals, the majority of which belong to phenolics, sterols, and triterpenoids. Other important compounds include 5-hydroxymethylfurfural, isohomoarbutin, and methyl salicylate (the only essential oil of the plant). Chimaphilin is the characteristic phytochemical of the plant. This review focuses on the phytochemistry of C. umbellata and digs into their chemical structures and attributes. It further discusses the challenges of working with C. umbellata including its alarming conservation status, problems with in-vitro cultivation, and research and development issues. This review concludes with recommendations based on biotechnology, bioinformatics, and their crucial interface.

Effect of Wide-Spectrum Monochromatic Lights on Growth, Phytochemistry, Nutraceuticals, and Antioxidant Potential of In Vitro Callus Cultures of Moringa oleifera.

Moringa oleifera, also called miracle tree, is a pharmaceutically important plant with a multitude of nutritional, medicinal, and therapeutic attributes. In the current study, an in-vitro-based elicitation approach was used to enhance the commercially viable bioactive compounds in an in vitro callus culture of M. oleifera. The callus culture was established and exposed to different monochromatic lights to assess the potentially interactive effects on biomass productions, biosynthesis of pharmaceutically valuable secondary metabolites, and antioxidant activity. Optimum biomass production (16.7 g/L dry weight), total phenolic contents (TPC: 18.03 mg/g), and flavonoid contents (TFC: 15.02 mg/g) were recorded in callus cultures placed under continuous white light (24 h), and of other light treatments. The highest antioxidant activity, i.e., ABTS (550.69 TEAC µM) and FRAP (365.37 TEAC µM), were also noted under white light (24 h). The analysis of phytochemicals confirmed the significant impact of white light exposures on the enhanced biosynthesis of plant secondary metabolites. The enhanced levels of secondary metabolites, i.e., kaempferol (1016.04 µg/g DW), neochlorogenic acid (998.38 µg/g DW), quercetin (959.92 µg/g DW), and minor compounds including luteolin, apigenin, and p-coumaric acid were observed as being highest in continuous white light (24 h with respect to the control (photoperiod). Similarly, blue light enhanced the chlorogenic acid accumulation. This study shows that differential spectral lights demonstrate a good approach for the enhancement of nutraceuticals along with novel pharmacologically important metabolites and antioxidants in the in vitro callus culture of M. oleifera.

Light Tailoring: Impact of UV-C Irradiation on Biosynthesis, Physiognomies, and Clinical Activities of Morus macroura-Mediated Monometallic (Ag and ZnO) and Bimetallic (Ag-ZnO) Nanoparticles.

A nano-revolution based on the green synthesis of nanomaterials could affect all areas of human life, and nanotechnology represents a propitious platform for various biomedical applications. During the synthesis of nanoparticles, various factors can control their physiognomies and clinical activities. Light is one of the major physical factors that can play an important role in tuning/refining the properties of nanoparticles. In this study, biocompatible monometallic (AgNPs and ZnONPs) and bimetallic Ag-ZnONPs (0.1/0.1 and 0.1/0.5) were synthesized under UV-C light irradiation from the leaf extract of Morus macroura, which possesses enriched TPC (4.238 ± 0.26 mg GAE/g DW) and TFC (1.073 ± 0.18 mg QE/g DW), as well as strong FRSA (82.39%). These green synthesized NPs were evaluated for their anti-diabetic, anti-glycation, and biocompatibility activities. Furthermore, their anti-cancerous activity against HepG2 cell lines was assessed in terms of cell viability, production of reactive oxygen/nitrogen species, mitochondrial membrane potential, and apoptotic caspase-3/7 expression and activity. Synthesized NPs were characterized by techniques including ultraviolet-visible spectroscopy, SEM, EDX, FTIR, and XRD. UV-C mediated monometallic and bimetallic NPs showed well-defined characteristic shapes with a more disperse particle distribution, definite crystalline structures, and reduced sizes as compared to their respective controls. In the case of clinical activities, the highest anti-diabetic activity (67.77 ± 3.29% against α-amylase and 35.83 ± 2.40% against α-glucosidase) and anti-glycation activity (37.68 ± 3.34% against pentosidine-like AGEs and 67.87 ± 2.99% against vesperlysine-like AGEs) was shown by UV-C mediated AgNPs. The highest biocompatibility (IC50 = 14.23 ± 1.68 µg/mL against brine shrimp and 2.48 ± 0.32% hemolysis of human red blood cells) was shown by UV-C mediated ZnONPs. In the case of anti-cancerous activities, the lowest viability (23.45 ± 1.40%) with enhanced ROS/NOS production led to a significant disruption of mitochondrial membrane potential and greater caspase-3/7 gene expression and activity by UV-C mediated bimetallic Ag-ZnONPs (0.1/0.5). The present work highlights the positive effects of UV-C light on physico-chemical physiognomies as well as the clinical activities of NPs.

Chitosan Elicitation Impacts Flavonolignan Biosynthesis in Silybum marianum (L.) Gaertn Cell Suspension and Enhances Antioxidant and Anti-Inflammatory Activities of Cell Extracts.

Silybum marianum (L.) Gaertn is a rich source of antioxidants and anti-inflammatory flavonolignans with great potential for use in pharmaceutical and cosmetic products. Its biotechnological production using in vitro culture system has been proposed. Chitosan is a well-known elicitor that strongly affects both secondary metabolites and biomass production by plants. The effect of chitosan on S. marianum cell suspension is not known yet. In the present study, suspension cultures of S. marianum were exploited for their in vitro potential to produce bioactive flavonolignans in the presence of chitosan. Established cell suspension cultures were maintained on the same hormonal media supplemented with 0.5 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L NAA (α-naphthalene acetic acid) under photoperiod 16/8 h (light/dark) and exposed to various treatments of chitosan (ranging from 0.5 to 50.0 mg/L). The highest biomass production was observed for cell suspension treated with 5.0 mg/L chitosan, resulting in 123.3 ± 1.7 g/L fresh weight (FW) and 17.7 ± 0.5 g/L dry weight (DW) productions. All chitosan treatments resulted in an overall increase in the accumulation of total flavonoids (5.0 ± 0.1 mg/g DW for 5.0 mg/L chitosan), total phenolic compounds (11.0 ± 0.2 mg/g DW for 0.5 mg/L chitosan) and silymarin (9.9 ± 0.5 mg/g DW for 0.5 mg/L chitosan). In particular, higher accumulation levels of silybin B (6.3 ± 0.2 mg/g DW), silybin A (1.2 ± 0.1 mg/g DW) and silydianin (1.0 ± 0.0 mg/g DW) were recorded for 0.5 mg/L chitosan. The corresponding extracts displayed enhanced antioxidant and anti-inflammatory capacities: in particular, high ABTS antioxidant activity (741.5 ± 4.4 µM Trolox C equivalent antioxidant capacity) was recorded in extracts obtained in presence of 0.5 mg/L of chitosan, whereas highest inhibitions of cyclooxygenase 2 (COX-2, 30.5 ± 1.3 %), secretory phospholipase A2 (sPLA2, 33.9 ± 1.3 %) and 15-lipoxygenase (15-LOX-2, 31.6 ± 1.2 %) enzymes involved in inflammation process were measured in extracts obtained in the presence of 5.0 mg/L of chitosan. Taken together, these results highlight the high potential of the chitosan elicitation in the S. marianum cell suspension for enhanced production of antioxidant and anti-inflammatory silymarin-rich extracts.

Elicitation of Submerged Adventitious Root Cultures of Stevia rebaudiana with Cuscuta reflexa for Production of Biomass and Secondary Metabolites.

Stevia rebaudiana is an important medicinal plant that belongs to the Asteraceae family. The leaves of Stevia rebaudiana are a rich source of many health-promoting agents such as polyphenols, flavonoids, and steviol glycoside, which play a key role in controlling obesity and diabetes. New strategies such as the elicitation of culture media are needed to enhance the productivity of active components. Herein, the Cuscuta reflexa extracts were exploited as elicitors to enhance the productivity of active components. Cuscuta reflexa is one of the parasitic plants that has the ability to elongate very fast and cover the host plant. Consequently, it may be possible that the addition of Cuscuta reflexa extracts to adventitious root cultures (ADR) of Stevia rebaudiana may elongate the root more than control cultures to produce higher quantities of the desired secondary metabolites. Therefore, the main objective of the current study was to investigate the effect of Cuscuta reflexa extract as a biotic elicitor on the biomass accumulation and production of antioxidant secondary metabolite in submerged adventitious root cultures of Stevia rebaudiana. Ten different concentrations of Cuscuta reflexa were added to liquid media containing 0.5 mg/L naphthalene acetic acid (NAA). The growth kinetics of adventitious roots was investigated for a period of 49 days with an interval of 7 days. The maximum biomass accumulation (7.83 g/3 flasks) was observed on medium containing 10 mg/L extract of Cuscuta reflexa on day 49. As the concentration of extract increases in the culture media, the biomass gradually decreases after 49 days of inoculation. In this study, the higher total phenolics content (0.31 mg GAE/g-DW), total flavonoids content (0.22 mg QE/g-DW), and antioxidant activity (85.54%) were observed in 100 mg/L treated cultures. The higher concentration (100 mg/L) of Cuscuta reflexa extract considerably increased the total phenolics content (TPC), total phenolics production (TPP), total flavonoids content (TFC), total flavonoids production (TFP), total polyphenolics content (TPPC), and total polyphenolics production (TPPP). It was concluded that the extract of Cuscuta reflexa moderately improved biomass accumulation but enhanced the synthesis of phenolics, flavonoids, and antioxidant activities. Here, biomass's independent production of secondary metabolites was observed with the addition of extract. The present study will be helpful to scale up adventitious roots culture into a bioreactor for the production of secondary metabolites rather than biomass accumulation in medicinally important Stevia rebaudiana.

Chemical Elicitors-Induced Variation in Cellular Biomass, Biosynthesis of Secondary Cell Products, and Antioxidant System in Callus Cultures of Fagonia indica.

Fagonia indica is a rich source of pharmacologically active compounds. The variation in the metabolites of interest is one of the major issues in wild plants due to different environmental factors. The addition of chemical elicitors is one of the effective strategies to trigger the biosynthetic pathways for the release of a higher quantity of bioactive compounds. Therefore, this study was designed to investigate the effects of chemical elicitors, aluminum chloride (AlCl3) and cadmium chloride (CdCl2), on the biosynthesis of secondary metabolites, biomass, and the antioxidant system in callus cultures of F. indica. Among various treatments applied, AlCl3 (0.1 mM concentration) improved the highest in biomass accumulation (fresh weight (FW): 404.72 g/L) as compared to the control (FW: 269.85 g/L). The exposure of cultures to AlCl3 (0.01 mM) enhanced the accumulation of secondary metabolites, and the total phenolic contents (TPCs: 7.74 mg/g DW) and total flavonoid contents (TFCs: 1.07 mg/g DW) were higher than those of cultures exposed to CdCl2 (0.01 mM) with content levels (TPC: 5.60 and TFC: 0.97 mg/g) as compared to the control (TPC: 4.16 and TFC: 0.42 mg/g DW). Likewise, AlCl3 and CdCl2 also promoted the free radical scavenging activity (FRSA; 89.4% and 90%, respectively) at a concentration of 0.01 mM, as compared to the control (65.48%). For instance, the quantification of metabolites via high-performance liquid chromatography (HPLC) revealed an optimum production of myricetin (1.20 mg/g), apigenin (0.83 mg/g), isorhamnetin (0.70 mg/g), and kaempferol (0.64 mg/g). Cultures grown in the presence of AlCl3 triggered higher quantities of secondary metabolites than those grown in the presence of CdCl2 (0.79, 0.74, 0.57, and 0.67 mg/g). Moreover, AlCl3 at 0.1 mM enhanced the biosynthesis of superoxide dismutase (SOD: 0.08 nM/min/mg-FW) and peroxidase enzymes (POD: 2.37 nM/min/mg-FW), while CdCl2 resulted in an SOD activity up to 0.06 nM/min/mg-FW and POD: 2.72 nM/min/mg-FW. From these results, it is clear that AlCl3 is a better elicitor in terms of a higher and uniform productivity of biomass, secondary cell products, and antioxidant enzymes compared to CdCl2 and the control. It is possible to scale the current strategy to a bioreactor for a higher productivity of metabolites of interest for various pharmaceutical industries.

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.

Callus Culture of Thai Basil Is an Effective Biological System for the Production of Antioxidants.

Thai basil is a renowned medicinal plant and a rich source of bioactive antioxidant compounds with several health benefits, with actions to prevent of cancer, diabetes and cardiovascular disease. Plant cell and tissue culture technologies can be routinely established as an important, sustainable and low-cost biomass source to produce high-value phytochemicals. The current study aimed at developing an effective protocol to produce Thai basil leaf-derived callus cultures with sustainable and high production of biomass and antioxidants as an alternative of leaves production. MS basal medium with various concentrations of plant growth regulators (PGRs) compatible with nutraceutical applications (i.e., gibberellic acid (GA3) and 6-benzylaminopurine (BAP) either alone or in combination with naphthalene acetic acid (NAA)) were evaluated. Among all tested PGRs, the combination BAP:NAA (5 mg/L:1 mg/L) yields the maximum biomass accumulation (fresh weight (FW): 190 g/L and dry weight (DW): 13.05 g/L) as well as enhanced phenolic (346.08 mg/L) production. HPLC quantification analysis indicated high productions of chicoric acid (35.77 mg/g DW) and rosmarinic acid (7.35 mg/g DW) under optimized callus culture conditions. Antioxidant potential was assessed using both in vitro cell free and in vivo cellular antioxidant assays. Maximum in vitro antioxidant activity DPPH (93.2% of radical scavenging activity) and ABTS (1322 µM Trolox equivalent antioxidant capacity) was also observed for the extracts from callus cultures grown in optimal conditions. In vivo cellular antioxidant activity assay confirmed the effective protection against oxidative stress of the corresponding extract by the maximum inhibition of ROS and RNS production. Compared to commercial leaves, callus extracts showed higher production of chicoric acid and rosmarinic acid associated with higher antioxidant capacity. In addition, this biological system also has a large capacity for continuous biomass production, thus demonstrating its high potential for possible nutraceutical applications.

Interactive Effect of Melatonin and UV-C on Phenylpropanoid Metabolite Production and Antioxidant Potential in Callus Cultures of Purple Basil (Ocimum basilicum L. var.s purpurascens).

The present study evaluated the interactive effect of melatonin and UV-C on phenylpropanoid metabolites profile and antioxidant potential of Ocimum basilicum L. Callus was treated with varying concentrations of melatonin and UV-C radiations for different time durations, either alone and/or in combination. Individual treatments of both UV-C and melatonin proved to be more effective than combine treatments. Results indicated that UV-C (10 min) exposure increased rosmarinic acid (134.5 mg/g dry weight (DW)), which was 2.3-fold greater than control. Chichoric acid (51.52 mg/g DW) and anthocyanin (cyanide 0.50 mg/g DW) were almost 4.1-fold, while peonidin was found 2.7-fold higher in UV-C (50 min) exposure. In the case of melatonin, 1.0 mg/L concentrations showed maximum rosmarinic acid (79.4 mg/g DW) accumulation; i.e., 1.4-fold more, as compared to the control. However, 2 mg/L melatonin accumulate chichoric acid (39.99 mg/g DW) and anthocyanin (cyanide: 0.45 mg/g DW and peonidin: 0.22 mg/g DW); i.e., 3.2, 3.7 and 2.0-fold increase, as compared to the control, respectively. On the other hand, melatonin-combined treatment (melatonin (Mel) (4 mg/L) + UV-C (20 min)) was proved to be effective in caffeic acid elicitation, which was 1.9-fold greater than the control. Furthermore, antioxidant potential was evaluated by both in vitro (DPPH, ABTS and FRAP assays) and in cellulo methods. Maximum in vitro antioxidant activity (DPPH: 90.6% and ABTS: 1909.5 µM) was observed for UV-C (50 min)-treated cultures. The highest in vitro antioxidant activity measured with the ABTS assay as compared to the FRAP assay, suggesting the main contribution of antioxidants from basil callus extracts acting through a hydrogen atom transfer (HAT) over an electron transfer (ET)-based mechanism. Cellular antioxidant assay was evaluated by production of ROS/RNS species using yeast cell cultures and further confirmed the protective action of the corresponding callus extracts against oxidative stress. Overall, both melatonin and UV-C are here proved to be effective elicitors since a positive correlation between the induced production of phenolic compounds, and in cellulo antioxidant action of basil callus extracts were observed.

Interactive Effects of Wide-Spectrum Monochromatic Lights on Phytochemical Production, Antioxidant and Biological Activities of Solanum xanthocarpum Callus Cultures.

Solanum xanthocarpum is considered an important traditional medicinal herb because of its unique antioxidant, and anti-diabetic, anti-aging, and anti-inflammatory potential. Because of the over exploitation linked to its medicinal properties as well as destruction of its natural habitat, S. xanthocarpum is now becoming endangered and its supply is limited. Plant in vitro culture and elicitation are attractive alternative strategies to produce biomass and stimulate biosynthesis of medicinally important phytochemicals. Here, we investigated the potential influence of seven different monochromatic light treatments on biomass and secondary metabolites accumulation in callus culture of S. xanthocarpum as well as associated biological activities of the corresponding extracts. Among different light treatments, highest biomass accumulation was observed in white light-treated callus culture. Optimum accumulation of total flavonoid contents (TFC) and total phenolic contents (TPC) were observed in callus culture kept under continuous white and blue light respectively than control. Quantification of phytochemicals through HPLC revealed that optimum production of caffeic acid (0.57 ± 0.06 mg/g DW), methyl-caffeate (17.19 mg/g ± 1.79 DW), scopoletin (2.28 ± 0.13 mg/g DW), and esculetin (0.68 ± 0.07 mg/g DW) was observed under blue light callus cultures. Compared to the classic photoperiod condition, caffeic acid, methyl-caffeate, scopoletin, and esculetin were accumulated 1.7, 2.5, 1.1, and 1.09-folds higher, respectively. Moreover, high in vitro cell free antioxidant, anti-diabetic, anti-aging, and anti-inflammatory activities were closely associated with the production of these secondary metabolites. These results clearly showed the interest to apply multispectral light as elicitor of in vitro callus cultures S. xanthocarpum to promote the production of important phytochemicals, and allow us to propose this system as an alternative for the collection of this endangered species from the wild.

Effect of Ultraviolet-C Radiation and Melatonin Stress on Biosynthesis of Antioxidant and Antidiabetic Metabolites Produced in In Vitro Callus Cultures of Lepidium sativum L.

Lepidium sativum L. is a rich source of polyphenols that have huge medicinal and pharmaceutical applications. In the current study, an effective abiotic elicitation strategy was designed for enhanced biosynthesis of polyphenols in callus culture of L. sativum. Callus was exposed to UV-C radiations for different time intervals and various concentrations of melatonin. Secondary metabolites were quantified by using high-performance liquid chromatography (HPLC). Results indicated the total secondary metabolite accumulation of nine quantified compounds was almost three fold higher (36.36 mg/g dry weight (DW)) in melatonin (20 µM) treated cultures, whereas, in response to UV-C (60 min), a 2.5 fold increase (32.33 mg/g DW) was recorded compared to control (13.94 mg/g DW). Metabolic profiling revealed the presence of three major phytochemicals, i.e., chlorogenic acid, kaemferol, and quercetin, in callus culture of L. sativum. Furthermore, antioxidant, antidiabetic, and enzymatic activities of callus cultures were significantly enhanced. Maximum antidiabetic activities (α-glucosidase: 57.84%; α-amylase: 62.66%) were recorded in melatonin (20 µM) treated callus cultures. Overall, melatonin proved to be an effect elicitor compared to UV-C and a positive correlation in these biological activities and phytochemical accumulation was observed. The present study provides a better comparison of both elicitors and their role in the initiation of physiological pathways for enhanced metabolites biosynthesis in vitro callus culture of L. sativum.

Isodon rugosus (Wall. ex Benth.) Codd In Vitro Cultures: Establishment, Phytochemical Characterization and In Vitro Antioxidant and Anti-Aging Activities.

Isodon rugosus (Wall. ex Benth.) Codd accumulates large amounts of phenolics and pentacyclic triterpenes. The present study deals with the in vitro callus induction from stem and leaf explants of I. rugosus under various plant growth regulators (PGRs) for the production of antioxidant and anti-ageing compounds. Among all the tested PGRs, thidiazuron (TDZ) used alone or in conjunction with α-napthalene acetic acid (NAA) induced highest callogenesis in stem-derived explants, as compared to leaf-derived explants. Stem-derived callus culture displayed maximum total phenolic content and antioxidant activity under optimum hormonal combination (3.0 mg/L TDZ + 1.0 mg/L NAA). HPLC analysis revealed the presence of plectranthoic acid (373.92 µg/g DW), oleanolic acid (287.58 µg/g DW), betulinic acid (90.51 µg/g DW), caffeic acid (91.71 µg/g DW), and rosmarinic acid (1732.61 µg/g DW). Complete antioxidant and anti-aging potential of extracts with very contrasting phytochemical profiles were investigated. Correlation analyses revealed rosmarinic acid as the main contributor for antioxidant activity and anti-aging hyaluronidase, advance glycation end-products inhibitions and SIRT1 activation, whereas, pentacyclic triterpenoids were correlated with elastase, collagenase, and tyrosinase inhibitions. Altogether, these results clearly evidenced the great valorization potential of I. rugosus calli for the production of antioxidant and anti-aging bioactive extracts for cosmetic applications.

Interactive Effects of Light and Melatonin on Biosynthesis of Silymarin and Anti-Inflammatory Potential in Callus Cultures of Silybum marianum (L.) Gaertn.

Silybum marianum (L.) Gaertn. is a well-known medicinal herb, primarily used in liver protection. Light strongly affects several physiological processes along with secondary metabolites biosynthesis in plants. Herein, S. marianum was exploited for in vitro potential under different light regimes in the presence of melatonin. The optimal callogenic response occurred in the combination of 1.0 mg/L α-naphthalene acetic acid and 0.5 mg/L 6-benzylaminopurine under photoperiod. Continuous light associated with melatonin treatment increased total flavonoid content (TFC), total phenolic content (TPC) and antioxidant potential, followed by photoperiod and dark treatments. The increased level of melatonin has a synergistic effect on biomass accumulation under continuous light and photoperiod, while an adverse effect was observed under dark conditions. More detailed phytochemical analysis showed maximum total silymarin content (11.92 mg/g dry weight (DW)) when placed under continuous light + 1.0 mg/L melatonin. Individually, the level of silybins (A and B), silydianin, isolsilychristin and silychristin was found highest under continuous light. Anti-inflammatory activities were also studied and highest percent inhibition was recorded against 15-lipoxygenase (15-LOX) for cultures cultivated under continuous light (42.33%). The current study helps us to better understand the influence of melatonin and different light regimes on silymarin production as well as antioxidant and anti-inflammatory activities in S. marianum callus extracts.

Investigation of Linum flavum (L.) Hairy Root Cultures for the Production of Anticancer Aryltetralin Lignans.

Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.

Exogenous melatonin trigger biomass accumulation and production of stress enzymes during callogenesis in medicinally important Prunella vulgaris L. (Selfheal).

The objective of the current study was to monitor the variations caused by the application of exogenous melatonin on growth kinetics and production of stress enzymes in Prunella vulgaris. Leaf and petiole explants were used for callogenesis. These explants were inoculated on Murashige and Skoog media containing various concentrations of melatonin alone or in combination with 2.0 mg/l naphthalene acetic acid. Herein, a maximum of 3.18-g/100 ml fresh biomass accumulation was observed on day 35 during log phase of growth kinetics at 1.0 mg/l melatonin concentration from leaf explants. While 0.5 and 1.0 mg/l melatonin enhanced the biomass accumulation from petiole explants. Moreover, the synergistic combination of melatonin and naphthalene acetic acid also promoted growth from leaf and petiole explants. Leaf derived callus cultures treated with 1.0 mg/l melatonin induced the production of total protein content (90.47 µg BSAE/mg FW) and protease activity (4.77 U/g FW). While the calli obtained from petiole explants have shown highest content of total protein (160.8 µg BSAE/mg FW) and protease activity (5.35 U/g FW) on media containing 0.5 mg/l melatonin. Similarly, 0.5 mg/l melatonin enhanced superoxide dismutase (3.011 nM/min/mg FW) and peroxidase (1.73 nM/min/mg FW) enzymes from leaf derived callus cultures. The combination of 1.0 and 1.5 mg/l naphthalene acetic acid enhanced content of total protein and protease activity in leaf and petiole derived cultures. These results suggested that the application of melatonin play a positive role in biomass accumulation and production of stress enzymes in P. vulgaris.

Strategies to enhance biologically active-secondary metabolites in cell cultures of Artemisia - current trends.

The genus Artemisia has been utilized worldwide due to its immense potential for protection against various diseases, especially malaria. Artemisia absinthium, previously renowned for its utilization in the popular beverage absinthe, is gaining resurgence due to its extensive pharmacological activities. Like A. annua, this species exhibits strong biological activities like antimalarial, anticancer and antioxidant. Although artemisinin was found to be the major metabolite for its antimalarial effects, several flavonoids and terpenoids are considered to possess biological activities when used alone and also to synergistically boost the bioavailability of artemisinin. However, due to the limited quantities of these metabolites in wild plants, in vitro cultures were established and strategies have been adopted to enhance medicinally important secondary metabolites in these cultures. This review elaborates on the traditional medicinal uses of Artemisia species and explains current trends to establish cell cultures of A. annua and A. absinthium for enhanced production of medicinally important secondary metabolites.

Sucrose-enhanced biosynthesis of medicinally important antioxidant secondary metabolites in cell suspension cultures of Artemisia absinthium L.

Natural products are gaining tremendous importance in pharmaceutical industry and attention has been focused on the applications of in vitro technologies to enhance yield and productivity of such products. In this study, we investigated the accumulation of biomass and antioxidant secondary metabolites in response to different carbohydrate sources (sucrose, maltose, fructose and glucose) and sucrose concentrations (1, 3, 5, 7 and 9 %). Moreover, the effects of 3 % repeated sucrose feeding (day-12, -18 and -24) were also investigated. The results showed the superiority of disaccharides over monosaccharides for maximum biomass and secondary metabolites accumulation. Comparable profiles for maximum biomass were observed in response to sucrose and maltose and initial sucrose concentrations of 3 and 5 %. Maximum total phenolic and total flavonoid contents were displayed by cultures treated with sucrose and maltose; however, initial sucrose concentrations of 5 and 7 % were optimum for both classes of metabolites, respectively. Following 3 % extra sucrose feeding, cultures fed on day-24 (late-log phase) showed higher biomass, total phenolic and total flavonoid contents as compared to control cultures. Highest antioxidant activity was exhibited by maltose-treated cultures. Moreover, sucrose-treated cultures displayed positive correlation of antioxidant activity with total phenolics and total flavonoids production. This work describes the stimulatory role of disaccharides and sucrose feeding strategy for higher accumulation of phenolics and flavonoids, which could be potentially scaled up to bioreactor level for the bulk production of these metabolites in suspension cultures of A. absinthium.

Effect of different in vitro culture extracts of black pepper (Piper nigrum L.) on toxic metabolites-producing strains.

In the present study, the effect of different in vitro cultures (callus, in vitro shoots) and commercially available peppercorn extract was investigated for its activity against toxic metabolite-producing strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Candida albicans). These in vitro cultures were extracted with ethanol, hexane, and chloroform, and the antipathogenic activity was determined by well-diffusion method. Hexane extract of callus showed 22 mm zone of inhibition against B. cereus, 23 mm against S. aureus, while regenerated shoots and seeds have shown 24.3 and 26 mm zones of inhibition. The ethanolic extracts of regenerated Piper shoots have shown 25 mm activity against S. aureus, 21 mm against B. cereus, and 16 mm in the case of C. albicans in comparison with standard antibiotics. Peppercorn extracts in chloroform and ethanol had shown activities against B. cereus (23.6 mm) and B. subtilis (23.5 mm). During in vitro organogenesis and morphogenesis, cells and tissues produced a comparable phytochemicals profile like mother plant. Morphogenesis is critically controlled by the application of exogenous plant-growth regulators. Such addition alters the hormonal transduction pathways, and cells under in vitro conditions regenerate tissues, which are dependant on the physiological state of cells, and finally enhance the production of secondary metabolites. To the best of our knowledge, this is the first report to compare the antimicrobial potential of in vitro regenerated tissues and peppercorn with standard antibiotics. In conclusion, most of the extracts showed pronounced activities against all the pathogenic microbes. This is a preliminary work, and the minimum inhibitory concentration values needs to be further explored. Regenerated tissues of P. nigrum are a good source of biologically active metabolites for antimicrobial activities, and callus culture presented itself as a good candidate for such activities.

Free radical scavenging activity in in vitro-derived tissues of Eruca sativa.

Feasible regeneration protocol for economically important plant Eruca sativa was established and 1, 1-diphenyl-2-picrylhydrazyl scavenging activity of regenerated tissues was evaluated and compared with plant material collected from the wild. Leaf portions inoculated onto Murashige and Skoog (MS) medium responded to all plant growth regulators exploited. Optimum callus production was achieved on a combination of 2.0 mg l(-1) 6-benzyladenine (BA) + 1.0 mg l(-1) α-naphthalene acetic acid (NAA) and the lowest response was recorded for 0.5 mg l(-1) gibberellic acid (GA3) + 1.0 mg l(-1) NAA. The callus was subcultured on similar composition/concentrations of plant growth regulators after 4 weeks of culture time. A 5.0 mg l(-1) 6-BA + 1.0 mg l(-1) NAA produced optimum percentage shoot organogenesis after 4 weeks of subculturing. However, optimum number of shoots per explant was recorded for moderate concentrations (1.0 and 2.0 mg l(-1)) of kinetin. Incorporation of NAA into MS medium-containing GA3 also produced a feasible number of shoots/explant. Similar mean shoot length was recorded for 2.0 mg l(-1) kinetin + 1.0 mg l(-1) NAA and optimum concentrations (2.0, 5.0, and 10.0 mg l(-1)) of GA3 + 1.0 mg l(-1) NAA. In vitro generated shoots were shifted to MS medium augmented with indole acetic acid (IAA) for rooting after 4 weeks of subculturing. Moderate concentrations (5.0 mg l(-1)) of IAA produced feasible rooting. Investigation of radical scavenging activity showed that callus possesses higher levels of radical scavengers than other plant tissues tested. Phenolics and glucosides are reported to be active components of Eruca sativa phytochemistry.