Ocimum basilicum L. and its derived products are primarily consumed by humans; hence, agrochemical use seems inappropriate for its cultivation. However, farmers are accustomed to using rampant inorganic fertilizers to augment crop productivity, which has unintendedly engendered severe environmental perturbations. Concomitantly, farmers will soon have to confront the challenges of growing crops under suboptimal conditions driven by global climate change. Consequently, to develop a clean, sustainable, and resilient production technology, field experiments spanning over two years (2020 and 2021) were conducted, comprising three biostimulants, viz., vermicompost , biofertilizer (uninoculated and inoculated), and liquid seaweed extract (without and at 7 ml L−1) in the Indian western Himalaya for the first time. Soil health indicators, leaf photosynthetic pigments, gaseous exchange, mineral contents, essential oil (EO) quantity, and composition were evaluated. Soil microbial respiration (SMR), microbial biomass carbon (MBC), organic carbon (OC), dehydrogenase (DHA), alkaline phosphatase (ALP), and β-glucosidase activities were increased by 36.23, 83.98, 30.61, 42.69, 34.00, and 40.57%, respectively, when compared with the initial soil status. The net photosynthetic rate (Pn) was significantly increased with the highest (8 Mg ha−1) and moderate (4 Mg ha−1) vermicompost dosages by 13.96% and 4.56%, respectively, as compared with the unfertilized control (0 Mg ha−1). Likewise, the biofertilizer and seaweed extract also enhanced Pn by 15.09% and 10.09%, respectively. The crop’s key EO constituents, viz., methyl chavicol and linalool, were significantly improved with the highest and moderate vermicompost rates of 2.71, 9.85%, and 1.18, 5.03%, respectively. Similarly, biofertilization and seaweed application also boosted methyl chavicol and linalool by 3.29, 8.67%, and 1.93, 3.66%, respectively. In both years, significantly higher herbage (8.86 and 11.25 Mg ha−1) and EO yield (113.78 and 154.87 kg ha−1) were recorded with a congregate treatment of the highest vermicompost dose, biofertilizer, and liquid seaweed extract. In conclusion, the integrated use of biostimulants having complementary properties can sustainably maximize the quantity and quality of O. basilicum and concomitantly ameliorate soil health. This study can inspire scientific communities and industries to develop second-generation biostimulant products, delivering better sustainability and resilience for a renaissance in agriculture. Meanwhiosystems . LikewisOcimum basilicum L.) is an annual, essential oil (EO) producing medicinal aromatic plant (MAP), predominantly cultivated for direct human consumption and chemical use is inappropriate for its production (−1) with an estimated commercial market value of ~US $700 million which is also forecasted to expand in the coming years , iron (Fe), zinc (Zn), and manganese (Mn) at optimal levels with better acquisition. Additionally, these can supply organic carbon (OC), which improves soil edaphic conditions and ultimately increases crop yield. Among composts, vermicompost is considered the best fertilizer, having higher humic substances (HSs), being homogenous, microbiologically active, and less phytotoxic  and smoke-derived compound (karrikinolide) showed antagonistic effects also (Owing to the plant growth promoting (PGP) characteristics and biostimulatory response of vermicompost, biofertilizer, and seaweed extracts , we hypocts also . Neverthcts also . As cropcts also . MoreoveBased on the available literature, the synergistic and additive properties between biostimulant categories are fascinating and indicative of their intricate biostimulatory mechanisms in determining plant growth, performance, and resilience. Consequently, it is indispensable to decipher the sole and combined effects of biostimulants to invent the next generation of biostimulant products having harmonious properties for improved crop growth, yield, quality, and resilience. Additionally, with improved mechanistic clarity, it will be possible to design deliberate combinations of non-microbial and microbial biostimulants that would interact synergistically to provide the required results in terms of acceptable yield and quality products in a sustainable manner. Therefore, accentuating the inextricable and serious collateral problems of using chemical fertilizers, coupled with their forthcoming shortages, and the context of sustainable production of MAPs, the current study was designed to evaluate the synergistic/additive effect of diverse biostimulants on growth, productivity, secondary metabolite accumulation, and composition in sweet basil. The salient objectives of the study were: (i) to analyze the effect of biostimulants on the physiological performance of sweet basil under field conditions; (ii) to elicit the amount and key constituents of sweet basil’s EO and elucidate its plausible mechanism; and (iii) to evaluate the effect of organic biostimulant amendments on soil physical, chemical, and biological properties.−1); the second factor was comprised of two levels of biofertilizer , and the third factor consisted of two levels of seaweed extract  (−1. The composition and attributes of biostimulants used in the current study are provided in A field study was conducted for two consecutive cropping years (2020 and 2021) at the experimental farm of CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India . The first factor was composed of three levels of vermicompost  Table 1.Arthrobacter psychrochitiniphilus IHB B 13602 (KU160185) isolated from the rhizosphere of agricultural land situated at the cold desert region of the Lahaul valley and ii) B. altitudinis IHBT 705 (CP074101) harnessed from the native agricultural field of Palampur, Himachal Pradesh, India. These PGPR strains were selected based on their multiple PGP traits like IAA synthesis , siderophore production , phosphate solubilization , and ACCD (1-aminocyclopropane-1-carboxylate deaminase) activity  at the seedling transplanting stage ; ii) at the first weeding event; 20 days after transplanting (DAT) after the establishment of plants in the main field.The Indian sweet basil seeds of accession IHBT/OC-1, having 98% vigor, were manually planted in pots comprising sand, soil, and farmyard manure in a 1:1:1 ratio within a depth of 1–2 cm during the first week of June in both years. Seeds were germinated 7–14 days after sowing, and thirty-day-old seedlings (30 DOS) having an average root length of 11.93 cm and a shoot length of 15.76 cm  and Sargassum swartzii  at 7 ml L−1 was done using a Knapsack sprayer with a constant flow until complete canopy runoff during a cloudless sunny day (2O5 (7.60 ± 0.87 kg ha−1), moderate in available N (139.10 ± 2.39 kg ha−1), and rich in available K2O (318.00 ± 3.19 kg ha−1) (The first inoculation of biofertilizer was achieved through root dipping  of disinunny day  at vegetkg ha−1)  and have2Cr2O7; 10 ml, 0.5 M), and deionized water (150 ml) to 0.5 g of homogenized soil. Diphenylamine was added followed by titration using 1 N ammonium iron (III) sulfate hexahydrate and expressed in percentage (OC%). The soil texture was determined by the hydrometer method while, pH was detected with soil and water (1:2) suspension by a glass electrode pH meter. A macro Kjeldahl method was adopted for the determination of available N by digesting 0.5 g of soil in a Kjeldahl digestion tube with concentrated sulfuric acid (10 ml), and the N concentration was determined by an N analyzer . Available phosphorous (P) was determined with a soil solution extracted with 0.5 M sodium bicarbonate (NaHCO3) at 470 nm using a spectrophotometer . At the same time, the amount of exchangeable K was quantified with 1 N extractable ammonium acetate (NH4OAc) by flame atomic emission spectrometry  and the rhizosphere of five plants from each plot, respectively. At each sampling event, 10–20 g of soil was collected, and the specimens were mixed to get a composite sample depicting the whole field. Soil biological, physicochemical, and enzymatic properties were analyzed. To assess the biological features such as soil microbial respiration (SMR), microbial biomass carbon (MBC), bacterial population count (BPC), and soil enzymatic activities some soil subsamples were put in zip-lock plastic bags and immediately kept inside a cold room (8°C), rest samples were shade dried, sieved with a 2 mm mesh and kept at room temperature until analysis. The OC was evaluated by adding potassium dichromate solution  solution was placed inside each flask to capture the CO2, followed by incubation at 27°C for 48 h. The remaining NaOH solution was titrated with HCl (0.25 M), BaCl2 (0.5 M), and phenolphthalein. Simultaneously, 10 g of soil was dried at 105°C, and SMR was calculated using a standard equation and expressed as mg CO2 g−1 dry soil day−1.The MBC and SMR were quantified using a modified fumigated and alkali absorption extraction techniques, respectively . Uniform−1 dry soil h−1. For determination of urease activity, soil (5 g) was taken in a flask (100 ml) and 2.5 ml of urea solution (0.08 M) was poured over. Then, the flask was stoppered and allowed to incubate at 37°C for 2 h, followed by the addition of 50 ml of potassium chloride solution (1 N KCl in 0.01 N HCl) and vigorously shaken for 30 min. Then the solution was filtered, and the clean filtrate (1 ml) was pipetted into another flask (50 ml), followed by the addition of deionized water (9 ml), sodium salicylate/sodium hydroxide solution (5 ml), and 2 ml of sodium dichloro-isocyanurate solution. The contents of flasks were incubated at room temperature for 30 min, followed by quantification of ammonium (NH4⁺) using a spectrophotometer at 690 nm, and urease activity was expressed as μg N-NH4+ g−1 dry soil h−1.Soil enzymatic activities like dehydrogenase (DHA), urease, alkaline phosphatase (ALP), and β-glucosidase were accessed as per the standard procedures  with app−1 dry soil h−1. The β-glucosidase activity was assessed by taking uniformly soil (1 g) in a test tube followed by the addition of toluene (0.25 ml). Thereafter, 4 ml of MUB (pH 6) and 1 ml of p-nitrophenyl-β-D-glucoside (25 mM PNG) were added, followed by proper mixing. The suspension was incubated at 37°C for 1 h, followed by the addition of 1 ml of calcium chloride (0.5 M) and tris solution (4 ml) followed by filtration, and OD was taken at 400 nm. A calibration curve of standard p-nitrophenyl-β-D-glucoside (PNG) was prepared and the activity was expressed as μg PNG g−1 dry soil h−1.For determination of ALP uniformly, 1 g of soil was taken in a flask (50 ml), followed by the addition of 0.25 ml toluene, 4 ml modified universal buffer (MUB) (pH 11), and 1 ml p-nitrophenyl phosphate solution (PNP). The flask was stoppered, followed by the mixing of all contents, and allowed to incubate at 37°C for 1 h. Afterward, 0.5 M calcium chloride (1 ml) and 0.5 M sodium hydroxide (4 ml) were added and again mixed to form a suspension. The suspension was then filtered, the filtrate’s OD was measured at 400 nm, and the amount of soil ALP activity was expressed as μg PNP gNon-peripheral net plants (22) were considered and selected for various observations in all treatments to avoid the border effect. Five plants from each plot were tagged for observation of growth attributes like plant height, plant spread, and primary branches at 50 and 100 DAT. Leaf, stem, and inflorescence weight, number of inflorescences, average length of inflorescence, herbage yield, EO content, and yield were recorded at harvest. In addition, a few healthy leaves were also taken at 50 and 100 DAT to determine the photosynthetic pigments and leaf mineral concentration, respectively.g for 5 min to collect the supernatant. The OD of the collected supernatant was measured at 663, 646, and 470 nm for Chla, Chlb, and carotenoids, respectively, using a spectrophotometer. Acetone (80%) was used as a blank for the procedure, and concentrations of Chla, Chlb, and carotenoids (mg g−1) were calculated according to formulas previously described by 2SO4 and a mixture of concentrated H2SO4:HClO4 (5:1) for estimation of N and P, respectively. The contents of N were estimated with an N analyzer while P was quantified by a spectrophotometer.Uniformly, 1 g of fresh leaf sample was washed with deionized water. It was followed by homogenization with 80% acetone and centrifugation at 6,000×n), stomatal conductance (Gs), transpiration rate (Tr), intracellular CO2 (Ci), and leaf vapor pressure deficit (VPDleaf) using a portable leaf chamber of 2 cm × 3 cm (6 cm2) having a red‐blue LED light source and an infrared gas analyzer . Before all measurements, the instrument was warmed up for ~30 min and an auto program was run to determine the light dependence of СО2 gaseous exchange in leaves with ten log points, and a light intensity curve was established by giving PAR ranging from 0 to 2,000 μmol photons m−2 s−1  using a CO2 mixer with a flow rate of 500 μmol s−1, relative humidity of 50%–60%, and PAR was set at 1,300 mmol m−2 s−1 as determined by light calibration curve  and the ratio of intracellular CO2 to ambient CO2 were calculated as Pn/Tr and Ci/Ca, respectively.Three individual plants were selected from each plot for the measurement of gas exchange-associated characteristics like net photosynthetic rate (P2SO4) and kept in a sealed amber glass vial at 4°C for further analysis. The EO weight  of aboveground dried parts was used to compute the EO content (% w/w). The EO yield was estimated using the following formula and expressed in kg ha−1.A uniform weight  of 24 h shade-dried aboveground parts (leaf and inflorescence) excluding stems from every plot was hydro-distilled for 3 h in the Clevenger apparatus. The extracted EO was treated with anhydrous sodium sulfate  was set at 1 scan−1. The GC peak was used to compute the number of volatile constituents and their amounts, which were then sorted by the order of GC elution. The retention index (RI) was calculated without correction factors using a series of hydrocarbons. The constituents of EO were recognized by a comparison of the experimental RIs with RIs described in the literature  through a flame ionization detector (FID)  fitted with an AOC 5000 Plus auto-injector comprised of Zebron ZB-5 MS capillary column . The extracted EO (10 μl) was dissolved in dichloromethane (2 ml), followed by injection in split mode (2 μl each). Nitrogen gas was used as a carrier at a 1.5 ml minterature . Additioterature .n = 3) ± SE. Data were analyzed by comparing means using Fisher’s LSD (least significant difference) at a P = 0.05 confidence level . A co-relationship between crop traits was also determined by using Pearson’s coefficient (PAST-4).Datasets of two years were pooled and used to evaluate the effects of the cropping year and used biostimulants on growth, yield, EO amount and composition, leaf nutrient uptake, and gaseous exchange using a four-factor analysis of variance (ANOVA). The results were represented as the mean of three replications  when compared with moderate (4 Mg ha−1) and control (0 Mg ha−1) at 50 and 100 DAT, respectively. Likewise, biofertilizer significantly benefited all treated plants, leading to a 7.21 11.19% enhancement in plant height and branching by 39.96 24.32% at 50 and 100 DAT, respectively. On the contrary, foliar application of liquid seaweed extract at 7 ml L−1 showed a significant effect on plant height and branches at 100 DAT only.Significantly higher plant height  and primary branches  at 50, and 100 DAT, respectively, were recorded during the second year compared with the first Table 3.a, Chlb, and carotenoids were significantly enhanced by 2.43, 5.14, and 3.52%, respectively, in the second year when compared with the first . Similarly, biofertilizer and seaweed extracts have also significantly increased Chla, Chlb, and carotenoids by 16.97, 16.12, 12.90%, and 4.68, 8.06, and 6.69%, respectively. The moderate and highest vermicompost dosages also significantly influenced the rate of photosynthesis by 4.56 and 13.96%, respectively, when compared with the unfertilized control. On the contrary, biofertilizer and seaweed extract significantly enhanced both Pn and WUEint by 15.09, 5.90%, and 4.46, 7.58%, respectively (Concentrations of photosynthetic pigments Chl−1) in the second year in contrast with the first year (6.01 Mg ha−1) (The herbage yield (leaf + inflorescence) was significantly higher  when compared with the first , respectively  but remained statistically at par with the moderate dose (4 Mg ha−1). Likewise, biofertilization and foliar application of seaweed extract also significantly affected EO content and yield by 45.35, 67.95%, and 18.48, 31.33%, respectively. The major constituents of EO, methyl chavicol and linalool, significantly increased by 2.84 and 5.68%, respectively, in the second year as compared with the first year  and showed a positive correlation of EO yield with herb yield (r = 0.91), EO content (r = 0.94), dry matter content (r = 0.97), leaf + inflorescence/stem ratio (r = 0.81); number of inflorescence plants−1 (r = 0.94); average length of panicle (r = 0.95); net rate of photosynthesis (r = 0.72). Furthermore, the methyl chavicol content was also positively correlated at P = 0.01 with all chosen traits, except for dry matter content, which was significant at P = 0.05 (r = 0.62). Similarly, the herbage yield was positively correlated (P = 0.01) with all considered attributes, excluding the net photosynthetic rate, which was significant at P = 0.05 (r = 0.67). Biomass and EO yield were both significantly influenced by the interaction effect of all three involved factors, viz., vermicompost (V), biofertilizer (B), and liquid seaweed extract (S) during both years  receiving congregate treatment of all three biostimulants  in seaweed extract , solubilization of insoluble compounds like calcium di, tri-phosphates, and siderophore production  in Zea mays L. upregulates genes related to root growth, PGR signaling, stress responses, plant nutrition, and transport, including DNA repair under drought conditions  induces the photosynthesis and starch biosynthesis associated genes in Z. mays, leading to higher biomass production (n)  Walp after treatment with seaweed extract (Kelpak®) and vermicompost leachate under drought stress  are substantially oxidized and their excitation energy is used in electron transport, resulting in the increased synthesis of energy molecules (ATP and NADPH). These energy molecules are further used for carbon assimilation during the C3 cycle and eventually increase crop biomass . Therefore, the increased SMR  Dunal after inoculation with cell pellets and metabolites of PGPRs like B. subtilis and Streptomyces sp. at 10 ml plant−1 pot−1 under a poly greenhouse condition (Bacopa monnieri (L.) Pennell inoculated with chitinolytic microbes as of alteration in the biosynthetic pathway of bacoside A (via an induced systemic resistance (ISR) mechanism, leading to activation of the host plant’s chemical defense cascades like jasmonic acid (JA) and ethylene (ETH), as many studies have successfully demonstrated the occurrence of ISR in MAPs after biostimulation  alteration of rhizomicrobiome through the production of enzymes and organic compounds. Likewise, the seaweed extract can augment plant growth and development through hormonal homeostasis, upregulation of nutrient transporter genes, stimulation of photosynthesis, and increased stress tolerance due to antioxidant stimulation and decreased lipid peroxidation, including reactive oxygen species (ROS) levels , and recruits beneficial microflora in the phyllo and rhizosphere. Likewise, vermicompost’s bio-stimulatory action is due to the presence of HSs, PGRs, PGPRs, and other advantageous substances in its composition. The biostimulatory activity of HSs has been attributed to numerous mechanisms (i) increasing WHC and CEC, neutralizing soil pH, and meliorating the soil structure; (ii) improving P solubility by preventing its precipitation and also preventing leaching, thereby augmenting the availability of nutrients, (iii) act like auxin and improve lateral root induction thereby, trigger the plasma membrane H+-ATPase activity, and (iv) stimulates nitrate assimilation via upregulation of the target genes/enzymes. Additionally, HSs can decrease hydrogen peroxide (H2O2) and lipid peroxidation, which consequently raises the proline concentration and recruits beneficial stress-resilient microbial communities in the rhizosphere  levels . Additiozosphere .viz., calcium ion (Ca2+), JA, salicylic acid (SA), nitric oxide (NO), and ROS, leading to enhanced secondary metabolite (SM) accumulation in MAPs  activity and converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). As a result, phosphorylated protein kinase A (PKA) stimulates transcriptional factors (TFs) of the target gene (TG). Furthermore, inositol trisphosphate (IP3) is produced by phospholipase C (PLC) activity stimulated by G-protein activation. This secondary messenger increases intracellular Ca2+ by activating Ca2+ channel efflux and its binding to a particular receptor on the endoplasmic reticulum (ER). The augmented intracellular Ca2+ concentrations cause phosphorylation of certain proteins like proteins kinase (PK), which regulates gene expression by activating TFs. Additionally, activated PLC is also implicated in generating oxidative bursts by producing H2O2, hydroxy free radical (OH), singlet oxygen (1O2), and superoxide anion (O2) through swapping between O2 and H2O2 depending on NADPH oxidase and peroxidase activity in the plasma membrane. Consequently, H2O2 generation in the plasma membrane leads to stimulation of ER through increased cytosolic Ca2+ concentration. Furthermore, these molecules also trigger secondary messengers like JA, SA, NO, PK, ETH, and ROS that can modify the epigenetic pathways regulating gene expression. Likewise, various secondary messengers regulate the expression of genes by transforming the methylation state of DNA into a hypo-methylation state to permit access to TFs and RNA polymerases to the promoter region. Moreover, secondary messengers can also relax the structure of chromatin by shifting it to a euchromatin state, which also allows access of TFs and RNA polymerases to the promoter region. These epigenetic state transitions mediated by secondary messengers allow gene expression flexible in the presence of elicitor(s). In this manner, the plant cell genome incorporates elicitor(s) signals via epigenomic variations and thereby increases the production of secondary metabolites for its defense  are associated with the response to biostimulants and other cellular processes related to growth, hormonal signaling, and defensive responses. The elicitor(s) produced by biostimulants serve as ligands and are identified by PRR (pattern-recognition receptors) to create a ligand–receptor complex. In this manner, a plant cell cognizes the signal produced by the elicitor(s) . Upon re defense .−1) was recorded, when the crop was treated with a congregate dose of vermicompost (8 Mg ha−1), biofertilizer, and foliar application of liquid seaweed extract (7 ml L−1). This production technology can be considered inexpensive and environmentally sustainable, having low carbon footprints and no harmful field emissions. Therefore, it can be concluded that the combination of microbial and non-microbial biostimulant categories can act synergistically and maximize the soil health and quality production of sweet basil EO, particularly under the rainfed conditions of the Indian western Himalayas. Nevertheless, more rigorous field evaluations are required to evaluate and legitimize the interaction effects of the diverse range of biostimulants, especially on MAPs in a range of agroclimatic conditions. In addition, deciphering the combinative effects of biostimulants on functional rhizomicrobiomes would allow us to have a better comprehension of the soil–microbe–plant nexus. Moreover, the exact mechanism of biostimulation is not fully understood and further investigations are required to discern the fine-tuning between biostimulants and the host plant by evaluating the global changes in the abundance of mRNA transcripts using a comparative high-throughput RNA-seq approach.The pursuit of cleaner crop production is to improve nutrient availability and nutrient use efficiency while preserving soil health and producing quality crop products to slacken reliance on agrochemicals. In this regard, the current study unequivocally alludes that the congregate use of vermicompost, biofertilizer and seaweed extract can be practiced by MAP growers and stakeholders for the cleaner and environment-friendly production of Indian sweet basil EO. In essence, the current field experiment suggests that the combinatorial application of organic biostimulants having harmonizing properties not only significantly enhanced crop yield but also improved soil health. Based on two years of crop productivity, averred maximum EO  is also acknowledged.The authors are grateful to the Director, CSIR-IHBT, Palampur, India, for providing the necessary facilities for study. This manuscript represents the CSIR-IHBT communication number 5082.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Unfortunately, we detected anerror in our calculation of cumulative greenhouse gas emissions fromour composting experiment. The error occurred during the conversionof cumulative gas emission in terms of wet feedstock to dry feedstock.Unfortunately, we made a mistake in this conversion as we used thewrong moisture content of the initial feedstocks.4 kg–1 dry feedstock, 451 g CO2 kg–1 dry feedstock, and 0.060 mg N2O kg–1 dry feedstock. The correct cumulative greenhouse gas emissions fromthe biochar-compost pile are as follows: 0.81 g CH4 kg–1 dry feedstock, 280 g CO2 kg–1 dry feedstock, and 0.119 mg N2O kg–1 dry feedstock.This mistakeimpacted only the value of the cumulative gas emissionsfor the treatment and control, which are both too low by a factorof ∼2. The correct cumulative greenhouse gas emissions fromthe manure-only pile are as follows: 5.03 g CHThere is also a slight difference in cumulativemethane reductioncompared to that in the original article. The biochar-compost treatmentreduced cumulative methane by 83.9% relative to the control, ratherthan the 79% reduction reported in the original article. This differencein methane reduction does not affect any of our work’s mainfindings, discussion, or conclusions. Our life-cycle assessment andscaling-up analyses remain unaffected by this error, as they werecalculated without the incorrect moisture contents.Supplementary Figures 4, 6, 7, and 9.


Sallow and/or dull skin appearance is greatly attributable to the yellow components of skin tone. Bilirubin is a yellow chromophore known to be made in the liver and/or spleen and is transported throughout the body via the blood stream. Recent publications suggest bilirubin may be synthesized in other cells/organs, including the skin. We found human keratinocytes express the transcripts involved in bilirubin biosynthesis. In parallel, we also found human keratinocytes could indeed synthesize bilirubin in monolayer keratinocytes and in a 3D human skin-equivalent model. The synthesized amount was substantial enough to contribute to skin yellowness. In addition, oxidative stress enhanced bilirubin production. Using UnaG, a protein that forms a fluorescent species upon binding to bilirubin, we also visualized the intracellular expression of bilirubin in keratinocytes. Finally, we screened a compound library and discovered that the sucrose laurate/dilaurate (SDL) combination significantly reduced bilirubin levels, as well as bilirubin-mediated yellowness. In conclusion, bilirubin is indeed synthesized in epidermal keratinocytes and can be upregulated by oxidative stress, which could contribute to chronic or transient yellow skin tone appearance. Application of SDL diminishes bilirubin generation and may be a potential solution to mitigate yellowish and/or dull skin appearance. Yellow skin color is frequently associated with sallow and/or dull skin appearance, which is considered a barometer of poor health. Coincidentally, dull skin appearance can be exacerbated by conditions that commonly lead to increased inflammation and oxidative stress, such as sleep deprivation, acute and/or chronic mental or physical stresses, and poor diet ,2,3,4,5.HMOX1), and a constitutively expressed form, heme oxygenase-2 (HMOX2) [2O2) [Bilirubin is a metabolite of senescent red blood cells largely produced in the spleen, bone marrow, and/or liver. Bilirubin is transported in the blood stream, mostly in a form tightly bound to albumin. Free or unbound bilirubin is reported to have the ability to diffuse out of blood vessels and into tissues, where it can act beneficially as an antioxidant . However (HMOX2) ,9,10,11. (HMOX2) ,13. Bili2) [2O2) ,15.HMOX1 mRNA expression in keratinocytes in association with bilirubin accumulation in the stratum corneum of human epidermis [HMOX1 induction under stressed conditions.In 1950, London and Gray estimated that 10–20% of bilirubin excreted by humans originates extrahepatically ,17. It hHere, we hypothesize that human skin keratinocytes house the machinery to perform de novo bilirubin synthesis, especially under oxidative stress, and the synthesized bilirubin may contribute to yellow skin appearance. We also highlight the potential usefulness of the combination of sucrose laurate and dilaurate (SDL) as a novel cosmetic ingredient in managing bilirubin-mediated yellowness.ALAS1, HMOX1, and BLVRB) is shown in ALAS), uroporphyrinogen III synthase (UROS), uroporphyrinogen III decarboxylase (UROD), oxygen-dependent protoporphyrinogen IX oxidase (PPO), and ferrochelatase (FECH), as well as de novo bilirubin synthetic pathway gene expression, including biliverdin reductase , HMOX1, and HMOX2  . A simplnd HMOX2 B. Upon segulated B. These HMOX1 and HMOX2 , an inhibitor of ALAS, which is a rate-limiting heme biosynthetic enzyme ,22 FiguA. As expThe effect of SA on intracellular bilirubin expression was further tested with the UnaG-mCherry sensor assay in living keratinocytes. SA inhibited the intracellular expression of bilirubin B,C. Thes2O2 for 48 h, followed by bilirubin quantification using HPLC-MS/MS. The noncytotoxic levels of H2O2 significantly upregulated bilirubin production , a commercially available, reconstituted, three-dimensional (3D) human skin-equivalent model. No bilirubin was detected from fresh medium. Bilirubin was readily detected in untreated control skin-equivalents  for 44 h in a vehicle of DPBS buffer. Afterward, skin samples were equilibrated in DPBS buffer without bilirubin for 138 h. Changes in yellowness scores from baseline  were measured at 0, 22, 44, 68, and 138 h after the initiation of culture . BilirubBilirubin is well known to act as a chemical antioxidant and is thought to offer cytoprotection . On the 2O2-induced bilirubin production , hemin (Cat. No.: H9039-1G), and 4,6-dioxoheptanoic acid (succinyl acetone (SA)) (Cat. No.: D1415-500 mg) were purchased from Sigma . Bilirubin (Cat. No.: 17161) and bilirubin conjugate , were purchased from Cayman Chemicals . CellTiter-Glo® luminescent cell viability assay kit (Cat. No.: G7571) was purchased from Promega . SDL (combination of sucrose laurate and sucrose dilaurate at approximately 6:4 ratio) was supplied from BASF .Culture medium and supplements were all purchased from Thermo Fisher Scientific , including EpiLife calcium-free phenol red-free medium (Cat. No.: MEPI500CA), gentamicin/amphotericin B , calcium chloride (Cat. No.: 50-9703), HKGS , trypsin/EDTA solution , trypsin neutralizer solution , penicillin/streptomycin , and DPBS (Cat. No.: 14190250). The keratinocyte cell line tKC was a kind gift from Dr. Shay  . ViaStai2 and 95% humidity for 24 h before treatment. All treatments were made in MEPI500CA medium that contained 1 mM added calcium chloride and 0.1% DMSO. After treatment, the cultures were maintained at 37 °C under 5% CO2 and 95% humidity for 48 h before harvesting. When harvesting, the culture supernatant was transferred onto a fresh 24-well plate and stored on dry ice immediately and shielded from light. Cell layers were also stored on dry ice immediately and shielded from light. Both the cell layer plate and medium plate were subsequently frozen at −80 °C until quantifying for bilirubin.tKCs were plated in 24-well tissue culture plates using MEPI500CA media supplemented with HKGS and gentamicin/amphotericin B at 200,000 cells/well. The cells were incubated at 37 °C under 5% CO® luminescent cell viability assay was used according to the manufacturer’s instructions. Briefly, CellTiter-Glo® reagent was mixed with an equal volume of cell culture medium and vortexed to create a homogeneous Cell GLO viability working solution. After removing cell culture media, tKCs were treated with 300 µL of the Cell GLO viability working solution. The plate was covered with aluminum foil and agitated on an orbital shaker at 150 rpm for 10 min. The plate was then read with a SpectroMax microplate reader  for chemiluminescence intensity.For viability assessment, the CellTiter-Glo2 for 20 h to equilibrate. A baseline group of 4 tissues and corresponding culture supernatant were harvested on dry ice and shielded from light before transferring to −80 °C for storage. The rest of the tissues were subject to either medium treatment as control groups or 30 µM H2O2 treatment as oxidatively stressed groups (n = 4 per group). All tissues were incubated at 37 °C with 95% humidity and 5% CO2 until the designated harvesting time points: 24 h, 48 h, and 72 h.EpiDerm™ 3D human skin-equivalent cultures were purchased from MatTek corporation  and cultured according to the manufacturer’s recommendations. EpiDerm™ cultures contain highly differentiated human-derived epidermal keratinocytes. The EpiDerm™ 3D cultures were immediately incubated with phenol red-free medium (Part No.: EPI-100-ASY-PRF) at 37 °C with 95% humidity and 5% COUnaG is a Japanese Eel muscle protein. It binds bilirubin, forming a complex that emits green fluorescence, making it a good bilirubin sensor . The UnaThe effect of SDL on bilirubin level was assessed with 20 h incubation in a cell-free system. Three replicates of each test sample were prepared on a 96-well plate  at a total volume of 250 μL/well. Positive control wells contained 25 µg/mL bilirubin in PBS buffer , while negative/vehicle control wells contained DMSO and PBS buffer. SDL was dissolved in DMSO. Each test sample well contained 25 µg/mL bilirubin with SDL added to 0.01% in PBS buffer. The plates were covered with aluminum foil and placed on top of a microplate shaker , followed by incubation at room temperature for 20 h with constant shaking at 150 rpm. Bilirubin was then quantified after incubation using a commercially available bilirubin quantification kit . Briefly, quantitation is based on the Jendrassik–Grof method using diazotized sulfanilic acid to react with bilirubin to form azobilirubin, detected at an OD of 540 nm. A standard bilirubin curve is generated. Bilirubin concentrations of all testing legs are calculated using linear regression against the bilirubin standard curve. Bilirubin reduction activity was determined by comparing bilirubin levels of treatments vs. the bilirubin-positive control leg.2 squares, placed in 1 M NaCl plus 10× penicillin/streptomycin (Invitrogen), and incubated overnight at 37 °C. The following day, the epidermis was carefully peeled off with forceps and stored in phosphate-buffered saline (PBS) plus 2× penicillin/streptomycin and stored at 4 °C before use . To assess bilirubin’s impact on skin tone appearance, the epidermal explants were cultured in individual inserts that sat on top of 6-well culture plates. Tissues were simultaneously treated both topically (50 µL) and in the medium 6-well plates (2.3 mL per well) with one of the 3 treatments: control vehicle , 1.4 µg/mL conjugated bilirubin solution, and 2.1 µg/mL conjugated bilirubin  solution, respectively. Epidermal explant tissues were imaged with a Spectroshade imaging device  at designated time points and b* values (yellowness score) were quantified from the images using built-in software.Twelve-millimeter punch biopsies of human abdominal explant  were procured from cosmetic procedures with an IRB-approved protocol . After fat removal, the skin was cut into 1.25 cmg using a benchtop centrifuge for 10 min with the supernatants used for analysis. The standards and the samples were analyzed using gradient high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Bilirubin can be separated by a reverse-phase column . Bilirubin and the corresponding stable isotope-labeled internal standard (ISTD) were monitored by electrospray ionization (ESI) in positive mode using the selected reaction monitoring schemes below.Biological samples were extracted with 0.1% triethylamine (TEA) to recover synthesized bilirubin. Briefly, 0.1% TEA was added into 3D human skin-equivalent tissue samples (120 µL per tissue) or monolayer cells in the well plate (110 µL per well of a 24-well cell culture plate), transferred to low-binding Eppendorf tubes, followed by vortexing for 10 min at room temperature. Samples were then centrifuged at 16,000× -Multiple reaction monitoring (MRM) transitions for analytes (bilirubin: 585.2 → 299.2);-Their corresponding stable isotope-labeled internal standards (d4-bilirubin: 589.2 → 301.2).The Used ISTD was d4-bilirubin . A standard curve was constructed by plotting the signal, defined here as the peak area ratio , for each standard versus the concentration of each analyte for the corresponding standard. The concentration of bilirubin in the calibration standards and biological samples were then calculated using the generated regression equation.2 × 0.005 cm = 0.002513 cm3 = 0.002513 mL. Upon normalizing the sum of bilirubin produced by each tissue, i.e., factor in bilirubin quantified from both tissue itself and corresponding culture supernatant/medium, the mean bilirubin concentration per milliliter tissue volume of each group was calculated.The diameter of each 3D human skin-equivalent model tissue is 8 mm, and the thickness is about 50 µm. By using such dimensions, an individual 3D human skin tissue volume was calculated as: π × (0.4 cm)2 incubator, the tKCs were treated with a media control vehicle and hydrogen peroxide at 250 µM for 1 h, respectively, before harvesting 6 h later for microarray analysis. To investigate the impact of bilirubin treatment on human primary keratinocytes, a control vehicle (10% water) and conjugated bilirubin at 1.4 μg/mL, 5 μg/mL, or 14 μg/mL, were added respectively, before harvesting for microarray analysis at 6 h and 24 h. Samples were collected in RNAlater® buffer, flash-frozen, and stored at −80 °C prior to RNA extraction. RNA was extracted and purified using the RNeasy kit . Purified RNA was converted to biotin-labeled complementary RNA copies using the HT 3′ IVT Plus kit , per the manufacturer’s protocol. Biotinylated cRNA was fragmented by limited alkaline hydrolysis and then hybridized overnight to Affymetrix GeneTitan U219 array plates using the Affymetrix GeneTitan instrument and protocol. Probe set expression values were calculated by quartile normalization and PLIER summarization algorithms. Differentially expressed genes were analyzed using the ANOVA model implemented in the R limma package [To investigate the impact of ROS stress, tKC cells were plated out at a density of 250,000 cells/well into 12-well plates. After growing for 24 h at 37 °C in a CO package . Gene Se package  against 2. UnaG was excited using a 488 nm laser and emission captured by the Airyscan detector with a 495–550 nm bandpass filter. mCherry was excited using a 594 nm laser and emission captured by the Airyscan detector with a 645 nm long pass filter. Mean fluorescent intensity of cells was measured using ImageJ. Individual cells were isolated by ROI and the mean fluorescence intensity of each channel measured. UnaG intensity values are expressed as a proportion of the mCherry signal to normalize and control for variation in probe expression.HaCaT keratinocytes were cultured in EpiLife medium (Thermo Fisher). At 72 h prior to imaging, cells were seeded in 35 mm quad No1.5 polymer coverslip-bottomed dishes (Ibidi) and grown to 70% confluency at the time of transfection. At 48 h prior to imaging, the cells were transfected with the CMV-UnaG-mCherry construct B using LA 10 µm amount of fresh frozen human skin sections was dried at RT for 30 min, followed by rehydration with PBS. Sections were incubated either with or without (negative control) 50 µg/mL bilirubin 1 h at RT in the dark, followed by washing with PBS. Sections were then treated with either (1) vehicle—0.5% DMSO in PBS or (2) 0.01% SDL in vehicle overnight at RT in the dark, washed in PBS, and DAPI counterstained using NucBlue fixed cell stain Ready Probes reagent . For comparison, fluorescent images were captured with a Zeiss Observer.Z1 microscope  at equal gamma values, pixel ranges, and exposures.t-test unless stated otherwise. Values of p < 0.05 were considered statistically significant.Statistical significance for all in vitro experiments, except for the transcriptome analysis, was determined by Student’s


Atriplex dimorphostegia  is an annual halophytic shrub that is widely distributed across various parts of Asia. The current study is the first to report the metabolites profile of the total ethanol extract of the aerial parts of A. dimorphostegia (TEAD), and its anabolic activity together with the isolated 20-hydroxyecdysone (20-HE) in orchidectomized male rats. TEAD was analyzed and standardized utilizing UPLC-PDA-ESI–MS/MS and UPLC-PDA-UV techniques, resulting in tentative identification of fifty compounds including polyphenols, steroids and triterpenoids. In addition, 20-HE was quantified, representing 26.79 μg/mg of the extract. Phytochemical investigation of TEAD resulted in the isolation of 20-HE from the ethyl acetate fraction (EFAD) and was identified by conventional spectroscopic methods of analysis. Furthermore, the anabolic effect of the isolated 20-HE and TEAD was then evaluated using in silico and in vivo models. Molecular docking experiments revealed in vitro selectivity of 20-HE towards estrogen receptors (ERs), specifically ERβ over ERα and androgenic receptor (AR). The anabolic efficacy of TEAD and 20-HE was studied in orchidectomized immature male Wistar rats using the weight of gastrocnemius and soleus muscles. The weights of ventral prostate and seminal vesicles were used as indicators for androgenic activity. Rats administered 20-HE and TEAD showed a significant increase (p = 0.0006 and p < 0.0001) in the net muscle mass compared to the negative control, while the group receiving TEAD showed the highest percentage among all groups at p < 0.0001. Histopathological investigation of skeletal muscle fibers showed normal morphological structures, and the group administered 20-HE showed an increase in cross sectional area of muscle fibers comparable to methandienone and testosterone groups at p > 0.99. A. dimorphostegia exhibited promising anabolic activity with minimal androgenic side effects. Since the discovery of testosterone in 1935, many derivatives have been synthesized to prolong its biological activity, rendering them orally active and developing products referred to as anabolic androgenic steroids (AAS) with maximum anabolic effects and minimum androgenic side effects ,3. TheirInterestingly, ecdysteroids, a class of hormones found in arthropods responsible for molting and reproduction, have shown anabolic effects in vertebrates and humans ,6,7. WhePhytoecdysteroids are the plant derived analogues of ecdysteroids, possessing a 14-α-hydroxy-7-en-6-one chromophore and a 27–29 carbon skeleton  EcdysterAtriplex belonging to the family Chenopodiaceae . Fi. Fi1H NMterature ,38,39. F1H NMR 0 MHz spe. Fifty compounds were tentatively identified, including phenolic acids and their derivatives, flavonoids, and their derivatives − ion at m/z 451.12 showing fragmentation peaks at m/z 353, indicating the presence of caffeoyl-quinic acid or chlorogenic acid. The peak at m/z 191 is due to quinic acid and was identified as an unknown chlorogenic acid derivative − at m/z 593.4 and fragment ions at m/z 353 [(M−H)−(120+120)]− and at m/z 383 [(M−H)−(90+120)]− indicates apigenin and two hexose moieties [5 showed a parent ion [M−H]− at m/z 417.01 and an MS2 fragment ion at m/z 255 , and was assigned liquiritin [6 was assigned ferulic acid with [M−H]− at m/z 193.1 and a fragment ion peak at m/z 178 [M−H−15]− due to the loss of a methyl group. The product ion at m/z 134 [M−H−15−44]− is due to the loss of methyl and carboxyl groups [7 presented with a base ion peak [M−H]− at m/z 431.04 and MS2 ion at m/z 285 [M−H−146]− due to the loss of a rhamnose moiety, and was assigned kaempferol rhamnoside [8 has a [M−H]− peak at m/z 153 characteristic of dihydroxybenzoic acid, while the ion at m/z 108 is due to the expulsion of the carboxyl moiety − at m/z 179.01 and a fragment ion at m/z 135 corresponding to [M−H−CO2]− − at m/z 487.08, was identified as caffeoyl-hexose-deoxyhexoside. The fragment ion peak at m/z 308 is due to the loss of caffeic acid moiety and at m/z 179 is due to the expulsion of hexose and deoxyhexose [11 exhibiting a molecular ion [M−H]− at m/z 595.2 was assigned quercetin pentosyl hexoside. The MS2 fragment ion at m/z 463 [M−H−132]− is due to the loss of pentose moiety and quercetin aglycone at m/z 301 [M−H−162−132]− − at m/z 521.1, and characteristic fragment ions at m/z 359 (rosmarinic acid) and at m/z 162 (hexoside moiety) − ion at m/z 297 and MS2 fragments at m/z 282 and 267 due to loss of methyl and methoxyl groups, was identified as retusin methyl ether [14 was identified as a caffeic acid ester derivative with a base ion peak at m/z 295. The fragment ion [M-116]−m/z 179 is formed after the ester breakdown and the ion peak at m/z 135 [M-116-CO2]− is characteristic of caffeic acid [15 was identified as a ferulic acid ester derivative with [M−H]− at m/z 309.53. The fragment ion at m/z 192 (ferulic acid) resulted from the ester breakdown and the loss of a residue [M−H−116]−. The fragment ion peak [M−H−116−CH3]− at m/z 177 resulted from the loss of the methyl of the methoxy group − at m/z 273, were assigned tetrahydroxyflavan (afzelechin), with the diagnostic product ion at m/z 179 due to the loss of the dihydroxybenzene moiety − at m/z 315.2 and fragment ions at m/z 301 and 272 characteristic of isorhamnetin [19 and 46 with a molecular ion [M−H]− at m/z 317 were tentatively identified as myricetin. The MS2 fragment ions at m/z 179 and 151 corresponds to the retrocyclization on the A–C ring and the loss of CO, respectively − ion at m/z 316.1, and a base peak at m/z 153 corresponding to dihydroxybenzoic acid [M−H−162]− and m/z 108 due to the loss of the carboxyl group of benzoic acid − at m/z 418.5, has a product ion at m/z 163 due to the loss of hydroxypalmatic acid moiety [M−H−256]−. The MS2 fragmentation pattern of m/z 163, 145, 119 and 93 is characteristic of the p-coumaroyl moiety. Thus, compound 22 was identified as coumaroyl-hydroxy-palmitic acid − molecular ion at m/z 585, was identified as quercetin-galloyl-pentoside. In the MS2 spectrum, fragment ions at m/z 153 and m/z 301 correspond to the galloyl unit and quercetin aglycone units, respectively [24 was identified as coumaroyl-hexose with the molecular ion [M−H]− at m/z 325.65 and the MS2 ion at m/z 163 resulting from the loss of glucose or galactose [25 showed an [M−H]− peak at m/z 577 and a base peak at m/z 285 [M−H−292]− owing to the loss of two hexoside moieties, and was suggested to be kaempferol-dideoxyhexoside (kaempferitrin) [26 was identified as dicaffeoyl spermidine with a molecular ion peak [M−H]− at m/z 468.2, a fragment ion at m/z 305 due to the loss of one caffeoyl moiety, and at m/z 145 corresponding to sperimidine with the loss of the two caffeoyl moeities [M−H−(162+162)]− [27 was identified as syringetin rutinoside with a molecular ion peak [M−H]− at m/z 579.2 and a fragment ion peak at m/z 345 owing to the loss of the rutinoside moiety [M−H−(308)]− [28 showed an [M−H]− peak at m/z 635.2 and a base peak at m/z 284 [M−H−351]− owing to kaempferol, and was tentatively identified as acetylated kaempferol deoxyhexosyl hexoside [29 with a [M−H]− peak at m/z 593.17 was tentatively identified as kaempferol deoxyhexosyl hexoside. The base peak at m/z 285 [M−H−308]− is due to the loss of the deoxyhexosyl moiety − and a base peak at m/z 315, attributed to the loss of hexose moiety producing the aglycone isorhamnetin [31 was identified as quercetin deoxyhexoside with a base ion peak [M−H]− at m/z 447, which produced a [M−H−146]− ion at m/z 301 due to the loss of deoxyhexosyl moiety − in addition to fragment ions at m/z 341 [M−H−90]− and 311 [M−H−120]− and a molecular ion peak [M−H]− at m/z 431.01, identical to apigenin-C-hexoside [33 showed a molecular ion peak [M+H]+ at m/z 577 representing daucosterol (sitosterol-glucoside), with a fragment ion [M+H−162]+ at m/z 413 due to the loss of the hexose moiety corresponding to sitosterol, and the fragment ion at m/z 369 is due to the loss of glucosyl moiety followed by dehydration − ion at m/z 623.3 and a MS2 ion at m/z 315 (isorhamnetin) after the removal of the rutinose moiety, was identified as isorhamnetin deoxhexosyl hexoside [35 showed a [M−H]− ion at m/z 461.07, and further fragmentation showed ions at m/z 285 and 175 corresponding to the aglycone kaempferol and glucouronide moiety. This compound was assigned kaempferol glucouronide − at m/z 385, was identified as sinapic acid hexoside. The base peak at m/z 223 [M−H−162]− was attributed to the loss of a hexosyl moeity [37 showed a [M−H]− molecular ion peak at m/z 623.2 and a base peak at m/z 315 (tamerexetin), and was tentatively identified as tamerexetin deoxyhexosyl hexoside [39 was identified as caffeoyl-coumaroyl spermidine with a molecular ion peak [M−H]− at m/z 452.4 + at m/z 397, was identified as β-sitosterol based on the fragments in the MS2 chromatogram [41 had a [M−H]− ion at m/z 519.5 showing MS2 peaks a at m/z 357 (pinoresinol), and the prominent ion at m/z 151 was due to the cleavage of the tetrahydrofuran ring − ion at m/z 237 and based on the spectral data, was identified as portulasoid (butoxyseptanoside) [49 was identified as ursolic acid with a parent ion peak [M+H]+ at m/z 457 and a fragment ion peak [M−H2O-COOH+H]+ at m/z 393 + at m/z 481 was tentatively identified as 20-hydroxyecdysone. The MS2 chromatogram showed sequential loss of water with a base ion peak M+H−2H2O]+ at m/z 445 and molecular ion peaks M+H−3H2O]+, M+H−4H2O]+ and [M+H−5H2O]+ at m/z 427, 409 and 391, respectively [53 was identified as lupeol. The molecular ion peak [M+H]+ at m/z 427.5 showed a fragmentation of [M+H−H2O]+ at m/z 409 and at m/z 397 due to the loss of two methyl groups [54 had a molecular ion peak [M+H]+ at m/z 413.2, showing fragment ions [M+H−H2O]+ and [M+H−2H]+ at m/z 395 and 411, respectively [55 was identified as olenoloic acid with a molecular ion peak [M+H]+ at m/z 457. The MS2 fragment ions were [M+H−H2O]+ at m/z 439, [M-COOH+ H]+ at m/z 411 and [M-COOH-H2O+H]+ at m/z 393 [56 showed a peak [M−H]− at m/z 657.2 and was identified as septanoecdysone − ,51. Compxyhexose . Compoun62−132]− . Compounxy group . Compounaglycone ,55. Comphamnetin . Compounectively . Compounuercetin . Compounoic acid . Compouneritrin) . Compoun [(M−H)−1+120]− anl moiety . Compounhamnetin . Compounydration . Compounhexoside  Compoundl moeity . Compounmatogram ,67. Compectively . Compounectively . Compounl groups ,72,73. Cectively  CompoundTEAD was analyzed using Ultra Performance Liquid Chromatography (UPLC). The PDA chromatogram of TEAD was recorded at 213 nm a and shoThe docked pose of 20-HE in the binding site of ERβ reveals a binding mode similar to that of the co-crystallized ligand, estradiol. The inhibitor occupies the hydrophobic central cavity of the binding pocket. The inhibitor is then anchored to the binding pocket via an array of hydrogen bonds. The cyclic end of the inhibitor is hydrogen bonded to the side chain of Arg346 and the backbone carbonyl of Leu339. Although the hydroxyl group of 20-HE occupies a virtually nearly identical position in space as the hydroxyl group of estradiol, it forms hydrogen bonds to different residues. The estradiol hydroxyl group is hydrogen bonded to the side chain of Glu305. Extending to the opposite side of the binding cavity, the aliphatic chain of 20-HE also contains a hydroxyl group that occupies a very similar position to that of the other hydroxyl group of estradiol. This hydroxyl group hydrogen bonds to the carbonyl of Gly472. Overall, the positioning of 20-HE is very similar to that of estradiol in the binding cavity of ERβ .α and AR immediately points out a critical determinant for selectivity towards ERβ  using SRB-U assay for 24 h, human SkMC was subjected to 20-HE and TEAD at concentrations of 0.01, 0.1, 1, 10 and 100 µg/mL . 20-HE ap = 0.54) increase in total body weight among all groups. However, groups receiving methandienone and testosterone showed a significant (p < 0.0001) increase in the mass of muscle bundle compared to the negative control, both by 80%. Similarly, the administration of 20-HE and TEAD increased the net mass of muscles significantly (p = 0.0006 and p < 0.0001) compared to the negative control by 76% and 112%, respectively. There was no significant (p > 0.5) difference between 20-HE and TEAD  increase in the mass of prostate and seminal vesicles, a 14-fold increase in comparison with the negative control. Moreover, when comparing group C to groups receiving 20-HE and TEAD, the mass of prostate and seminal vesicles also increased significantly (p < 0.0001), by 17-folds , significantly different from the methandienone group at p values of <0.0001 and 0.0005, respectively.The anabolic to androgenic ratio of each group was calculated first using the weight of the muscle bundle  and thenp values of 0.002 and <0.0001 respectively. There is no significant (p > 0.99) difference between the group receiving TEAD when compared to methandienone, and neither is there a significant difference when compared to 20-HE (p > 0.99). When comparing the group receiving 20-HE compared to the negative control, the difference is not significant, but the difference compared to testosterone is significant at p = 0.0036. Although the group receiving total extract showed a higher ratio when compared to 20-HE, the difference was not significant (p > 0.1).The muscle bundle to prostate and seminal vesicles ratio showed a significant difference in the group receiving TEAD when compared to the negative control and testosterone groups at The muscle mass percentage was calculated using the mean mass of the muscle bundle of both right and left hindlimbs against the mean final body weight of rats in each group at the end of the study . The groAll samples stained with H&E demonstrated almost normal morphological structures of skeletal muscle fibers, without abnormal subcellular alterations with normal intact intermuscular fine connective tissue, without abnormal inflammatory cell infiltrates and with normal vasculatures .p < 0.0001) in all groups compared to the negative control (p = 0.67) difference when compared to 20-HE (941 ± 168 µm²). Similarly, no significant difference (p > 0.99) when comparing methandienone (946 ± 132 µm²) to 20-HE treatments. However, TEAD treatment (683 ± 86 µm²) showed significant difference when compared to testosterone (p < 0.0001) and methandienone (p = 0.0004).The size of muscles increased ( control . While tp < 0.0001 (p < 0.0001. There was no significant difference (p = 0.6) between the groups receiving 20-HE and TEAD. The number of muscle fibers increased in all groups when compared to the negative control at < 0.0001 . The groA. dimorphostegia. TEAD was found to contain 20-HE (26.79 µg/mg), a phytoecdysteroid that was proven to have low acute toxicity [The purpose of the current study was to evaluate the anabolic effect of toxicity  and an an 20-HE 2.79 µg/mgtoxicity ,76. Sevetoxicity ,16. Howetoxicity . Atriplex species [A. dimorphostegia. The UPLC-PDA-MS/MS profiling of TEAD highlighted the presence of a range of metabolites including polyphenols, phenolic acids, flavonoid glycosides and aglycones. Examples include coumaroyl hydroxy-palmitic acid, quercetin galloyl pentoside, acetylated kaempferol hexoxyl-rhamnoside and quercetin rhamnoside. In addition to 20-HE as the major identified compound, sterols, steroids and triterpenoids such as β-sitosterol, stigmasterol and β/α-amyrin were also identified. This comes in context with previous reports which refers to the occurrence of different types of polyphenols and steroids previously identified from different  species ,50,67,73Several studies reported that polyphenols such as apigenin quercetin and phenolic acids are highly effective in attenuating muscle atrophy and enhancing muscle health. Apigenin reduced oxidative stress and inhibited hyperactive autophagy and apoptosis, alleviating age-related skeletal muscle atrophy in rats ,78. KanzTo understand the practical in vitro selectivity pattern of 20-HE towards ERβ over ERα and the AR, molecular docking experiments were conducted using all three receptors. In these experiments, the protein data bank (PDB) entries 3OLL, 3UUD and 2AM9 were used for ERβ, ERα and AR, respectively ,85,86. Tp = 0.0006 and p < 0.0001) in the net mass of muscles compared to the negative control, while the group receiving the TEAD showed the highest percentage (0.64%) among all groups. Tóth et al. proved that 20-HE increased fiber cross-sectional area in the regenerating soleus muscle, suggesting a beneficial effect on muscle regeneration without exhibiting the androgenic effects of steroids [p > 0.99). An effect even exceeding methandienone was found in vitro [The anabolic efficacy of TEAD and the isolated 20-HE was evaluated using testosterone enanthate and methandienone as positive controls in orchidectomized immature male Wistar rats . The grosteroids . The anain vitro . A 10-wein vitro .50 (>100 µg/mL).It could be concluded that TEAD is a potential anabolic agent containing 20-HE together with other secondary metabolites which previously reported to have anabolic effects and activities that promote muscle health ,76,77,78A. dimorphostegia Kar. & Kir were harvested from Wadi Al-Hilali, Arar-Dammam International North Road (50 km from Arar) in March 2018. The position from which the plant was harvested was determined using the global positioning system having coordinates 30°49′47.6″ N 41°14′44.1″ E. The plant collected was identified by Prof. Dr. Ahmed K. Othman, Biology Department, Faculty of Science, Northern Border University, Saudi Arabia. Plant material was then air-dried at room temperature and reduced to a fine powder. A voucher specimen was placed at the herbarium (PHG-P-AM-245) at the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Egypt. All solvents were of analytical grade, while those used in the preparative HPLC, and UPLC/PDA/ESI/MS assays were of HPLC grade. Methandienone and testosterone enanthate were obtained from Novector labs .The aerial parts of A. dimorphostegia aerial parts were extracted by maceration in 5 L of absolute ethanol for 24 h at room temperature, followed by filtration. The same process was repeated for four more days until exhaustion. The extracts were collected and dried under reduced pressure at 45 °C yielding 100 g residue. Ten grams of the dried alcohol extract was reserved for further analysis, labeled as TEAD and freeze-dried. The remaining 90 g of the dried extract was suspended in 500 mL distilled water. Fractionation was carried out successively using solvents of increasing polarities: n-hexane followed by ethyl acetate. Each fraction was dried completely under reduced pressure to yield n-hexane (38 g) and ethyl acetate (14 g) residues, respectively. The residues were then freeze-dried for further investigations.Two kilograms of the air-dried 2O: MeOH): 0–100% step-gradient with an increment of 20% methanol every 1000 mL. The column was flushed with 200 mL of 100% methanol for the last three fractions. Elution was monitored using TLC and (DCM: MeOH) (9:1) as the mobile phase. Eluents from 0–40%, 60–80% and 100% methanol were pooled into fractions 1, 2 and 3, respectively. Fraction 2 (60–80%) was further refined using column chromatography. The fraction weighing 1 g was dissolved in 5 mL DCM and chromatographed using 50 g Silica gel 60  column (1.5 × 100 cm). DCM and MeOH were used as eluents with a ratio of 9.5:0.5. Elution was monitored using TLC in a solvent system of DCM and MeOH (9:1). Fractions 101–153 showed a single green band after spraying with p-anisaldehyde/H2SO4, suggesting the presence of triterpenoid skeleton. Thus, it was pooled and dried under reduced pressure at 45 °C, yielding 55 mg white crystalline solid. EFAD was subjected to column chromatography. EFAD (4 g) was dissolved in 20 mL of distilled water and chromatographed over a 160 g Diaion HP-20  column (2.5 × 100 cm). Fractions were eluted using water and methanol  were measured in ppm, relative to TMS and 13C NMR chemical shifts to DMSO-d6, and were converted to the TMS scale by adding 39.49. NMR spectra were conducted using a Bruker Avance 400 MHz NMR spectrometer using DMSO-18 column with dimensions of 2.1 mm × 50 mm, 1.7 μm  was used. 20-HE was dissolved in methanol at a concentration of 100 μg/mL, then filtered using a 0.2 micropore filter. A gradient elution program was conducted using eluent A (0.1% formic acid in water) and eluent B (0.1% formic acid in methanol). The total run time was 32 min and a flow rate was kept at 0.2 mL/min. For mass spectrometric detection, a XEVO TQD triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source  was used. Mass spectra were detected in the ESI between m/z 100–1000. The peaks and spectra were processed using the Maslynx 4.1 software . LC-ESI-MS spectrometrical analysis of 20-HE Waters ™ ACQUITY Xevo ™ TQD system . A C18 column with dimensions of 100 mm × 2.1 mm   was used. The sample was dissolved in methanol at a concentration of 1 mg/mL and was then filtered using a 0.2 µm micropore filter. The instrument settings and the gradient elution program were conducted using the method described earlier by El Zahar et al. using eluent A (0.1% formic acid in water) and eluent B (0.1% formic acid in acetonitrile) [The profiling of TEAD was performed on a Waters™ ACQUITY Xevo ™ TQD system . A Cnitrile) . The floChromatographic analysis of alcohol extract was performed using an UPLC system Thermo Fisher UHPLC Dionex Ultimate 3000 . The system is equipped with a (ISO-3100SD) pump, (WPS 3000 SL) autosampler, (TCC-3000SD) column thermostat and (DAD-3000 RS) diode array detector . The software used was Chromeleon 6.8 . A column of Hypersil Gold was used with a 5 μm particle size and dimensions of 250 × 4.6 mm. The solvents used were 1% orthophosphoric acid in water (A) and acetonitrile (B). The separation was performed for a total of 70 min using gradient elution from 10% to 75% B in A for the first 35 min at a flow rate 0.5 mL/min, and then increased to 0.8 mL/min for the whole run. Then, the elution went from 75% to 100% B in A for 15 min and was kept isocratic for another 15 min. For the next 15 min, the elution was from 100% to 40% B in A and for the last 10 min kept isocratic from 10% B in A. UV detection was at 213 nm, column temperature at 30 °C and the injection volume was 20 μL. The isolated 20-HE (1 mg) was dissolved in 1 mL methanol, serial dilutions were performed using five concentrations  and a calibration curve was generated using the same method .The molecular docking experiments were carried out using protein crystal structures obtained from the Protein Data Bank (PDB) . The strThe human skeletal muscle cell line was cultivated as adherent cultures on Petri dishes and kept at 37 °C. The SRB-U assay was used to assess cell viability ,95 ThreeHandling of animals and experimental design were approved and conducted according to the guidelines of the Research Ethics Committee of the Faculty of Pharmacy, British University in Egypt, Cairo. . Twenty-five immature (21 days old) male Wistar rats weighing approximately 60 g were obtained from Nile Co. for Pharmaceutical and Chemical Industries, Cairo, Egypt. Animals were housed in a temperature-controlled environment at 25 °C with alternating 12 h light and dark cycles. They were kept on a standard diet and water ad libitum.Orchidectomy was performed using the technique described earlier . GeneralTwenty-three rats with an average weight of 62.25 ± 10.26 g were randomly divided into five groups and treated with test compounds by gavage and subcutaneous (SC) injection . Group AOn day 10, rats were killed by cervical dislocation. The seminal vesicles (S. vesicles), ventral prostate and levator ani were dissected out and weighed to the nearest 0.1 mg. The seminal vesicles were squeezed before weighing to remove any fluids. The soleus and gastrocnemius muscles were then dissected together as a bundle (muscle bundle) from the right and left hind limb of rats, weighed immediately and then stored in 10% neutral buffered formalin for 72 h for further investigation.Samples were trimmed and processed in serial grades of ethanol, cleared in Xylene, synthetic paraffin wax infiltration and embedded into Paraplast tissue embedding media. Then, 4 μm thick cross sections were cut by rotatory microtome. The sections were stained with Harris hematoxylin and eosin as a general tissue examination staining method. All methods of tissue samples processing and staining were as outlined before . Nine raAll statistical calculations and generation of graphs and the cumulative data of weight gain in rats, secondary sex organs and muscle fibers, as well as the muscle fiber’s cross-sectional area, were compared by using the t-test for unpaired samples and one-way ANOVA analysis followed by Tukey–Kramer as a post hoc test. All tests were performed using GraphPad Prism version 7.00. The data are expressed as means ± SD.Atriplex dimorphostegia total ethanol extract proved anabolic potency. This was represented by the increase in net muscle mass of gastrocnemius and soleus muscle, as well as an increase in the cross-sectional area of muscle fibers. Moreover, both 20-HE and TEAD exhibited a low androgenic effect on sex organs without increasing the weights of prostate and seminal vesicles of rats after administration for ten consecutive days. These findings indicate that A. dimorphostegia is a promising candidate for use as an anabolic agent and warrants further clinical investigation. Moreover, future study of the pharmacokinetics of TEAD and 2-HE in rats, as well as the lifespan, biodistribution, and muscle tissue retention, will be conducted to evaluate the biological effects of indicated doses.The


Drought stress usually causes huge economic losses for tobacco industries. Drought stress exhibits multifaceted impacts on tobacco systems through inducing changes at different levels, such as physiological and chemical changes, changes of gene transcription and metabolic changes. Understanding how plants respond and adapt to drought stress helps generate engineered plants with enhanced drought resistance. In this study, we conducted multiple time point-related physiological, biochemical,transcriptomic and metabolic assays using K326 and its derived mutant 28 (M28) with contrasting drought tolerance. Through integrative analyses of transcriptome and metabolome,we observed dramatic changes of gene expression and metabolic profiles between M28 and K326 before and after drought treatment. we found that some of DEGs function as key enzymes responsible for ABA biosynthesis and metabolic pathway, thereby mitigating impairment of drought stress through ABA signaling dependent pathways. Four DEGs were involved in nitrogen metabolism, leading to synthesis of glutamate (Glu) starting from NO−3 /NO−2 that serves as an indicator for stress responses. Importantly, through regulatory network analyses, we detected several drought induced TFs that regulate expression of genes responsible for ABA biosynthesis through network, indicating direct and indirect involvement of TFs in drought responses in tobacco. Thus, our study sheds some mechanistic insights into how plant responding to drought stress through transcriptomic and metabolic changes in tobacco. It also provides some key TF or non-TF gene candidates for engineering manipulation for breeding new tobacco varieties with enhanced drought tolerance. Drought stress is one of key restricting environmental factors that adversely affect plant growth and development. In particular, it dramatically reduces global crop productivity in arid and semi-arid regions, depending on its severity and duration . It attrBrassica rapa  is also a popular model plant for functional genomic studies  with contrasting drought tolerance. Through integration of RNA-seq with metabolic data, and construction of gene regulatory networks, we revealed that some key genes directly or indirectly regulate differential gene expression or accumulation of metabolites potentially involved in drought responses.Seeds of K326 and its derived EMS mutant, M28 with enhanced drought resistance, were sterilized and pretreated at 4°C for 2 days. The pretreated seeds were put into pots with nutrient soil for germination. The germinated seedlings with fully expanded cotyledons were transferred into small pots. Each pot contained the exactly same amount (120g) of nutrient soil, 10 seedlings per pot for each replicate, total three replicates for K326 or M28. All pots were randomly put into the incubator to grow under 22 ± 1°C,16h/8h light-dark cycle and 70% humidity. At the fully expanded five-leaf stage, each pot with 3 similar-sized seedlings was fully watered, and then all pots with K326 or M28 were randomly divided into 3 parts for drought treatment, natural drought (ND) treatment and 200 mM mannitol for simulated drought (SD) treatment. According to visible phenotypic changes (4h/24h for SD and 7D/14D for ND), we decided to collect the third leaf of each plant from the top to the bottom at 7 and 14 days after ND, or at 1h and 4h post SD for the downstream physiological and biochemical assays. Considering that drought induced transcriptional changes occur prior to occurrence of phenotypic changes, we decided to collect leaves with ND and SD and the corresponding CK at 0, 1, 2, 4 and 8h, respectively, for the downstream RNA-seq and metabolome analyses.Leaf water content assay: The collected leaves from drought treatment and CK were measured with a balance for fresh weight (FW) followed by merging the leaves into ddH2O for 6-8h for maximizing turgidity. Completely turgid leaves were measured with a balance for saturated fresh weight (SFW) followed by oven-dried at 80 °C for 24 h for measuring dry weight (DW). Relative leaf water content (RWC) and water saturation deficit (WSD) were calculated according to the formulas:Relative conductivity: The leaves were submerged into 10 ml ddH2O in the centrifuge tube, and constantly shaking at RT for 24 h. The first conductivity (A) was measured followed by heating to 95 °C for 15 min, after cooling down to RT, the second conductivity (B) was measured. The relative conductivity was calculated as A/B*100%.Net photosynthetic rate, Stomatal conductance and Transpiration rate: Those three parameters were measured and recorded by using portable photosynthesis system , which was conducted from 9:30 AM to 11:00 AM at 26°C and photosynthetic photon flux as 600 μmol·m-2·s-1.Enzyme activity assays: Firstly, 0.2 g of fully expanded functional leaves were weighed and ground into powder using liquid nitrogen. 4 mL of pre-cooled 150 mmol/L phosphate buffer solution (pH 7.0) was added to mix well, and the homogenate was transferred into a 15 mL centrifuge tube. After centrifugation at 12,000 rpm at 4 °C for 20 min, the supernatant was collected and used as the crude enzyme solution. MDA content and POD activity were determined according to the method described in Hu  and Hydrogen Peroxide assay kit  respectively, according to the manufacturer’s instructions.ed in Hu . SOD actFor construction of the recombinant of pTRV2: NCED1, a fragment of about 300 bp of NCED1 was cloned and inserted into the restriction site of rDNA11 of pTRV2. pTRV2: PP2C-37L and pTRV2: P450 84A1L were constructed by using the same method. pTRV1 and the three vectors containing the target gene sequences were then transformed into a bacteria A. tumefaciens strain GV3101, respectively.Nicotiana benthamiana) for infection. After 24-hour dark acclimation, inoculated tobacco plants were transferred to normal culture conditions  for 14 days. They were then subjected to drought stress treatment for 14 days, after which it was rehydrated. The leaf samples were collected at 7 days of drought stress for RNA extraction.Tumefaciens positive clones were selected for culture in 500 μL LB liquid medium (containing 50 μg/ml kana and 25 μg/ml rifampicin) with constant shaking at 200 rpm, at 28°C overnight. They were further cultured with 50 mL the same LB medium with constant shaking at 28°C for 16-24 h with OD600 reaching about 0.6-0.8. The thalli were collected by centrifugation  and treated with infection buffer , adjusted to OD600 between 1.0-1.5. The Agrobacterium solution with equal concentration of pTRV1 and pTRV2  were mixed and left at room temperature for 3 h. A syringe without a needle was used to inject 400-600 μL of the prepared infection solution into the back of the leaf from 25-day-old tobacco  according to the instructions. qRT-PCR was conducted by using fluorescent intercalating dye PowerUp™ SYBR™ Green Master Mix  with the QuantStudio™ 3 Real-Time PCR Instrument . The gene-specific primers are listed in Total RNA was extracted using collected leaves with ND and SD at 0, 1, 2, 4 and 8h. After completely removal of genomic DNA contamination, mRNA was enriched for RNA-seq library preparation following the kit instruction.For RNA-seq analyses, the quality of raw reads was processed using FastQC . AdapterGene Ontology (GO) and KEGG analyses of DEGs were analyzed using AgriGO  and KOBAThe freeze-dried samples were crushed using a mixer mill  with a zirconia bead for 1.5 min at 30 Hz. 100 mg of lyophilized powder per sample was dissolved using 1.2 ml of 70% methanol solution, vortexed 30 seconds every 30 minutes for 6 times in total. The homogenized samples were placed in a refrigerator at 4°C overnight. After centrifugation at 12,000rpm for 10 min, the extracts were filtrated using millipore filter (0.22 μm pore size). The filtrates were analyzed using an UPLC-ESI-MS/MS system. The analytical conditions were conducted as follows: UPLC: column, Agilent SB-C18 , the mobile phase consisting of solvent A (pure water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Sample measurements were performed with a gradient program as below: the starting conditions as 95% A, 5% B, then a programmed linear gradient as 5% A, 95% B within 9 min, and a composition of 5% A, 95% B kept for 1 min; a composition of 95% A, 5.0% B subsequently adjusted within 1.10 min and kept for 2.9 min; the flow velocity set as 0.35ml per minute; the column oven set to 40°C. The injection volume was 4 μl. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), AB4500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, which was operated in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550°C; ion spray voltage (IS) 5500 V (positive ion mode)/-4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) were set at 50, 60, and 25.0 psi, respectively; the collision-activated dissociation (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision nitrogen gas set to medium. DP and CE for individual MRM transitions were done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this periodDrought induced differential metabolic changes in M28 or K326 or between M28 and K326 were analyzed using MetaboAnalyst5.0 . PCA andβ = 14) was determined by using the function pickSoftThreshold in the R package. The default parameters were used to construct the scale-free network. The module Hub genes were identified using the threshold as |kME| > 0.8.The FPKM matrix of gene expression was used as input data for filtering the genes with low variations of expression levels among 30 samples according to the median absolute deviation (MAD) value > 0.5 for each gene. The remaining 14,445 genes were used to construct the weighted gene co-expression network using the R package WGCNA (v1.69) . The besTF centered regulatory networks were predicted using all sample gene expression matrix exprMatr as input argument, which was conducted using the R package GENIE3 (v1.12.0) . The preThrough EMS (Ethylmethanesulfonate) treatment following by subsequent screening, we obtained one mutant, named as M28, with increased tolerance to drought stress as compared to the wild type K326. To assess drought responses between K326 and M28, we grew them in the growth chamber, three plants per pot, for two weeks followed by progressive natural drought (ND) stress without watering. As shown in 2O2 accumulation at 14 D post ND, and 4h/24h post SD was less in M28 than in K326 , and obtained well-correlated biological triplicates for each time point  showed a clear separation between M28 and K326. We noticed that, after drought treatment, M28 mainly exhibited the first principal component (PC1) changes, whereas K326 mainly exhibited the second principal component (PC2) changes  in M28 or K326, and between M28 and K326 using orthogonal partial least squares discriminant analysis (OPLS-DA)  score >1, an FC > 1.5 or <0.66 and We also conducted KEGG pathway analyses using DiffExp metabolites before and after drought treatment, including 8/13 up-regulated and 4/28 down-regulated metabolites corresponding to CK and post drought treatment, respectively  was involved in several key steps for phenylpropanoid biosynthesis such as cinnamic acid to p-coumaric acid, and p-coumaroyl quinic acid to chlorogenic acid, indicating that this gene plays important roles in controlling phenylpropanoid biosynthesis in tobacco.After associating DEGs- and DiffExp metabolites-related KEGG pathways, we detected 16 pathways shared between DEGs and DiffExp metabolites , Nitab4.5_0000777g0010.1 (glutamine synthetase) and glutamate synthase (GOGAT), were involved in nitrogen metabolism, leading to synthesis of glutamate (Glu) starting from Nitab4.5_0001454g0180.1 and Nitab4.5_0000123g0370.1 by using RT-qPCR assay, which were identified from metabolic pathways linked with RNA-seq. Both genes were more expressed at 1 h, 2 h and 4 h but less at 8 h in M28 as compared to K326 post drought stress  was involved in Acetoacetyl coenzyme A biosynthesis, which is the key component in terpenoid pathways for synthesis of ABA    in drought responses are mediated by their interactive genes, we performed Weighted Gene Co-Expression Network Analysis (WGCNA) with 2,568 DEGs. We obtained 13 modules containing co-expression genes according to the WGCNA package function  or SD (24h). Moreover, M28 had higher photosynthetic rate, less stomatal conductance and transpiration rate. These changes lead to less membrane damage and water loss and higher capacity for producing energy through photosynthesis, facilitating M28 to adapt to drought stress through alleviating drought related damage. Similar findings have been reported in tobacco or other plant species in response to drought or other stress factors such as salt, cold and heat .NtEXGT gene, encoding xyloglucan endotransglucosylases/hydrolases (XTHs) in Nicotiana tabacum L., has been reported to be involved in abiotic stress response through ABA-dependent signaling pathway , carotenoid biosynthesis KEGG pathway  Figure. Transcr pathway . Alanine pathway . ArgininMYB TFs, which can be induced by drought stress, play vital roles in plants responding to drought stress, including maintaining cellular or organ structures and functions, adjusting stomatal movement and the regulation of secondary metabolisms  generation in transgenic Nicotiana benthamiana  can regulate expression of genes responsible for ABA biosynthesis  can be found in the article/WZ and RH conceived the study. ZHe analyzed the data. YL, PY, CY, RH, ZHu and JY performed the experiments. XC and QW assisted data analyses. BG edited the manuscript. ZHe and WZ wrote the manuscript with contribution from all authors. All authors contributed to the article and approved the submitted version.


Together, our findings suggest that a multi-ingredient AOX supplement may attenuate the IR-induced cellular damage response and represents a feasible and cost-effective preventative supplement for at-risk populations of radiation exposure.Radiation exposure is an undeniable health threat encountered in various occupations and procedures. High energy waves in ionizing radiation cause DNA damage and induce reactive oxygen species (ROS) production, which further exacerbate DNA, protein, and lipid damage, increasing risk of mutations. Although endogenous antioxidants such as superoxide dismutase have evolved to upregulate and neutralize ROS, exogenous dietary antioxidants also have the potential to combat ionizing radiation (IR)-induced ROS production. We evaluated a cocktail of ingredients (AOX) purported to have antioxidant and mitochondrial protective properties on the acute effects of IR. We show that IR stimulates DNA damage through phosphorylation of DNA repair proteins in the heart, brain, and liver of mice. AOX showed partial protection in brain and liver, through a lack of significant activation in given repair proteins. In addition, AOX attenuated the IR-induced increase in NF-kβ mRNA and protein expression in brain and liver. Lastly, cytochrome  Radiation exposure is a concern for many populations and professions, as high energy waves, particularly ionizing radiation (IR), alter chemical structure in cells , induce The mutagenic effects and cellular damage associated with IR are primarily caused by two cellular events ,11,12. F2O+ and a free electron, which subsequently produces free radicals and ROS [12 [Secondly, IR-induced effects include the generation of ROS, which oxidize molecules and cause further damage to DNA, lipids, and proteins in the cell . The hig and ROS . Molecul and ROS . As prot and ROS . Exogeno and ROS ,19. Prev and ROS . However and ROS  or atten and ROS ,19,20, i and ROS ,23, asta and ROS , zeaxant and ROS , vitamin and ROS , vitamin ROS [12 , seleniu ROS [12 , folate  ROS [12 , CoQ10 [ ROS [12 ,31, α-li ROS [12 , and vit ROS [12 . Importa ROS [12 . Consequ ROS [12 ,36 and e ROS [12  dose of Thus, the aim of our study was to test the hypothesis that our multi-ingredient antioxidant supplement (AOX) would provide cellular protection to rodents exposed to a single high dose of IR (2 Gy). We employed varying molecular biology techniques to assess nuclear and mitochondrial DNA damage, inflammatory response, and mitochondrial stress response, following acute high dose IR in mice. We provide evidence that partial cellular protection may be achieved when ingesting AOX prior to IR exposure. This AOX represents a feasible, cost-effective therapeutic intervention that has the potential to reduce the increased risk in mutagenesis when exposed to IR. We specifically designed the study to interrogate the potential for the supplement to prophylactically protect against IR exposure that would be of relevance to patients undergoing diagnostic imaging, radiation workers and those at risk of nuclear accidents.ad libitum diet of standard chow throughout the experimental period. Mice were fasted for 12 h prior to gavage feeding and experimental procedures. An antioxidant cocktail . Animals in the acute and later cohorts were sacrificed at +30 min and +24 h post radiation exposure, respectively. The blood, liver, heart, and brain were rapidly removed, snap-frozen, and stored at −80 °C until analysis. Samples intended for flow cytometry were incubated in 1 mL of buffer containing 10 g/L collagenase B , 4 g/L Dispase II  and Ham’s F-10 media, at 37 °C.Animals and study design. All animal experiments and procedures were performed with the approval of McMaster University Animal Research Ethics Board and conformed to the standards of the Canadian Council on Animal Care (AUP#20-04-17). Male C57BL/6J mice were ordered from Jackson Laboratories  and randomly assigned to one of two separate cohorts, acute (+30 min) or later (+24 h) post-IR collection times. Furthermore, each of these two groups were further divided into one of three study groups: CON (no RAD nor AOX), RAD, or RAD + AOX. Animals were maintained in standard mouse cages (3 mice per cage) on an Flow cytometry. Flow cytometry technique was performed on liver, heart, and brain cells, as well as bone marrow (BMCs) and peripheral blood mononuclear cells (PBMCs). Prior to fixation, cells were stained with LIVE/DEAD® Fixable Dead Cell Stain Kit  viability dye for 10 min at 4 °C in the dark. Samples were then washed and fixed with BD Cytofix/Cytoperm Buffer™  to permeabilize the cells, incubating at 4 °C for 20 min. After fixation, cells were washed, centrifuged at 500× g for 5 min and cell pellets were resuspended in 100 μL BD Perm/Wash™ buffer . Cells were incubated with Phospho-Histone H2A.X (Ser139)  Rabbit mAb (Alexa Fluor® 488 Conjugate) antibody  for 1 h at 4 °C in the dark. Following incubation, cells were washed and resuspended in 400 µL PBS  and spun at 500× g for 5 min at 4 °C. Tissues with a prominent pellet were resuspended in up to 300 μL PBS while PBMCs and BMCs were resuspended in 150 μL PBS. The cell suspension was filtered through a 100 µm cell strainer prior to analysis. All samples were immediately analyzed using a flow cytometer .Immunoblotting. Approximately 20 mg of snap-frozen tissue was homogenized in Axygen™ Snaplock Microcentrifuge tubes  via the FastPrep-24 Tissue and Cell Homogenizer in chilled RIPA lysis and extraction buffer  supplemented with protease and phosphatase inhibitor cocktail . The homogenate was centrifuged for 20 min at 4 °C and 12,000× g, followed by removal of the supernatant. Total protein concentrations were determined using a BCA Protein Assay Kit  and protein samples were prepared in 1× Laemmli Buffer . Prior to gel electrophoresis, samples were heated at 95 °C for 5 min and micro-centrifuged. Immunoblots were resolved using a 4–20% Criterion TGX Precast Midi Protein Gel  with a PageRuler Plus Prestained Protein Ladder at 10 to 250 kDA . All blots were run in 1× Tris/Glycine/SDS buffer . Protein samples were transferred to a membrane using Trans-Blot Turbo Midi Nitrocellulose Transfer Packs  and subsequently stained with Ponceau S solution  for 5 min. Membranes were imaged using the ChemiDoc MP Imaging System (Bio-Rad Laboratories) and placed in blocking solution (5% BSA in 1× TBS) for 1 h. Blots were left to incubate in primary antibody at 4 °C overnight with constant agitation. All primary antibodies were prepared in 5% BSA in TBS  for 40 s at a speed of 6 m/s. All samples were incubated at room temperature for 5 min. 10µL chloroform was added to each sample, mixed vigorously for 30 s, incubated at RT for 3 min, and spun at 12,000× g for 15 min at 4 °C. The aqueous phase containing RNA was transferred to a new tube, precipitated with 250 µL isopropanol, incubated for 10 min at 4 °C, and centrifuged at 12,000× g for 10 min at 4 °C. The pellet was immediately resuspended in 500 µL 75% ethanol, vortexed briefly, and centrifuged at 7500× g for 5 min at 4 °C. Prior to solubilization, the pellet was air dried for 10 min. The pellet was resuspended in 30 µL RNase-free water. RNA concentration (ng/mL) and purity (260/280) were determined with the Nano-Drop 1000 Spectrophotometer (ThermoFisher Scientific).cDNA Synthesis. Reverse transcription was performed on 1 μg cell lysate of RNA using a SuperScript™ IV VILO™ Master Mix , as per the manufacturer’s protocol. The reaction mixtures were incubated in a T100 Thermal Cycler (Bio-Rad Laboratories) with thermocycling conditions of 25 °C for 10 min , 50 °C for 10 min (Elongation), and 85 °C for 5 min (Enzyme Inactivation). Synthesized cDNA was diluted with nuclease-free water to obtain a concentration of 5.5 ng/µL prior to storage at −80 °C.Quantitative PCR. Gene expression was determined by running 4.5 µL of cDNA template (5.5 ng/µL) to a final volume of 10 uL on a CFX384 Real-Time System (Bio-Rad Laboratories) using the TaqMan Fast Advanced Master Mix , using the following TaqMan cycling conditions: 20 s at 95 °C for initial denaturation, followed by 40 cycles with 3 s denaturation at 95 °C, and 30 s annealing and elongation at 60 °C. The fluorescence threshold was automatically set above the background level. Fold-increase in mRNA probes of interest . The absence of genomic contamination was ensured using an NTC control for each probe. All RT-qPCR reactions were run in duplicates.interest  was calcLORD-Q assay. Tissue fragments from heart, brain, and liver were homogenized in lysis buffer using the FastPrep-24 Tissue and Cell Homogenizer . DNA was then isolated and purified, as per manufacturer instructions with DNeasy Blood and Tissue Kit . DNA concentration (ng/mL) and purity (260/280) was determined by spectrometric analysis using the Nano-Drop 1000 Spectrophotometer (Thermo Scientific). Samples were then diluted to 7.5 ng/uL where 1 µL was used per 10 µL reaction. In addition, the rt-PCR reaction contained forward and reverse primers for mtDNA, 1× KAPA2G Fast Hot Start ReadyMix, and LightCycler 480 ResoLight dye. The ResoLight dye is provided at 20× concentration, which was optimized and diluted to 0.2×. Primers used for amplification of the mitochondrial short (S) and long (L) DNA fragment are listed in t (Cp) scores of short (CpS) and long fragments (CpL), using the formula below (1). Non-irradiated mice were used as the control (Ref). Amplification efficiencies (E) for both primer sets were determined through a standard curve with serial dilutions of 24, 16, 8, 4, 2, 1, 0.5 and 0.25 ng/uL. This protocol was adapted from Lehle et al. (2014), and Dannenmann et al. (2017) [. (2017) ,39.(1)LA higher percentage of γ-H2AX-positive cells was observed, as a result of IR, across multiple tissues C–G. In ip < 0.05). pATM expression was also higher in RAD + AOX animals compared to CON heart and brain tissue. Specifically, pATM expression was 3.6-fold higher in the heart (p < 0.05), and 4.6-fold in the brain (p < 0.05); whereas, in liver, there was no significant difference in pATM levels between RAD + AOX and CON.Higher pATM expression was found in RAD and RAD + AOX groups compared to CON across all three interrogated tissues H–K. The p = 0.082)  L. Import= 0.082) L. No sig= 0.082) M,N.NF-κβ, STAT3, and CDKN1A in heart, brain, and liver harvested 24 h following IR.To investigate the protective effect of AOX upon IR-induced DNA damage, we measured the mRNA abundance of inflammatory transcription factors Relative to CON, NF-κβ mRNA abundance was significantly higher in RAD animals in both the brain and liver by 2.3 and 2.0-fold, respectively. RAD + AOX animals showed no significant difference compared to CON B–D. RelaSTAT3 transcripts were significantly higher in brain tissue of RAD mice (2.3-fold), with no significant elevation in the RAD + AOX group. Heart and liver tissues did not show a significant difference in STAT3 transcription between groups  and brain (2.1-fold), with no radiation-induced elevations observed in RAD + AOX animals vs. CON (COX4I1), showed a similar pattern for both heart and brain, but did not reach statistical significance. No effect of IR was found on mRNA abundance for either COX1 or COX4I1 in the liver  is higher than the typical dose of exposure during nuclear medicine procedures (~0.02 Gy for PET/CT scan); however, it is known that prolonged and repeated IR exposure can nevertheless compound within the body and lead to adverse effects in the heart and brain ,40. Surv99mTc MDP) bone scans, an attenuation of the increase in percentage of γ-H2AX foci was observed in isolated PBMCs when compared to PBMCs isolated from individuals that did not preemptively supplement with antioxidants, suggesting that antioxidants taken prior to radiation exposure may prevent DNA injury [Indeed, earlier studies of patients undergoing diagnostic imaging have shown promising results. Velauthapillai and colleagues showed that when taking oral antioxidants prior to technetium-99m methylene diphosphonate  and trending (COX4I1) higher in heart and brain tissue of RAD animals, but not in those treated with AOX and exposed to IR when compared to CON. Numerous studies have shown induction of mtDNA lesions by radiation, both ionizing and ultraviolet [COX mRNA transcripts in heart and brain. In liver tissue, however, no changes in gene transcripts were detected following radiation. The liver possesses greater regenerative properties, with increased rate of proliferation [COX mRNA transcripts, whereas an investigation 12 h post radiation may have allowed for the observation of significant changes. In addition, although not significant, our results suggest a trend towards an increase in mtDNA lesions in the liver immediately after radiation. Interrogating an intermediary timepoint, possibly 12 h post radiation, may have allowed more time for mtDNA lesions to develop and be observed. Together, we provide evidence that our formula may be beneficial and protective against mitochondrial-related radiation-induced damage, in heart, brain and possibly liver given a more inclusive timeline.In addition to protecting against nuclear DNA damage, protection to mitochondrial DNA damage is crucial due to limited repair mechanisms of mtDNA and its increased rate of mutagenesis ,14. Inde2+ ,47, whicraviolet ,48, likeraviolet . Interesferation ,50, possNF-kβ was significantly higher in liver and brain of RAD mice 24 h post-IR. This translated into an upregulation in protein expression in liver specifically. It is well known that NF-kβ is activated following radiation [Furthermore, it stands to reason that combining several individual antioxidant supplements may have a synergistic effect, not only related to nuclear and mitochondrial DNA protection, but also other radiation-mediated consequences such as inflammation, and possibly even greater benefits with continuous administration of AOX. The inflammatory response is a protective mechanism by which the immune system responds to foreign pathogens and damaged cells . Herein,adiation . It has adiation . Inhibitadiation . Interesadiation . In addiadiation .Collectively, our study provides evidence that a single dose of a multi-ingredient antioxidant supplement can have some beneficial effects in vivo, by partially negating the consequences of radiation exposure. Although not tested in our current investigation, it is possible that multiple intermittent doses of antioxidants prior to exposure may result in greater protection. In addition, the timing of oral gavage feeding represents a challenge, as it is possible that different ingredients have different timing required to be bioavailable to tissue. Furthermore, studies of continuous administration of antioxidants following high exposure to radiation increased survival in mice ,56, suggPCT/CA2020/051439 filed at the European patent and trademark office (PCT) covers the formulation used in this research work.