Simultaneous determination of four phytoecdysteroids by LC-MS/MS: application to a comparative pharmacokinetic study in normal and adjuvant arthritis rats after oral administration of C. officinalis Kuan phytoecdysteroids extract.

C. officinalis Kuan is the dry root of Cyathula officinalis Kuan. Clinically, it is used for fall and flutter injury, rheumatism and arthralgia. Phytoecdysteroids have significant anti-inflammatory effects, and the phytoecdysteroids present in C. officinalis Kuan exhibit potential for treating rheumatoid arthritis.This study first developed a selective, accurate and efficient LC-MS/MS method for 12-day pharmacokinetic studies regarding the simultaneous determination of cyasterone, 25-epi-28-epi-cyasterone, precyasterone and capitasterone from C. officinalis Kuan phytoecdysteroids extract in normal and adjuvant arthritis rats.An Agilent Eclipse Plus C18 RRHD column (1.8 µm, 50mm × 2.1 mm) with a gradient mobile phase consisting of water (A) and acetonitrile (B) was used for analysis. The mass analysis was performed in an Agilent 6430 QQQ-MS mass spectrometer with positive mode multiple reaction monitoring (MRM).The results indicated that the AUC0-t and AUC0-∞ values of the four phytoecdysteroids in adjuvant arthritis rats were different from those in normal rats on the first day, which could provide a helpful reference for pharmacological and toxicological studies, as well as clinical applications of C. officinalis Kuan in the treatment of rheumatoid arthritis.

1. C. officinalis Kuan is the dry root of Cyathula officinalis Kuan which has been used for the treatment of flapping injury, rheumatism arthralgia, foot flaccidity, and tendon contracture thousands of years in China, and has been officially included in the Chinese Pharmacopoeia.2. A highly accurate, stable, and sensitive ultra-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method was first established and validated for simultaneously determination four phytoecdysteroids: cyasterone, 25-epi-28-epi-cyasterone, precyasterone and capitasterone in normal and adjuvant arthritis rats plasma samples 12 days of continuous gavage of C. officinalis Kuan phytoecdysteroids extract.3. The phytoecdysteroids is the important component of C. officinalis Kuan, which is difficult to separated. And there is no report for the pharmacokinetic study of phytoecdysteroids from C. officinalis Kuan. And the method provides a good reference for the follow-up studies clinical medication of the phytoecdysteroids from C. officinalis Kuan.

De Novo Transcriptome Assembly of Two Microsorum Fern Species Identifies Enzymes Required for Two Upstream Pathways of Phytoecdysteroids.

Microsorum species produce a high amount of phytoecdysteroids (PEs), which are widely used in traditional medicine in the Pacific islands. The PEs in two different Microsorum species, M. punctatum (MP) and M. scolopendria (MS), were examined using high-performance liquid chromatography (HPLC). In particular, MS produces a high amount of 20-hydroxyecdysone, which is the main active compound in PEs. To identify genes for PE biosynthesis, we generated reference transcriptomes from sterile frond tissues using the NovaSeq 6000 system. De novo transcriptome assembly after deleting contaminants resulted in 57,252 and 54,618 clean transcripts for MP and MS, respectively. The clean Microsorum transcripts for each species were annotated according to gene ontology terms, UniProt pathways, and the clusters of the orthologous group protein database using the MEGAN6 and Sma3s programs. In total, 1852 and 1980 transcription factors were identified for MP and MS, respectively. We obtained transcripts encoding for 38 and 32 enzymes for MP and MS, respectively, potentially involved in mevalonate and sterol biosynthetic pathways, which produce precursors for PE biosynthesis. Phylogenetic analyses revealed many redundant and unique enzymes between the two species. Overall, this study provides two Microsorum reference transcriptomes that might be useful for further studies regarding PE biosynthesis in Microsorum species.

[Physiological and biochemical studies as a necessary component of the algorithm for assessing the effectiveness of food minor biologically active substances].

Preclinical physiological and biochemical studies of the effectiveness of the tested biologically active substances in the conditions of simulated pathology in laboratory animals are an important stage preceding the assessment of the clinical effectiveness of the created specialized foods for therapeutic or preventive nutrition. The aim of this work is a brief review of the developed algorithm for preclinical assessment of the effectiveness of specialized foods, which includes a set of sequential stages of testing the safety, stability, bioavailability and effectiveness of biologically active substances in experiments on laboratory animals. Results. The paper presents a brief review of methodological approaches for in vivo preclinical assessment of the effectiveness of minor plant biologically active substances - polyphenols from bilberry fruits and leaves and phytoecdysteroids extracted from spinach. A beneficial effect of bilberry leaves' polyphenols on carbohydrate and lipid metabolism of male Zucker and Wistar rats was shown, which determined the necessity to increase the effectiveness of the developed polyphenolic ingredient by sorption on a food carrier. When evaluating the in vivo effects of polyphenols from blueberries and leaves sorbed on buckwheat flour, a pronounced hypoglycemic effect was found. The experiments aimed to the assessment of the adaptogenic effects of phytoecdysteroids from spinach leaves showed a significant decrease in the content of the main biomarkers of the stress system, which indicates a «smoothing¼ of the response of the animal organism to a strong stress impact. Conclusion. The inclusion of minor biologically active substances into the composition of foods for preventive and therapeutic nutrition is the necessary condition to improve their effectiveness. The scientific and practical significance of the preclinical evaluation to a decisive extent depends on the presence of a correctly selected biomodel. Thus, the adequacy of the assessment of the effects of minor biologically active substances as functional food ingredients in the composition of the specialized food products depends on the reproducibility of pathological processes (clinical, biochemical and morphological disorders) characteristic for the studied disease on the selected in vivo model.

[Spinach and quinoa - prospective food sources of biologically active substances].

Formally, phytoadaptogens extracted mainly from medicinal plants are not food substances, and their use in preventive nutrition is significantly limited. However, screening studies can detect phytoadaptogens in food plants, however in much smaller quantities. For example, phytoecdysteroids are biologically active substances (BAS), used to relieve chronic fatigue syndrome, reduce nervous and muscle fatigue, improve memory and attention processes. Phytoecdysteroids can be isolated not only from various medicinal plants, but also from food plants like spinach (Spinacia oleracea) and quinoa (Chenopodium quinoa). Researchers show interest in possible synergies in the manifestation of a wide range of pharmacological effects, determined by a combination of phytoecdysteroids and other biologically active substances of these food plants. The aim of this research is a brief analytical review of publications characterizing the spectrum of minor biologically active substances of spinach and quinoa, as well as those devoted to the experimental assessment of their pharmacological activity in experiments on laboratory animals. Results. An analysis of the publications presented in the review indicates that spinach and its extracts are promising food sources of phytoadaptogens, carotenoids, phenolic compounds, in particular quercetin, kempferol, phenolic acids (p-coumaric, ferulic), as well as lignans metabolized into biologically active phytoestrogens (enterolactone and enterodiol). The possible prophylactic effect of spinach is being actively studied in vivo in laboratory animals when modeling diseases such as diabetes mellitus, cardiovascular and cancer diseases. Numerous adaptogenic effects and the neuroprotective effect of spinach and its extracts are shown, expressed in improving the memory and learning of animals. Publications of the last decade indicate an increased interest in such a promising food source of high-grade protein and a wide spectrum of biologically active substances, including phytoecdysteroids, as quinoa grains. More than 20 phenolic compounds in free or conjugated forms were found in quinoa grains; tannins, saponins, sterols, phytic acid, and ecdysteroids are present in small quantities. The consumptionof quinoa can have a beneficial effect on the organism, normalizing carbohydrate and lipid metabolism, reducing body weight and preventing excessive lipid peroxidation. The anti-inflammatory effects and antioxidant effects of quinoa are widely discussed. Conclusion. Spinach, quinoa and their extracts are promising food sources of biologically active substances, their pharmacological action is confirmed from the standpoint of evidence-based medicine by in vivo experimental studies. Accordingly, it is advisable to expand the use of these food plants for inclusion in biologically active dietary supplements, functional food ingredients and specialized food products for various purposes.

[Phyto pharmacological correction of functional state, hormonal status and memory productivity in patients of different ages with organic changes of brain vessels.]

The course intake of the nutritional supplement «Kardisten¼ normalizes increased levels of cortisol in the blood, optimizes the activity of the vessels of the brain, improving the functional state and leading to an increase in the productivity of short-term and long-term memory, more pronounced in the group of persons up to 50. At initial organic changes in the brain vessels, «Kardisten¼ significantly reduces the level of cortisol, increases to a greater extent the productivity of memory than at atherosclerosis. «Kardisten¼ activates the natural mechanisms of increasing the functional capabilities of an organism, exerting a beneficial effect on the body of adults and the elderly people, and can be recommended for the correction of physical, mental health and preservation of active longevity.

Isoprenoid-derived plant signaling molecules: biosynthesis and biological importance.

MAIN CONCLUSION: The present review summarizes current knowledge of the biosynthesis and biological importance of isoprenoid-derived plant signaling compounds. Cellular organisms use chemical signals for intercellular communication to coordinate their growth, development, and responses to environmental cues. The skeletons of majority of plant signaling molecules, mediators of plant intercellular 'broadcasting', are built from C5 units of isoprene and therefore belong to a huge and diverse group of natural substances called isoprenoids (terpenoids). They fill many important roles in nature. This review summarizes current knowledge of the biosynthesis and biological importance of a group of isoprenoid-derived plant signaling compounds.

Chromatographic separation, determination and identification of ecdysteroids: Focus on Maral root (Rhaponticum carthamoides, Leuzea carthamoides).

The review presents general principles for choosing optimal conditions for ecdysteroid separation, identification, and isolation using HPLC/TLC techniques in RP, NP-HILIC or NP modes. Analytics of ecdyteroids pose a still insufficiently resolved problem. Plant-derived ecdysteroids are a point of interest of pharmaceutical industry and sport medicine due to their postulated adaptogenic and anabolic properties. In insects, ecdysteroids regulate larval transformation. Maral root (Rhaponticum carthamoides, Leuzea carthamoides), traditional Siberian folk-medicine plant used as stimulant to boost overall health and fitness, is a particularly rich source of a wide variety of phytoecdysteroids. The similarity of molecular structures of ecdysteroids present in its extracts together with high content of unrelated compounds of similar chromatographic characteristics makes optimization of separation, identification and isolation of ecdysteroids a difficult analytical task. In that respect, two-dimensional separations, two-dimensional separations, 2D HPLC or 2D TLC, could be of use. For identification, the hyphenated techniques are particularly important. Thus, comprehensive overview of MS spectral parameters of ecdysteroids is provided. Described principles could easily be applied for separation of ecdysteroids in extracts from other sources. They are also useful for development of separation procedures for isolation of ecysteroids in preparative-scale applications.

Plant ecdysteroids: plant sterols with intriguing distributions, biological effects and relations to plant hormones.

MAIN CONCLUSION: The present review summarises current knowledge of phytoecdysteroids' biosynthesis, distribution within plants, biological importance and relations to plant hormones. Plant ecdysteroids (phytoecdysteroids) are natural polyhydroxylated compounds that have a four-ringed skeleton, usually composed of either 27 carbon atoms or 28-29 carbon atoms (biosynthetically derived from cholesterol or other plant sterols, respectively). Their physiological roles in plants have not yet been confirmed and their occurrence is not universal. Nevertheless, they are present at high concentrations in various plant species, including commonly consumed vegetables, and have a broad spectrum of pharmacological and medicinal properties in mammals, including hepatoprotective and hypoglycaemic effects, and anabolic effects on skeletal muscle, without androgenic side-effects. Furthermore, phytoecdysteroids can enhance stress resistance by promoting vitality and enhancing physical performance; thus, they are considered adaptogens. This review summarises current knowledge of phytoecdysteroids' biosynthesis, distribution within plants, biological importance and relations to plant hormones.

20-hydroxyecdysone accumulation and regulation in Ajuga lobata D. Don suspension culture.

Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid 20-hydroxyecdysone (20E) which can regulate the molting process of larvae. We characterized the culture conditions to optimize 20E production. Growth of A. lobata D. Don cells fits the logistic equation curve with a growth cycle of 19 days. Medium conductivity was negatively correlated with dry cell weight and 20E accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced 20E synthesis, but supplementing medium with 20E precursors mevalonic (MVA), α-Pinene, and nitric oxide (NO) can significantly promote cell growth and influence 20E accumulation. Combination of α-Pinene, MVA, and SNP significantly elevated 20E accumulation, thus may synergistically enhance 20E synthesis in A. lobata D. Don. The optimal concentrations of α-Pinene, MVA, and NO donor SNP in suspension culture were 50 µL L(-1), 10 mg L(-1), and 80 µmol L(-1).

Evaluation of Ajuga bracteosa for antioxidant, anti-inflammatory, analgesic, antidepressant and anticoagulant activities.

BACKGROUND: Ajuga bracteosa has been extensively used traditionally for the treatment of a variety of diseases. The aim of the study was to scientifically validate the wide-scale exploitation of A. bracteosa in folk medicine various in vitro and in vivo assays. Moreover, these activities were related to the intrinsic biologically active phytoecdysteroids of A. bracteosa. METHODS: Aerial and root parts of A. bracteosa were first extracted separately with chloroform (AbCA and AbCR) and the residue was again extracted with methanol (AbMA and AbMR). Total flavonoid and phenolic contents were assayed as quercetin (QE) and gallic acid equivalents (GAE), respectively. These extracts were analyzed for in vitro antioxidant assessment including DPPH and H2O2 (% inhibition of free radicals), and reducing power and phosphomolybdenum methods (ascorbic acid equivalents AAE mg/g DW). Further, these extracts were assayed in vivo in separate groups of Sprague-Dawley rats for carrageenan induced rat paw edema inhibition, hotplate antinociception, forced swim antidepression and anticoagulation. Dose of each crude extract and standard drug given to rats was 200 mg/Kg- and 10 mg/10 mL/Kg body weight respectively. Plant extracts and standard drugs were administered orally, 60 min prior to the conduction of assays. Moreover, biologically active phytoecdysteroids were screened in A. bracteosa with the help of RP-HPLC. RESULTS: AbMA represented highest values of flavonoids (QE 1.98 % DW) and phenolic contents (GAE 5.94 % DW), significantly scavenged DPPH radicles (IC50 36.9) and reduced ferric ions with 718.4 mg ascorbic acid equivalent/g (AAE). Highest total antioxidant capacity was expressed by AbMR (927 mg AAE) with an IC50 value 19.1 µg/mL. The extracts which were found potent anti-oxidants, were also good at in vivo activities. AbMA significantly reduced edema in all the three hours of treatment (67.9, 70.3 and 74.3 %). AbMA also showed maximum nociceptor suppression in analgesic assay by delaying the time to start licking of paws in rats (57.7 ± 4.9 s). In addition, maximum anti-coagulation was also exhibited by AbMA (89.3 s), while all extracts were found strong antidepressants (≤15.66 s immobility time). Screening of biologically active phytoecdysteroids revealed the presence of 20-hydroxyecdysone (20-HE), makisterone (MKA), cyasterone (CYP) and ajujalactone (AJL). Total phytoecdysteroid content found in A. bracteosa was 1232.5 µg/g DW and 20-HE was most abundant (1232.5 µg/g DW) as compared to other phytoecdysteroids. CONCLUSION: Based on the tested in vitro and in vivo activities, AbMA was found to be a promising bioactive extract. These activities can be attributed to the intrinsic polyphenols and phytoecdysteroids contents of A. bracteosa.

Ecdysterone and Turkesterone-Compounds with Prominent Potential in Sport and Healthy Nutrition.

The naturally occurring compounds ecdysterone and turkesterone, which are present in plants, including Rhaponticum carthamoides Willd. (Iljin), Spinacia oleracea L., Chenopodium quinoa Willd., and Ajuga turkestanica (Regel) Briq, are widely recognized due to their possible advantages for both general health and athletic performance. The current review investigates the beneficial biological effects of ecdysterone and turkesterone in nutrition, highlighting their roles not only in enhancing athletic performance but also in the management of various health problems. Plant-based diets, associated with various health benefits and environmental sustainability, often include sources rich in phytoecdysteroids. However, the therapeutic potential of phytoecdysteroid-rich extracts extends beyond sports nutrition, with promising applications in treating chronic fatigue, cardiovascular diseases, and neurodegenerative disorders.

Phytoecdysteroids from Dioscorea dumetorum (Kunth) Pax. and their antioxidant and antidiabetic activities.

Diabetes is a significant global health challenge, affecting circa 540 million adults worldwide. Dioscorea dumetorum, a Nigerian folkloric antidiabetic plant is severely understudied in terms of its bioactive phytochemical constituents. Antidiabetic guided isolation of the tubers and peels of D. dumetorum afforded three phytoecdysteroids bearing a cis-fused A/B ring junction including two new ones: 24-hydroxymuristerone A (1) and 24-hydroxykaladasterone (2), alongside the known muristerone A (3). Additionally, 2,2',7,7'-tetramethoxy-[1,1'-biphenanthrene]-4,4',6,6'-tetraol (4), batatasin I (5), and dihydroresveratrol (6) were isolated. Structural elucidation relied on spectroscopic, spectrometric methods, and comparison with existing literature. The ethyl acetate extracts of both the tubers and peels of D. dumetorum exhibited the highest phenolic content, correlating with potent antioxidant activity. Compounds 4 (IC50 = 0.10 mg/mL) and 6 (IC50 = 0.22 mg/mL) demonstrated superior inhibitory effects against α-glucosidase compared to acarbose (IC50 = 0.63 mg/mL). In contrast, compounds 3, 4, and 5 showed reduced α-amylase inhibition, with IC50 values of 2.58, 3.78, and 1.12 mg/mL, respectively, compared to acarbose (IC50 = 0.42 mg/mL). These observed bioactivities validate the traditional use of D. dumetorum and contribute valuable phytochemical data to the scientific literature of the species.

Comparative identification of phytoecdysteroids in Achyranthes bidentata Blume and its three analogous species and application in differentiation between processing products from different species.

The differentiation of raw herbal products from similar species have been achieved by plant metabolomics. However, the distinguishment on various processed products with improved activities and wide clinical utilization from similar species is still tricky due to obscure composition variations during processing. In this study, a comprehensive analysis of phytoecdysteroids in Achyranthes bidentata Blume (AB) and its three analogous species, which were all called Niuxi in Chinese, was conducted on UPLC-HRMS by integrating dynamic exclusion acquisition with data post-processing of targeted multilateral mass defect filter. Two most frequently used species, AB and Cyathula officinalis Kuan (CO) were systematically compared with plant metabolomics methods. And the differential components from the raw materials were evaluated on the ability of distinguishing processed products. The substitution of hydroxyl groups on C-21, C-20, C-22 and C-25 were determined by characteristic mass differences, leading to systematical characterization of 281 phytoecdysteroids. In plant metabolomics studies of raw AB and CO, 16 potential markers were filtered by VIP value > 1, and displayed satisfactory differentiation on the processed AB and CO. The results facilitated the quality control of the four species, especially the processed products of AB and CO, also provided a reference method for the quality control of other processed products.

Prumnopitys Andina Fruit Extract Activates Liver X Receptors after In Vitro Digestion.

SCOPE: 20-Hydroxyecdysone (20E) is the main phytochemical present in the fresh arils of Prumnopitys andina. 20E is reported to have anabolic effects by modulation of gene transcription by interaction with nuclear receptors. Our aim is to evaluate the in vitro bioaccessibility, transepithelial transport of 20E, and the capacity of P. andina fruit extract and 20E to activate selected mammalian nuclear receptors in transiently transfected human cells after simulated gastrointestinal digestion. RESULTS: 20E shows good stability, solubility, and micellization after in vitro digestion. 20E is taken up by Caco-2 cells, but poorly transported through the epithelial cell membrane, possibly due to P-glycoprotein-mediated efflux. In transiently transfected HepG2 cells, the fruit extract significantly induces the signal intensity for the liver X receptor (LXR)-α and -ß by 1.6 and 1.4-fold, respectively. In contrast, the treatment with 20E, irrespective of its concentration, did not change the activity of both LXR receptors. No effects are observed for the pregnane X receptor or the constitutive androstane receptor. CONCLUSION: Our findings show that components of the digested P. andina extract other than 20E are responsible for the effects on LXR-α and -ß. Our findings open new perspectives on the potential role of P. andina fruits in cholesterol metabolism and inflammatory diseases.

20-Hydroxyecdysone bioavailability and pharmacokinetics in Gerbillus tarabuli, a gerbil model for metabolic syndrome studies.

Phytoecdysteroids are active natural compounds that have demonstrated many beneficial pharmacological effects on mammals, including Humans. 20-Hydroxyecdysone (20E) is the major phytoecdysteroid present in plants, and gerbils are particularly good responders to the addition of 20E to their diet. We have examined the oral bioavailability of 20E in the desert gerbil, Gerbillus tarabuli. 20E (5 and 50 mg.kg-1 body weight) was administered to gerbils by intra-peritoneal injection and oral gavage, respectively. Plasma samples were collected over 8 h and analyzed by HPLC-MS/MS to determine the 20E concentrations. The calculated oral bioavailability of 20E is approx. 12%, with a half-life of 30.6 and 33 min after per os administration or intra-peritoneal injection, respectively. This bioavailabilitty is much higher than that observed in laboratory rodents (ca. 1%). It is proposed that this unexpectedly high oral bioavailability of 20E in gerbils contributes to its high efficacy in this animal.

Pfaffia glomerata polyploid accession compromises male fertility and fetal development.

ETHNOPHARMACOLOGICAL RELEVANCE: Pfaffia glomerata (Spreng.) Pedersen has traditionally been used as a tonic and a stimulant by the Brazilian population. It shows higher biomass accumulation and production of secondary compounds, such as the phytosterol 20-hydroxyecdysone. AIMS: The present study aimed to evaluate the effects of the hydroalcoholic extract of the root of tetraploid P. glomerata (BGEt) on testicular parenchyma, and its implications on fertility. MATERIAL AND METHODS: Adult Swiss mice were divided as: control (water) and sildenafil citrate (7 mg/kg), BGEt at 100, 200, and 400 mg/kg, and BGEtD 200 mg/kg (treated with BGE every three days). Males (n = 4/group) were mated with normal untreated adult females to assess fertility rates, while other animals (n = 6/group) were euthanized for testis, epididymis, and oxidative stress analyses. RESULTS: Increase in tubule diameter and epithelium height in the discontinuous group, in addition to an increase in the proportion of tubules with moderate pathologies was observed. The pre-implantation loss was lower in all treated groups. The post-implantation loss was significantly increased in all treated groups, except for the lowest BGEt dose. BGEt intake caused a decrease in daily sperm production, along with the number and quality of sperm in the epididymis. Changes were observed in protein carbonylation and hydrogen peroxide and nitric oxide levels, characterizing oxidative stress. CONCLUSIONS: The hydroalcoholic extract of P. glomerata tetraploid altered sperm and testicular parameters, compromising embryonic development after implantation.

Physiological, epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine.

Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40 days in Murashige and Skoog medium supplemented with NaCl (50 mM), 5-azaC (25 µM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.

Phytoecdysteroids from Dianthus superbus L.: Structures and anti-neuroinflammatory evaluation.

Six undescribed C27-phytoecdysteroid derivatives, named superecdysones A-F, and ten known analogs were extracted from the whole plant of Dianthus superbus L. Their structures were identified by extensive spectroscopy, mass spectrometric methods, chemical transformations, chiral HPLC analysis, and the single-crystal X-ray diffraction analysis. Superecdysones A and B possess a tetrahydrofuran ring in the side chain and superecdysones C-E are rare phytoecdysones containing a (R)-lactic acid moiety, whereas superecdysone F is an uncommon B-ring-modified ecdysone. Notably, based on the variable temperature (from 333 K to 253 K) NMR experiments of superecdysone C, the missing carbon signals were visible at 253 K and assigned. The neuroinflammatory bioassay of all compounds were evaluated, and 22-acetyl-2-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, 20-hydroxyecdysone, ecdysterone-22-O-benzoate, 20-hydroxyecdysone-20,22-O-R-ethylidene, and acetonide derivative 20-hydroxyecdysterone-20, 22-acetonide significantly suppressed the LPS-induced nitric oxide generation in microglia cells (BV-2), with IC50 values ranging from 6.9 to 23.0 µM. Structure-activity relationships were also discussed. Molecular docking simulations of the active compounds confirmed the possible mechanism of action against neuroinflammations. Furthermore, none compounds showed cytotoxicity against HepG2 and MCF-7. It is the first report about the occurrence and anti-neuroinflammatory activity of the phytoecdysteroids in the genus Dianthus. Our findings demonstrated that ecdysteroids may be used as potential anti-inflammatory drugs.

Sustainable Production of Ajuga Bioactive Metabolites Using Cell Culture Technologies: A Review.

The genus Ajuga (Lamiaceae) is rich in medicinally important species with biological activities ranging from anti-inflammatory, antitumor, neuroprotective, and antidiabetic to antibacterial, antiviral, cytotoxic, and insecticidal effects. Every species contains a unique and complex mixture of bioactive metabolites-phytoecdysteroids (PEs), iridoid glycosides, withanolides, neo-clerodane terpenoids, flavonoids, phenolics, and other chemicals with high therapeutic potential. Phytoecdysteroids, the main compounds of interest, are natural anabolic and adaptogenic agents that are widely used as components of dietary supplements. Wild plants remain the main source of Ajuga bioactive metabolites, particularly PEs, which leads to frequent overexploitation of their natural resources. Cell culture biotechnologies offer a sustainable approach to the production of vegetative biomass and individual phytochemicals specific for Ajuga genus. Cell cultures developed from eight Ajuga taxa were capable of producing PEs, a variety of phenolics and flavonoids, anthocyanins, volatile compounds, phenyletanoid glycosides, iridoids, and fatty acids, and demonstrated antioxidant, antimicrobial, and anti-inflammatory activities. The most abundant PEs in the cell cultures was 20-hydroxyecdysone, followed by turkesterone and cyasterone. The PE content in the cell cultures was comparable or higher than in wild or greenhouse plants, in vitro-grown shoots, and root cultures. Elicitation with methyl jasmonate (50-125 µM) or mevalonate and induced mutagenesis were the most effective strategies that stimulated cell culture biosynthetic capacity. This review summarizes the current progress in cell culture application for the production of pharmacologically important Ajuga metabolites, discusses various approaches to improve the compound yield, and highlights the potential directions for future interventions.

Isolation, structural elucidation of new phytoecdysteroids from Ajuga decumbens Thunb.

Five new phytoecdysteroids (1, 2, 3, 4, and 5) were isolated from Ajuga decumbens Thunb. planted at Pingtan island of Fujian Province. Their structures were elucidated by NMR(1H NMR, 13C NMR, DEPT, HMBC, HSQC, 1H-1H COSY, ROESY), and HRESIMS. Especially, compound 2 is the epimer of Cyasterone, which was directly confirmed using single crystal X-ray diffraction analysis.