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There is no cause -based treatment for Medication-Related Osteonecrosis of the Jaw (MRONJ). MRONJ is a morbid condition including exposed, infected bone and mandibular fractures in osteoporotic individuals and metastatic cancers patients treated with nitrogen containing bisphosphonates (NBP). NBPs inhibit farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, depriving osteoclasts and other bone cells of small GTPases necessary for their function and survival. We test the hypothesis that geranylgeraniol (GGOH),a metabolite downstream of FDPS, when incorporated into a bone cement pellet, enhances osteoclast function and promotes local bone healing in in vitro and in a proven animal model of MRONJ. 3H labelled GGOH (2 mM) was incorporated into a Hydroset bone cement pellet and release from the cement was assessed over time. To assess the effect on bone cell function, the GGOH-loaded cement was placed in a porous filter above cultured osteoclasts treated with bisphosphonate and the effect on osteoclast survival and function were measured. In a pilot study the effect of GGOH on osteotomy microstructure was measured in a rat model of MRONJ using a split mouth design. The release of GGOH from bone cement increased osteoclast survival/metabolic activity, and promoted resorption of the calcified substrate. In vivo released GGOH limited the effects of the bisphosphonate and promoted healing. In an animal pilot study, GGOH from the infused cement carrier stabilizes bone structure and restores the ability of osteoclasts to remodel. These initial findings point to GGOH in a bone cement carrier as a useful therapeutic approach to prevent or mitigate the pathogenesis of MRONJ. [Display omitted] [ABSTRACT FROM AUTHOR]

The interest in natural colorants derived from sustainable processes has prompted research into obtaining bixin from defatted annatto (Bixa orellana L.) seeds. Bixin is a compound that imparts yellow-orange-red coloration, known for its high biodegradability, low toxicity, and wide industrial applicability. Meanwhile, high-intensity ultrasound (HIUS) technology has emerged as a promising method for extracting natural colorants, offering higher yields through shorter processes and minimizing thermal degradation. Although some studies have demonstrated the efficiency of HIUS technology in bixin extraction, research on the effects of acoustic cavitation on the properties of the colorant remains limited. Therefore, this study aimed to investigate the influence of HIUS-specific energy levels (0.02, 0.04, 0.12, and 0.20 kJ/g) on the chemical, physical, and morphological characteristics of annatto extracts containing bixin and geranylgeraniol. Single-step extractions of bixin using ethanol as a solvent were evaluated at various acoustic powers (4.6, 8.5, 14.5, and 20 W) and extraction times (0.5, 1, 3, and 5 min) to determine their impact on the yield of natural colorant extraction. Increasing the acoustic power from 4.6 to 20 W and extending the extraction time from 0.5 to 5 min resulted in higher yields of natural colorant, likely due to the effects of acoustic cavitation and increased heat under more intense conditions. However, elevated levels of mechanical and thermal energy did not affect the chemical properties of the colorant, as indicated by UV–Vis and FTIR spectra. Conversely, higher specific energies yielded colorants with a more intense red hue, consistent with increased bixin content, and altered the microstructure and physical state, as observed in X-ray diffractograms. Nevertheless, these alterations did not impact the solubility of the colorant. Therefore, employing a cleaner extraction procedure aided by one-step ultrasound facilitated the recovery of natural colorants and contributed to the biorefining of annatto seeds, enabling the production of a rich geranylgeraniol colorant through a sustainable approach.

To counteract the effect of zoledronate and decrease the risk of osteonecrosis of the jaw (BRONJ) development in patients undergoing guided bone regeneration surgery, the use of geranylgeraniol (GGOH) has been proposed. Collagen membranes may act as biomimetical drug carriers. The objective of this study was to determine the capacity of collagen-based membranes doped with GGOH to revert the negative impact of zoledronate on the growth and differentiation of human osteoblasts. MG-63 cells were cultured on collagen membranes. Two groups were established: (1) undoped membranes and (2) membranes doped with geranylgeraniol. Osteoblasts were cultured with or without zoledronate (50 μM). Cell proliferation was evaluated at 48 h using the MTT colorimetric method. Differentiation was tested by staining mineralization nodules with alizarin red and by gene expression analysis of bone morphogenetic proteins 2 and 7, alkaline phosphatase (ALP), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), type I collagen (Col-I), osterix (OSX), osteocalcin (OSC), osteoprotegerin (OPG), receptor for RANK (RANKL), runt-related transcription factor 2 (Runx-2), TGF-β1 and TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3), and vascular endothelial growth factor (VEGF) with real-time PCR. One-way ANOVA or Kruskal–Wallis and post hoc Bonferroni tests were applied (p < 0.05). Scanning electron microscopy (SEM) observations were also performed. Treatment of osteoblasts with 50 μM zoledronate produced a significant decrease in cell proliferation, mineralization capacity, and gene expression of several differentiation markers if compared to the control (p < 0.001). When osteoblasts were treated with zoledronate and cultured on GGOH-doped membranes, these variables were, in general, similar to the control group (p > 0.05). GGOH applied on collagen membranes is able to reverse the negative impact of zoledronate on the proliferation, differentiation, and gene expression of different osteoblasts' markers. [ABSTRACT FROM AUTHOR]

Nitrogen-containing bisphosphonates lead to the depletion of geranylgeranyl pyrophosphate involved in the mevalonate pathway. The effect of geranylgeraniol (GGOH) on human osteoblast and osteoclast activities suppressed by zoledronate was investigated in this study. The effect of GGOH on human osteoblasts and osteoclasts subjected to treatment with zoledronate was analyzed by assessing cell viability, osteoclast differentiation, resorption ability, gene expression, and protein synthesis. Cell viability suppressed by bisphosphonates in osteoblasts and osteoprogenitor cells was restored with GGOH. Osteoclast differentiation was analyzed by vitronectin receptor immunofluorescence staining, and the addition of GGOH to zoledronate significantly increased osteoclast differentiation compared with zoledronate alone. A trend of reversal of osteoclast resorption by GGOH was observed; however, it was not significant in all groups. The expression of ALP, type 1 collagen, and RUNX2 in osteoblasts was recovered by the addition of GGOH. Only CALCR expression in osteoclasts was significantly recovered by GGOH addition in the zoledronate group. Although the activities of osteoblasts and osteoclasts were not entirely restored, the possibility that the topical application of GGOH in MRONJ patients or patients with dental problems and bisphosphonates might lessen the risk of development and recurrence of MRONJ is shown. [ABSTRACT FROM AUTHOR]

Juvenile hormone (JH) plays pivotal roles in several critical developmental processes in insects, including metamorphosis and reproduction. JH-biosynthetic pathway enzymes are considered highly promising targets for discovering novel insecticides. The oxidation of farnesol to farnesal, catalysed by farnesol dehydrogenase (FDL), represents a rate-limiting step in JH biosynthesis. Here, we report farnesol dehydrogenase (HaFDL) from H. armigera as a promising insecticidal target. The inhibitory potential of natural substrate analogue geranylgeraniol (GGol) was tested in vitro, wherein it showed a high binding affinity (kd 595 µM) for HaFDL in isothermal titration calorimetry (ITC) and subsequently exhibited dose-dependent enzyme inhibition in GC–MS coupled qualitative enzyme inhibition assay. Moreover, the experimentally determined inhibitory activity of GGol was augmented by the in silico molecular docking simulation which showed that GGol formed a stable complex with HaFDL, occupied the active site pocket and interacted with key active site residues (Ser147 and Tyr162) as well as other residues that are crucial in determining the active site architecture. Further, the diet-incorporated oral feeding of GGol caused detrimental effects on larval growth and development, exhibiting a significantly reduced rate of larval weight gain (P < 0.01), aberrant pupal and adult morphogenesis, and a cumulative mortality of ~ 63%. To the best of our knowledge, the study presents the first report on evaluating GGol as a potential inhibitor for HaFDL. Overall, the findings revealed the suitability of HaFDL as a potential insecticidal target for the management H. armigera. [ABSTRACT FROM AUTHOR]

Dyslipidemia is a metabolic condition linked to increased morbidity. This study aimed to evaluate the effect of two new nutraceuticals derived from annatto (Bixa orellana), açaí (Euterpe oleracea), and soybean germ or red clover isoflavones (Trifolium pratense) against high-fat-induced dyslipidemia in female Wistar rats. The formulations were assessed through GC-MS and HPLC-UV/Vis. Next, female Wistar rats received daily administrations of coconut oil and were treated with Ormona® SI (OSI), Ormona® RC (ORC), soybean germ (SG), simvastatin (simv.), simvastatin + Ormona® SI (simv. + OSI), or only distilled water (control). Biochemical tests were performed using the animals’ blood, and their arteries were screened for atheroma formation. The results show abnormal TC, TG, LDL, and HDL levels caused by the high-fat diet, increased glucose levels, hepatotoxicity, nephrotoxicity, and atherogenesis. The nutraceuticals significantly ameliorated all conditions, with results that are similar to the group treated with simvastatin. Notably, the groups treated with either Ormona® or simvastatin + Ormona® had better liver protection than those treated only with SG or simvastatin; additionally, the nutraceuticals could prevent atherogenesis, unlike SG. The results indicate a high efficacy of the nutraceuticals in preventing dyslipidemia and its complications.

To counteract the effect of zoledronate and decrease the risk of osteonecrosis of the jaw (BRONJ) development in patients undergoing guided bone regeneration surgery, the use of geranylgeraniol (GGOH) has been proposed. Collagen membranes may act as biomimetical drug carriers. The objective of this study was to determine the capacity of collagen-based membranes doped with GGOH to revert the negative impact of zoledronate on the growth and differentiation of human osteoblasts. MG-63 cells were cultured on collagen membranes. Two groups were established: (1) undoped membranes and (2) membranes doped with geranylgeraniol. Osteoblasts were cultured with or without zoledronate (50 μM). Cell proliferation was evaluated at 48 h using the MTT colorimetric method. Differentiation was tested by staining mineralization nodules with alizarin red and by gene expression analysis of bone morphogenetic proteins 2 and 7, alkaline phosphatase (ALP), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), type I collagen (Col-I), osterix (OSX), osteocalcin (OSC), osteoprotegerin (OPG), receptor for RANK (RANKL), runt-related transcription factor 2 (Runx-2), TGF-β1 and TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3), and vascular endothelial growth factor (VEGF) with real-time PCR. One-way ANOVA or Kruskal–Wallis and post hoc Bonferroni tests were applied (p < 0.05). Scanning electron microscopy (SEM) observations were also performed. Treatment of osteoblasts with 50 μM zoledronate produced a significant decrease in cell proliferation, mineralization capacity, and gene expression of several differentiation markers if compared to the control (p < 0.001). When osteoblasts were treated with zoledronate and cultured on GGOH-doped membranes, these variables were, in general, similar to the control group (p > 0.05). GGOH applied on collagen membranes is able to reverse the negative impact of zoledronate on the proliferation, differentiation, and gene expression of different osteoblasts’ markers.

Isoprenoids are the output of the polymerization of five-carbon, branched isoprenic chains derived from isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP). Isoprene units are consecutively condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively), necessary for the biosynthesis of several metabolites. Polyprenyl transferases and synthases use polyprenyl pyrophosphates as their natural substrates; however, it is known that free polyprenols, such as farnesol (FOH), and geranylgeraniol (GGOH) can be incorporated into prenylated proteins, ubiquinone, cholesterol, and dolichols. Furthermore, FOH and GGOH have been shown to block the effects of isoprenoid biosynthesis inhibitors such as fosmidomycin, bisphosphonates, or statins in several organisms. This phenomenon is the consequence of a short pathway, which was observed for the first time more than 25 years ago: the polyprenol salvage pathway, which works via the phosphorylation of FOH and GGOH. Biochemical studies in bacteria, animals, and plants suggest that this pathway can be carried out by two enzymes: a polyprenol kinase and a polyprenyl-phosphate kinase. However, to date, only a few genes have been unequivocally identified to encode these enzymes in photosynthetic organisms. Nevertheless, pieces of evidence for the importance of this pathway abound in studies related to infectious diseases, cancer, dyslipidemias, and nutrition, and to the mitigation of the secondary effects of several drugs. Furthermore, nowadays it is known that both FOH and GGOH can be incorporated via dietary sources that produce various biological effects. This review presents, in a simplified but comprehensive manner, the most important data on the FOH and GGOH salvage pathway, stressing its biomedical importance The main objective of this review is to bring to light the need to discover and characterize the kinases associated with the isoprenoid salvage pathway in animals and pathogens. [ABSTRACT FROM AUTHOR]

Cyanobacteria are photosynthetic prokaryotes with strong potential to be used for industrial terpenoid production. However, the key enzymes forming the principal terpenoid building blocks, called short‐chain prenyltransferases (SPTs), are insufficiently characterized. Here, we examined SPTs in the model cyanobacteria Synechococcus elongatus sp. PCC 7942 and Synechocystis sp. PCC 6803. Each species has a single putative SPT (SeCrtE and SyCrtE, respectively). Sequence analysis identified these as type‐II geranylgeranyl pyrophosphate synthases (GGPPSs) with high homology to GGPPSs found in the plastids of green plants and other photosynthetic organisms. In vitro analysis demonstrated that SyCrtE is multifunctional, producing geranylgeranyl pyrophosphate (GGPP; C20) primarily but also significant amounts of farnesyl pyrophosphate (FPP, C15) and geranyl pyrophosphate (GPP, C10); whereas SeCrtE appears to produce only GGPP. The crystal structures were solved to 2.02 and 1.37 Å, respectively, and the superposition of the structures against the GGPPS of Synechococcus elongatus sp. PCC 7002 yield a root mean square deviation of 0.8 Å (SeCrtE) and 1.1 Å (SyCrtE). We also discovered that SeCrtE is co‐encoded in an operon with a functional GGPP phosphatase, suggesting metabolic pairing of these two activities and a putative function in tocopherol biosynthesis. This work sheds light on the activity of SPTs and terpenoid synthesis in cyanobacteria. Understanding native prenyl phosphate metabolism is an important step in developing approaches to engineering the production of different chain‐length terpenoids in cyanobacteria. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is the etiological agent of human malaria, one of the most widespread diseases in tropical and subtropical regions. Drug resistance is one of the biggest problems in controlling the disease, which leads to the need to discover new antimalarial compounds. One of the most promissory drugs purposed is fosmidomycin, an inhibitor of the biosynthesis of isoprene units by the methylerythritol 4-phosphate (MEP) pathway, which in some cases failed in clinical studies. Once formed, isoprene units are condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate, which are necessary for Heme O and A formation, ubiquinone, and dolichyl phosphate biosynthesis as well as for protein isoprenylation. Even though the natural substrates of polyprenyl transferases and synthases are polyprenyl pyrophosphates, it was already demonstrated that isoprenoid alcohols (polyprenols) such as farnesol (FOH) and geranylgeraniol (GGOH) can rescue parasites from fosmidomycin. This study better investigated how this rescue phenomenon occurs by performing drug-rescue assays. Similarly, to FOH and GGOH, it was observed that phytol (POH), a 20-carbon plant isoprenoid, as well as unsaponifiable lipid extracts from foods rescue parasites from the antimalarial effect of fosmidomycin. Contrarily, neither dolichols nor nonaprenol rescue parasites from fosmidomycin. Considering this, here we characterized the transport of FOH, GGOH, and POH. Once incorporated, it was observed that these substances are phosphorylated, condensed into longer isoprenoid alcohols, and incorporated into proteins and dolichyl phosphates. Through proteomic and radiolabelling approaches, it was found that prenylated proteins are naturally attached to several isoprenoids, derived from GGOH, dolichol, and POH if exogenously added. Furthermore, the results suggest the presence of at least two promiscuous protein prenyltransferases in the parasite: one enzyme which can use FPP among other unidentified substrates and another enzyme that can use GGPP, phytyl pyrophosphate (PPP), and dolichols, among other substrates not identified here. Thus, further evidence was obtained for dolichols and other isoprenoid products attached to proteins. This study helps to better understand the apicoplast-targeting antimalarial mechanism of action and a novel post-translational modification of proteins in P. falciparum.

Annona macroprophyllata Donn (A. macroprophyllata) is used in traditional Mexican medicine for the treatment of cancer, diabetes, inflammation, and pain. In this work, we evaluated the antitumor activity of three acyclic terpenoids obtained from A. macroprophyllata to assess their potential as antilymphoma agents. We identified the terpenoids farnesyl acetate (FA), phytol (PT) and geranylgeraniol (Gg) using gas chromatography–mass spectroscopy (GC-MS) and spectroscopic (1H, and 13C NMR) methods applied to petroleum ether extract of leaves from A. macroprophyllata (PEAm). We investigated antitumor potential in Balb/c mice inoculated with U-937 cells by assessing brine shrimp lethality (BSL), and cytotoxic activity in these cells. In addition, to assess the potential toxicity of PEAm, FA, PT and Gg in humans, we tested their acute oral toxicity in mice. Our results showed that the three terpenoids exhibited considerable antilymphoma and cytotoxic activity. In terms of lethality, we determined a median lethal dose (LD50) for thirteen isolated products of PEAm. Gg, PT and AF all exhibited a higher lethality with values of 1.41 ± 0.42, 3.03 ± 0.33 and 5.82 ± 0.58 µg mL−1, respectively. To assess cytotoxic activity against U-937 cells, we calculated the mean cytotoxic concentration (CC50) and found that FA and PT were closer in respect to the control drug methotrexate (MTX, 0.243 ± 0.007 µM). In terms of antilymphoma activity, we found that FA, PT and Gg considerably inhibited lymph node growth, with median effective doses (ED50) of 5.89 ± 0.39, 6.71 ± 0.31 and 7.22 ± 0.51 mg kg−1 in females and 5.09 ± 0.66, 5.83 ± 0.50 and 6.98 ± 0.57mg kg −1 in males, respectively. Regarding acute oral toxicity, we classified all three terpenoids as category IV, indicating a high safety margin for human administration. Finally, in a molecular docking study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, we found binding of terpenoids to some amino acids of the catalytic site, suggesting an effect upon activity with a resulting decrease in the synthesis of intermediates involved in the prenylation of proteins involved in cancer progression. Our findings suggest that the acyclic terpenoids FA, PT, and Gg may serve as scaffolds for the development of new treatments for non-Hodgkin's lymphoma. [ABSTRACT FROM AUTHOR]

Natural bioactive compounds are proposed as alternatives in mitigating obesity-associated skeletal muscle dysfunction. The objective of this study was to test the hypothesis that the combination of geranylgeraniol (GGOH) and green tea polyphenols (GTPs) can alleviate high-fat-diet (HFD)-induced muscle atrophy and alter gut microbiome composition. Male C57BL/6J mice fed an HFD were assigned to four groups (12 mice each) in a 2 (no GGOH vs. 400 mg GGOH/kg diet) × 2 (no GTPs vs. 0.5% weight/volume GTPs in water) factorial design. After 14 weeks of diet intervention, skeletal muscle and cecal samples were collected and examined. Compared to the control groups, the group that consumed a combination of GGOH and GTPs (GG + GTPs) had significantly decreased body and fat mass but increased skeletal muscle mass normalized by body weight and cross-sectional area. In soleus muscle, the GG + GTP diet increased citrate synthase activity but decreased lipid peroxidation. Gut microbiome beta-diversity analysis revealed a significant difference in the microbiome composition between diet groups. At the species level, the GG + GTP diet decreased the relative abundance of Dorea longicatena, Sporobacter termitidis, and Clostridium methylpentosum, and increased that of Akkermansia muciniphila and Subdoligranulum variabile. These results suggest that the addition of GGOH and GTPs to an HFD alleviates skeletal muscle atrophy, which is associated with changes in the gut microbiome composition. [ABSTRACT FROM AUTHOR]

Geranylgeraniol (GGOH) is a crucial component in fragrances and essential oils, and a valuable precursor of vitamin E. It is primarily extracted from the oleoresin of Bixa orellana , but is challenged by long plant growth cycles, severe environmental pollution, and low extraction efficiency. Chemically synthesized GGOH typically comprises a mix of isomers, making the separation process both challenging and costly. Advancements in synthetic biology have enabled the construction of microbial cell factories for GGOH production. In this study, Yarrowia lipolytica was engineered to efficiently synthesize GGOH by expressing heterologous phosphatase genes, enhancing precursor supplies of farnesyl diphosphate, geranylgeranyl pyrophosphate, and acetyl-CoA, and downregulating the squalene synthesis pathway by promoter engineering. Additionally, optimizing fermentation conditions and reducing reactive oxygen species significantly increased the GGOH titer to 3346.47 mg/L in a shake flask. To the best of our knowledge, this is the highest reported GGOH titer in shaking flasks to date, setting a new benchmark for terpenoid production.

Nitrogen-containing bisphosphonates lead to the depletion of geranylgeranyl pyrophosphate involved in the mevalonate pathway. The effect of geranylgeraniol (GGOH) on human osteoblast and osteoclast activities suppressed by zoledronate was investigated in this study. The effect of GGOH on human osteoblasts and osteoclasts subjected to treatment with zoledronate was analyzed by assessing cell viability, osteoclast differentiation, resorption ability, gene expression, and protein synthesis. Cell viability suppressed by bisphosphonates in osteoblasts and osteoprogenitor cells was restored with GGOH. Osteoclast differentiation was analyzed by vitronectin receptor immunofluorescence staining, and the addition of GGOH to zoledronate significantly increased osteoclast differentiation compared with zoledronate alone. A trend of reversal of osteoclast resorption by GGOH was observed; however, it was not significant in all groups. The expression of ALP, type 1 collagen, and RUNX2 in osteoblasts was recovered by the addition of GGOH. Only CALCR expression in osteoclasts was significantly recovered by GGOH addition in the zoledronate group. Although the activities of osteoblasts and osteoclasts were not entirely restored, the possibility that the topical application of GGOH in MRONJ patients or patients with dental problems and bisphosphonates might lessen the risk of development and recurrence of MRONJ is shown.

Medication-related osteonecrosis of the jaw (MRONJ) is an often-severe complication found in patients receiving bisphosphonates in the management of Paget's, osteoporosis and metastatic bone cancer. Mucosal breakdown with bone exposure is a primary clinical presentation of MRONJ linked to the inhibitory effect of nitrogen-containing bisphosphonates (N-BP) on the mevalonate pathway. Geranylgeraniol (GGOH) has demonstrated a rescue effect on N-BP-treated osteoclasts but the biological effects on oral soft tissues and cells remain unclear. This study aimed to determine whether GGOH could prevent bisphosphonate induced toxicity to oral mucosa cells in vitro. Primary oral fibroblasts and keratinocytes were exposed to different GGOH concentrations or GGOH in combination with two nitrogen-containing bisphosphonates, zoledronic acid (ZA) or pamidronic acid (PA), for 72 h. The metabolic activity of each cell type was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GGOH without bisphosphonates significantly reduced the metabolic activity of oral mucosa cells. Fibroblasts treated with GGOH and ZA in combination showed a slight increase in metabolic status compared to fibroblasts treated with ZA alone, however this positive effect was not observed in keratinocytes. In the presence of PA, GGOH was unable to increase the metabolic activity of either cell type. These findings demonstrate that GGOH is toxic to oral mucosa cells and that GGOH was not able to prevent bisphosphonate induced toxicity. These data show that GGOH does not have therapeutic potential for bisphosphonate-induced soft tissue toxicity in MRONJ and the use of GGOH as an MRONJ treatment should be strongly reconsidered.

[Display omitted] • Ultrasound acoustic power (4.6 to 20 W) and extraction time (0.5 to 5 min) were evaluated. • The more intense ultrasound conditions allowed redder colorants consistent with higher bixin content. • Specific energies (0.02 to 0.20 kJ/g) affected the microstructure and X-ray diffractograms. • The extraction condition did not impact the colorant solubility. The interest in natural colorants derived from sustainable processes has prompted research into obtaining bixin from defatted annatto (Bixa orellana L.) seeds. Bixin is a compound that imparts yellow-orange-red coloration, known for its high biodegradability, low toxicity, and wide industrial applicability. Meanwhile, high-intensity ultrasound (HIUS) technology has emerged as a promising method for extracting natural colorants, offering higher yields through shorter processes and minimizing thermal degradation. Although some studies have demonstrated the efficiency of HIUS technology in bixin extraction, research on the effects of acoustic cavitation on the properties of the colorant remains limited. Therefore, this study aimed to investigate the influence of HIUS-specific energy levels (0.02, 0.04, 0.12, and 0.20 kJ/g) on the chemical, physical, and morphological characteristics of annatto extracts containing bixin and geranylgeraniol. Single-step extractions of bixin using ethanol as a solvent were evaluated at various acoustic powers (4.6, 8.5, 14.5, and 20 W) and extraction times (0.5, 1, 3, and 5 min) to determine their impact on the yield of natural colorant extraction. Increasing the acoustic power from 4.6 to 20 W and extending the extraction time from 0.5 to 5 min resulted in higher yields of natural colorant, likely due to the effects of acoustic cavitation and increased heat under more intense conditions. However, elevated levels of mechanical and thermal energy did not affect the chemical properties of the colorant, as indicated by UV–Vis and FTIR spectra. Conversely, higher specific energies yielded colorants with a more intense red hue, consistent with increased bixin content, and altered the microstructure and physical state, as observed in X-ray diffractograms. Nevertheless, these alterations did not impact the solubility of the colorant. Therefore, employing a cleaner extraction procedure aided by one-step ultrasound facilitated the recovery of natural colorants and contributed to the biorefining of annatto seeds, enabling the production of a rich geranylgeraniol colorant through a sustainable approach. [ABSTRACT FROM AUTHOR]

Bisphosphonate (BP) and geranylgeraniol (GGOH) are known drug/reagent affecting osteoblast shape and dimensions. Digital image analysis such as ImageJ can detect changes in cell dimensions. Commonly, fluorescence staining, a high-cost dye, is used for staining cells. Therefore, this study was attempted to investigate dimensional changes in cells stained with a simple and lowcost dye and measured by ImageJ analysis. Pre-osteoblast, MC3T3, cells were incubated with alendronate (ALN) and GGOH. Cells were stained with crystal violet and were recorded for quantitative measurements via ImageJ. The measurement procedure was defined to perform cell segmentation. Cell area, number, dimensions, and aspect ratio were measured. Crystal violet staining method exhibited similar results to fluorescence staining method in terms of cell area and aspect ratio. ImageJ distinguished the changes in cell numbers and cell sizes after treating with ALN and GGOH. Furthermore, the result obtaining from the automated ImageJ method was comparable to that of the manual ImageJ method in terms of aspect ratio. The staining and analyzing technique detected pre-osteoblast dimensional changes after ALN and GGOH treatments. It was practical and easy to use. It could be an alternative method for screening of cell changes after drug treatment. [ABSTRACT FROM AUTHOR]

This study evaluated the phytochemical characterization of Bixa orellana (BO extract) unsaponifiable extract and resulting fractions (F fraction - FF, Geranyl fraction – GF and R fraction- RF) obtained as by-products of an industrial process investigating in vitro antiproliferative activities in human tumoral cells. The main compounds identified by GC-MS for BO extract were Geranylgeraniol (61.51%); for FF: Geranylgeraniol (70.23%); for GF: Geranylgeraniol (78.92%) and for RF: β-cubebene (27.75%). Quantifications of geranylgeraniol by GC-FID presented the percentage content: BO 27.52%; FF 38.52%; GF 51.44% and RF 1.81%. BO extract showed a significant antiproliferative activity, with GI50 up to 4 µg/mL. All fractions had a remarkably similar antiproliferative activity profile (GI50 27-47 µg/mL). Data reported herein showed an important cytostatic effect for BO extract, nevertheless this activity is not attributed exclusively to geranylgeraniol. In conclusion, this by-product becomes of great value, being a potential candidate for development of new anti-tumor ingredients. [ABSTRACT FROM AUTHOR]

Isoprenoids are the output of the polymerization of five-carbon, branched isoprenic chains derived from isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP). Isoprene units are consecutively condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively), necessary for the biosynthesis of several metabolites. Polyprenyl transferases and synthases use polyprenyl pyrophosphates as their natural substrates; however, it is known that free polyprenols, such as farnesol (FOH), and geranylgeraniol (GGOH) can be incorporated into prenylated proteins, ubiquinone, cholesterol, and dolichols. Furthermore, FOH and GGOH have been shown to block the effects of isoprenoid biosynthesis inhibitors such as fosmidomycin, bisphosphonates, or statins in several organisms. This phenomenon is the consequence of a short pathway, which was observed for the first time more than 25 years ago: the polyprenol salvage pathway, which works via the phosphorylation of FOH and GGOH. Biochemical studies in bacteria, animals, and plants suggest that this pathway can be carried out by two enzymes: a polyprenol kinase and a polyprenyl-phosphate kinase. However, to date, only a few genes have been unequivocally identified to encode these enzymes in photosynthetic organisms. Nevertheless, pieces of evidence for the importance of this pathway abound in studies related to infectious diseases, cancer, dyslipidemias, and nutrition, and to the mitigation of the secondary effects of several drugs. Furthermore, nowadays it is known that both FOH and GGOH can be incorporated via dietary sources that produce various biological effects. This review presents, in a simplified but comprehensive manner, the most important data on the FOH and GGOH salvage pathway, stressing its biomedical importance The main objective of this review is to bring to light the need to discover and characterize the kinases associated with the isoprenoid salvage pathway in animals and pathogens.

The zebrafish is a popular organism to test the toxicity of compounds. Here, we evaluate the acute and reproductive toxicity of Ormona SI® (OSI) and RC® (ORC), two herbal products developed for menopausal women with tocotrienols, geranylgeraniol, isoflavones, and anthocyanins. The acute toxicity was evaluated by behavioral alterations, lethality, and tissue changes (intestine, liver, kidney) after oral treatment with high product doses (500, 1000, and 2000 mg/kg). The reproductive toxicity was evaluated after 21 days of oral treatment with OSI and ORC at 200 mg/kg. Our results show that the LD50 could not be assessed due to the low mortality rate even with the highest dose; the behavioral alterations were not different from those of the group treated only with the vehicle (2% DMSO). The tissue changes were minor in OSI and more severe in ORC at the highest (2000 mg/kg) dose, while no tissue abnormality was observed at 500 mg/kg. In the reproductive assessment, continuous treatment could decrease the maturation of the reproductive cells, which also significantly decreases the egg spawning. This effect was attributed to the estrogenic activity of the isoflavones. In conclusion, the acute toxicity analysis shows that the products did not elicit lethal or sublethal effects observed in the model when used up to 500 mg/kg. Regarding the reproductive toxicity, decreased fertility was observed, which was expected due to the presence of isoflavones (phytoestrogens). Considering that the product is intended for menopausal and postmenopausal women, the presence of isoflavones is beneficial. Further studies should be performed to corroborate these results in mammals.

• Geranylgeraniol (GGOH) alleviates obesity-associated glucose intolerance. • GGOH improved the bone microstructure in obese mice • GGOH decreased Dorea longicatena, the marker for insulin resistance. Geranylgeraniol (GGOH) is found in edible oils such as olive, linseed, and sunflower oils, which have favorable metabolic effects. However, it is unknown whether these physiological benefits are mediated through the gut microbiome. Thus, the purpose of this study was to test the hypothesis that GGOH supplementation would improve glucose homeostasis and benefit the bone microstructure in obese mice through suppression of inflammation and modification of gut microbiota composition. Thirty-six male C57BL/6J mice were divided into 3 groups: a low-fat diet, a high-fat diet (HFD), and an HFD supplemented with 800 mg GGOH/kg diet (GG) for 14 weeks. Glucose and insulin tolerance tests were measured at baseline and end of study. The concentrations of adipokine cytokines (resistin, leptin, monocyte chemoattractant protein-1, interleukin-6) were measured via ELISA. Bone microarchitecture and quality were measured by micro-CT. Microbiome analysis was performed using 16S rRNA amplicon sequencing on cecal content. Relative to the HFD group, the GG group: (1) improved glucose tolerance and insulin sensitivity; (2) reduced production of pro-inflammatory adipokines, (3) increased serum procollagen I intact N-terminal propeptide (bone formation marker) concentrations, while decreasing serum collagen type 1 cross-linked C-telopeptide (bone resorption marker) levels, and (4) increased stiffness at both femur and LV-4 and cortical thickness at femoral midshaft. Compared to the HFD group, the GG group had an increased abundance of Butyricicoccus pullicaecorum and decreased Dorea longicatena in the cecal microbiome. Collectively, GGOH improves glucose homeostasis and bone microstructure in obese mice, probably via suppression of pro-inflammation and modification of microbiome composition. [ABSTRACT FROM AUTHOR]

Annona macroprophyllata Donn (A. macroprophyllata) is used in traditional Mexican medicine for the treatment of cancer, diabetes, inflammation, and pain. In this work, we evaluated the antitumor activity of three acyclic terpenoids obtained from A. macroprophyllata to assess their potential as antilymphoma agents. We identified the terpenoids farnesyl acetate (FA), phytol (PT) and geranylgeraniol (Gg) using gas chromatography–mass spectroscopy (GC-MS) and spectroscopic (1H, and 13C NMR) methods applied to petroleum ether extract of leaves from A. macroprophyllata (PEAm). We investigated antitumor potential in Balb/c mice inoculated with U-937 cells by assessing brine shrimp lethality (BSL), and cytotoxic activity in these cells. In addition, to assess the potential toxicity of PEAm, FA, PT and Gg in humans, we tested their acute oral toxicity in mice. Our results showed that the three terpenoids exhibited considerable antilymphoma and cytotoxic activity. In terms of lethality, we determined a median lethal dose (LD50) for thirteen isolated products of PEAm. Gg, PT and AF all exhibited a higher lethality with values of 1.41 ± 0.42, 3.03 ± 0.33 and 5.82 ± 0.58 µg mL−1, respectively. To assess cytotoxic activity against U-937 cells, we calculated the mean cytotoxic concentration (CC50) and found that FA and PT were closer in respect to the control drug methotrexate (MTX, 0.243 ± 0.007 µM). In terms of antilymphoma activity, we found that FA, PT and Gg considerably inhibited lymph node growth, with median effective doses (ED50) of 5.89 ± 0.39, 6.71 ± 0.31 and 7.22 ± 0.51 mg kg−1 in females and 5.09 ± 0.66, 5.83 ± 0.50 and 6.98 ± 0.57mg kg −1 in males, respectively. Regarding acute oral toxicity, we classified all three terpenoids as category IV, indicating a high safety margin for human administration. Finally, in a molecular docking study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, we found binding of terpenoids to some amino acids of the catalytic site, suggesting an effect upon activity with a resulting decrease in the synthesis of intermediates involved in the prenylation of proteins involved in cancer progression. Our findings suggest that the acyclic terpenoids FA, PT, and Gg may serve as scaffolds for the development of new treatments for non-Hodgkin’s lymphoma.

Natural bioactive compounds are proposed as alternatives in mitigating obesity-associated skeletal muscle dysfunction. The objective of this study was to test the hypothesis that the combination of geranylgeraniol (GGOH) and green tea polyphenols (GTPs) can alleviate high-fat-diet (HFD)-induced muscle atrophy and alter gut microbiome composition. Male C57BL/6J mice fed an HFD were assigned to four groups (12 mice each) in a 2 (no GGOH vs. 400 mg GGOH/kg diet) × 2 (no GTPs vs. 0.5% weight/volume GTPs in water) factorial design. After 14 weeks of diet intervention, skeletal muscle and cecal samples were collected and examined. Compared to the control groups, the group that consumed a combination of GGOH and GTPs (GG + GTPs) had significantly decreased body and fat mass but increased skeletal muscle mass normalized by body weight and cross-sectional area. In soleus muscle, the GG + GTP diet increased citrate synthase activity but decreased lipid peroxidation. Gut microbiome beta-diversity analysis revealed a significant difference in the microbiome composition between diet groups. At the species level, the GG + GTP diet decreased the relative abundance of Dorea longicatena, Sporobacter termitidis, and Clostridium methylpentosum, and increased that of Akkermansia muciniphila and Subdoligranulum variabile. These results suggest that the addition of GGOH and GTPs to an HFD alleviates skeletal muscle atrophy, which is associated with changes in the gut microbiome composition.

Long-term use of zoledronic acid (ZA) increases the risk of medication-related osteonecrosis of the jaw (MRONJ). This may be attributed to ZA-mediated reduction of viable mesenchymal stem cells (MSCs). ZA inhibits protein geranylgeranylation, thus suppressing cell viability and proliferation. Geranylgeraniol (GGOH), which is a naturally found intermediate compound in the mevalonate pathway, has positive effects against ZA. However, precise mechanisms by which GGOH may help preserve stem cell viability against ZA are not fully understood. The objective of this study was to investigate the cytoprotective mechanisms of GGOH against ZA. The results showed that while ZA dramatically decreased the number of viable MSCs, GGOH prevented this negative effect. GGOH-rescued ZA-exposed MSCs formed mineralization comparable to that produced by normal MSCs. Mechanistically, GGOH preserved the number of viable MSCs by its reversal of ZA-mediated Ki67+ MSC number reduction, cell cycle arrest and apoptosis. Moreover, GGOH prevented ZA-suppressed RhoA activity and YAP activation. The results also established the involvement of Rho-dependent YAP and YAP-mediated CDK6 in the cytoprotective ability of GGOH against ZA. In conclusion, GGOH preserves a pool of viable MSCs with osteogenic potency against ZA by rescuing the activity of Rho-dependent YAP activation, suggesting GGOH as a promising agent and YAP as a potential therapeutic target for MRONJ.

Alendronate (ALN), a nitrogen-containing bisphosphonate, is prescribed to treat bone diseases. ALN acts as an inhibitor of enzymes in the mevalonate pathway, which results in reducing osteoblast viability and mineralization. Geranylgeraniol (GGOH) is a substrate in mevalonate pathway and mediates protein prenylation in the cells. To investigate the effects of GGOH on ALN-treated osteoblast activities in order to improve the application of GGOH. MC3T3 cells were treated with ALN. GGOH were added at different time points. Cell activities were examined using alizarin red S, MTT assay, alkaline phosphatase (ALP) activity, and quantitative polymerase chain reaction. ALN decreased mineralization. In the presence of ALN, GGOH addition at the first week of culture increased mineralization compared with the addition at other time points. ALN treatment for 7 days caused a reduction in osteoblast and pre-osteoblast viability compared with untreated cells. GGOH supplement partially rescued cell viability and increased total protein in cells treated with ALN. Furthermore, GGOH significantly upregulated gene expressions of Col I, OPN, VEGF, and VEGFR2. GGOH could be best applied at the early stage of osteogenesis since GGOH helped increasing cell viability and differentiation at the first 7 day of treatment. [ABSTRACT FROM AUTHOR]

Background: Alendronate (ALN) is a bisphosphonate, which is prescribed as an anti‐osteoporotic drug. ALN has been shown to increase osteoblast cell death and decrease bone mineralization. ALN inhibits a key regulatory enzyme in the mevalonate pathway, consequently reducing geranylgeranyl pyrophosphate (GGPP). Geranylgeraniol (GGOH) can be converted to GGPP. The aim of this study was to investigate the effects of exogenous GGOH on MC3T3 cell viability, cell cycle, osteoblast function, and cell cytoskeleton under ALN treatment. Methods: MC3T3 cells and osteoblast precursors, were incubated with ALN (0‐50 µmol/L) and GGOH (0‐50 µmol/L). After treatment, cells were evaluated for cell viability, cell cycle, osteoblast function, and cell cytoskeleton by MTT, flow cytometry, alizarin red S assay, and fluorescent microscopy, respectively. Results: ALN reduced cell viability and bone nodule formation in a dose‐dependent manner. GGOH partially inhibited the negative effects of ALN on cell viability and function. ALN increased the percentages of cell apoptosis and necrosis and arrested cells in G2M phase. Co‐incubation with GGOH partially reduced late cell apoptosis and rescued cell cycle arrest. Furthermore, ALN altered MC3T3 morphology and decreased cell area, actin stress fiber density as well as nuclear area. GGOH abolished the effect of ALN on cell area, actin stress fiber density, and nuclear area. Conclusions: GGOH partially inhibited negative effects of ALN on cell viability, cell cycle, function, and cell cytoskeleton. It might be an additional option for increasing osteoblast function and reducing apoptosis of osteoblasts in the condition treated with low bisphosphonate concentration. [ABSTRACT FROM AUTHOR]

Geranylgeranoic acid (GGA), developed as a preventive agent against second primary hepatoma, has been reported to be biosynthesized via the mevalonate pathway in human hepatoma-derived cells. Recently, we found that monoamine oxidase B (MAOB) catalyzed the oxidation of geranylgeraniol (GGOH) to produce geranylgeranial (GGal), a direct precursor of endogenous GGA in hepatoma cells, using tranylcypromine, an inhibitor of MAOs, and knockdown by MAOB siRNA. However, endogenous GGA level was unexpectedly unchanged in MAOB-knockout (KO) cells established using the CRISPR-Cas9 system, suggesting that some other latent metabolic pathways maintain endogenous GGA levels in the MAOB-KO cells. Here, we investigated the putative latent enzymes that oxidize GGOH in Hep3B/MAOB-KO cells. First, the broad-specific cytochrome P450 enzyme inhibitors decreased the amount of endogenous GGA in Hep3B/MAOB-KO cells in a dose-dependent manner. Second, among the eight members of cytochrome P450 superfamily that have been suggested to be involved in the oxidation of isoprenols and/or retinol in previous studies, only the CYP3A4 gene significantly upregulated its cellular mRNA level in Hep3B/MAOB-KO cells. Third, a commercially available recombinant human CYP3A4 enzyme was able to oxidize GGOH to GGal, and fourth, the knockdown of CYP3A4 by siRNA significantly reduced the amount of endogenous GGA in Hep3B/MAOB-KO cells. These results indicate that CYP3A4 can act as an alternative oxidase for GGOH when hepatic MAOB is deleted in the human hepatoma-derived cell line Hep3B, and that endogenous GGA levels are maintained by a multitude of enzymes.

Some significant compounds present in annatto are geranylgeraniol and tocotrienols. These compounds have beneficial effects against hyperlipidemia and chronic diseases, where oxidative stress and inflammation are present, but the exact mechanism of action of such activities is still a subject of research. This study aimed to evaluate possible mechanisms of action that could be underlying the activities of these molecules. For this, in silico approaches such as ligand topology (PASS and SEA servers) and molecular docking with the software GOLD were used. Additionally, we screened some pharmacokinetic and toxicological parameters using the servers PreADMET, SwissADME, and ProTox-II. The results corroborate the antidyslipidemia and anti-inflammatory activities of geranylgeraniol and tocotrienols. Notably, some new mechanisms of action were predicted to be potentially underlying the activities of these compounds, including inhibition of squalene monooxygenase, lanosterol synthase, and phospholipase A2. These results give new insight into new mechanisms of action involved in these molecules from annatto and Chronic®.

Background/Aim: Geranylgeraniol (GGOH), a C20 isoprenoid naturally occurs in several foods. We previously reported that GGOH treatment reduced the expression levels of Atrogin-1 which is involved in skeletal muscle degradation and stimulates the myogenic differentiation of C2C12 myoblasts. However, the effect of GGOH supplementation on skeletal muscle metabolism in vivo is unknown. Materials and Methods: Skeletal muscle atrophy was induced by denervation. The expression levels of Atrogin-1 were assessed by western blotting or real time PCR. Results: Intraoral administration of GGOH reduced the decrease in the cross-sectional area of muscle fibers and also suppressed the expression levels of Atrogin-1 in denervation induced muscle atrophy. Also, GGOH treatment suppressed the expression of Atrogin-1 and the decrease in skeletal muscle fiber size by glucocorticoid in vitro. Conclusion: Intraoral administration of GGOH rescues denervation-induced muscle atrophy via suppression of Atrogin-1. [ABSTRACT FROM AUTHOR]

Aim: The propouse of this study was to available the cytotoxicity, phagocytic activity, and leishmanicidal potential of extract standardized from the fruits of Pterodon emarginatus Vogel. Background: P. emarginatus is a Brazilian medicinal plant with several constituents shown to have anti-inflammatory, antinociceptive, antimicrobial, and antiproliferative activities. Materials and Methods: The standardized extract in geranylgeraniol obtained from the fruit of P. emarginatus was investigated for clastogenic and cytotoxic effects, as well as phagocytic activity against Leishmania guyanensis. Results: A 24-h exposure of Allium cepa roots to standardized geranylgeraniol extract (9.77 mg/mL) promoted clastogenic effects, with a mitotic index (MI) of 10.33 ± 0.61. Extending the exposure time to 48 or 72 h significantly reduced the MI (P < 0.05). The IC50for geranylgeraniol in 3T3 and RAW 264.7 cells was found to be 15.5 mg/mL and 12.2 mg/mL, respectively. The standardized extract in geranylgeraniol exhibited high phagocytic and microbicidal activities in Leishmania, promoting a >60% increase in macrophage infection, with greater internalization and destruction of the parasite. In silico study results suggest that geranylgeraniol acts in Leishmania by interfering with the biosynthesis of sterols through steric hindrance of the active site of the enzyme CYP51. Conclusion: The standardized extract in geranylgeraniol may be useful as an antiproliferative agent, macrophage immunomodulator, and as a cytotoxic treatment against infectious agents. [ABSTRACT FROM AUTHOR]

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication associated with bisphosphonate treatment. Zoledronic acid (ZA) is a commonly used bisphosphonate due to its effectiveness in increasing bone density and reducing skeletal events, with evidence that it alters angiogenesis. Replacement of the mevalonate pathway using geranylgeraniol (GGOH) was studied to determine the effects of ZA on angiogenic gene expression in primary human osteoclasts. Osteoclast cultures were generated from peripheral blood mononuclear cells of three patients using the peripheral blood mononuclear cell isolation. These cells were phenotyped by phase-contrast microscopy, tartrate-resistant acid phosphatase staining, and pit assays. Primary osteoclasts were found to express a number of key angiogenic molecules at very high levels. Gene expression levels for 84 human angiogenic factors were determined using PCR arrays. Three genes with significant fold regulation (FR) in response to ZA were as follows: tumor necrosis factor (FR = +2.57, P = 0.050), CXCL9 (FR = +39.48, P = 0.028), and CXCL10 (FR = +18.52, P = 0.0009). The co-addition of geranylgeraniol with ZA resulted in the significant down-regulation of these three genes along with CCL2, TGFBR1, ENG, and CXCL1. GGOH reversed the gene changes induced by ZA and may offer a promising treatment for BRONJ. [ABSTRACT FROM AUTHOR]

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K2 sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1β, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/β, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.

To counteract the effect of zoledronate and decrease the risk of osteonecrosis of the jaw (BRONJ) development in patients undergoing guided bone regeneration surgery, the use of geranylgeraniol (GGOH) has been proposed. Collagen membranes may act as biomimetical drug carriers. The objective of this study was to determine the capacity of collagen-based membranes doped with GGOH to revert the negative impact of zoledronate on the growth and differentiation of human osteoblasts. MG-63 cells were cultured on collagen membranes. Two groups were established: (1) undoped membranes and (2) membranes doped with geranylgeraniol. Osteoblasts were cultured with or without zoledronate (50 μM). Cell proliferation was evaluated at 48 h using the MTT colorimetric method. Differentiation was tested by staining mineralization nodules with alizarin red and by gene expression analysis of bone morphogenetic proteins 2 and 7, alkaline phosphatase (ALP), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), type I collagen (Col-I), osterix (OSX), osteocalcin (OSC), osteoprotegerin (OPG), receptor for RANK (RANKL), runt-related transcription factor 2 (Runx-2), TGF-β1 and TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3), and vascular endothelial growth factor (VEGF) with real-time PCR. One-way ANOVA or Kruskal-Wallis and post hoc Bonferroni tests were applied ( p < 0.05). Scanning electron microscopy (SEM) observations were also performed. Treatment of osteoblasts with 50 μM zoledronate produced a significant decrease in cell proliferation, mineralization capacity, and gene expression of several differentiation markers if compared to the control ( p < 0.001). When osteoblasts were treated with zoledronate and cultured on GGOH-doped membranes, these variables were, in general, similar to the control group ( p > 0.05). GGOH applied on collagen membranes is able to reverse the negative impact of zoledronate on the proliferation, differentiation, and gene expression of different osteoblasts' markers.

• A sequential CO 2 extraction process was proposed to separate annatto seed compounds. • A geranylgeraniol-rich fraction was recovered in the first step at low CO 2 density. • Tocotrienols were recovered in the second step at high CO 2 density. The present work proposed a two-step sequential scCO 2 extraction process to selectively extract the main bioactive compounds from annatto seeds, geranylgeraniol and tocols, aiming to diversify and to improve the added-value of annatto seeds products. Firstly, pressure (10, 17, 24 and 31 MPa) and temperature (40 and 60 °C) conditions resulting CO 2 densities ranging from 290 kg/m3 to 915 kg/m3 were investigated to verify its effect on the chemical composition of the extracts. Extracts obtained at 290 kg/m3 presented the highest geranylgeraniol content (39 ± 9%) and the lowest tocotrienols content (0.95 ± 0.04%). CO 2 densities higher than 629 kg/m3 were needed to recover tocotrienols-rich fractions. Therefore, a two-step sequential scCO 2 extraction process was carried out using 60 °C and 10 MPa (290 kg/m3) in the first step to recover the geranylgeraniol-rich fraction, and 40 °C and 20 MPa (840 kg/m3) in the second step to recover the tocotrienols-rich fraction. [ABSTRACT FROM AUTHOR]

Phytol, the prenyl side chain of chlorophyll, is derived from geranylgeraniol by reduction of three double bonds. Recent results demonstrated that the conversion of geranylgeraniol to phytol is linked to chlorophyll synthesis, which is catalyzed by protein complexes associated with the thylakoid membranes. One of these complexes contains light harvesting chlorophyll binding like proteins (LIL3), enzymes of chlorophyll synthesis (protoporphyrinogen oxidoreductase, POR; chlorophyll synthase, CHLG) and geranylgeranyl reductase (GGR). Phytol is not only employed for the synthesis of chlorophyll, but also for tocopherol (vitamin E), phylloquinol (vitamin K) and fatty acid phytyl ester production. Previously, it was believed that phytol is derived from reduction of geranylgeranyl-diphosphate originating from the 4-methylerythritol-5-phosphate (MEP) pathway. The identification and characterization of two kinases, VTE5 and VTE6, involved in phytol and phytyl-phosphate phosphorylation, respectively, indicated that most phytol employed for tocopherol synthesis is derived from reduction of geranylgeranylated chlorophyll to (phytol-) chlorophyll. After hydrolysis from chlorophyll, free phytol is phosphorylated by the two kinases, and phytyl-diphosphate employed for the synthesis of tocopherol and phylloquinol. The reason why some chloroplast lipids, i.e. chlorophyll, tocopherol and phylloquinol, are derived from phytol, while others, i.e. carotenoids and tocotrienols (in some plant species) are synthesized from geranylgeraniol, remains unclear. [ABSTRACT FROM AUTHOR]

Strategies for enhancing GGOH production in Croton stellatopilosus callus culture included additions of precursors (sodium acetate-NA, sodium pyruvate-NP, mevalonic acid lactone-MVA) and elicitors (methyl jasmonate-MJ, acetylsalicylic acid-ASA, yeast extract-YE). Treated cells were evaluated for their GGOH contents by GC-FID and compared with the nontreated cells as controls. Additions of NA (25 mg/L), NP (50 mg/L) and MVA (100 mg/L) resulted in an enhancement of GGOH productivity to 0.61 mg/g DW, 0.52 mg/g DW and 0.70 mg/g DW, respectively, compared to the control culture (0.29 mg/g DW). Callus cultures elicited with MJ at 30 mg/L for 24 h stimulated GGOH production to 0.35 mg/g DW compared to the control culture (0.07 mg/g DW). Cells also responded to ASA (20 mg/L, 2 days) and YE (0.25 g/L, 4 days) and produced GGOH contents of 0.46 mg/g DW and 1.37 mg/g DW, respectively. This study has shown that isoprenoid precursors and conventional elicitors enhanced GGOH production in the C. stellatopilosus callus culture.

The seeds of Bixa orellana L. is widely used in food industry as a natural colorant. A major technological challenge for its utilization on aqueous food products is the low water solubility. Therefore, the present study aimed to obtain aqueous nanodispersions using this natural raw material by using a simple organic solvent-free and low energy method. Bixin and geranylgeraniol were found on the extract. The nanodispersions were prepared by addition of water on a mixture of extract and surfactant (s). The effect of the surfactants and dilution on the nanodispersions were also evaluated. Best results were achieved using polysorbate 80/sorbitan monooleate (HLB 13) (diluted in water, 1:10). The results shows the potential of this ecofriendly approach, in contrast to common methods that make use of potentially toxic organic solvents and high input of energy, which elevate the costs for further industrialization. [ABSTRACT FROM AUTHOR]

Isoprenoids play widely differing roles in various physiological processes in animals and plants. Geranylgeraniol (GGOH) is an isoprenoid found in plants, and is an important metabolic derivative in the isoprenoid/cholesterol synthesis pathway. Earlier studies focused on GGOH's ability to improve the side effects of bisphosphonate therapy by regulating the mevalonate pathway. More recently, the mevalonate pathway-independent effects of GGOH have been described, including anti-inflammatory, anti-tumorigenic, and neuroprotective activities. It is noteworthy that GGOH regulates the steroidogenesis pathway in testis-derived I-10 tumor cells. Testosterone is a hormone produced via steroidogenesis in testicles and plays a role in fetal development and the male reproductive system. GGOH enhanced testosterone and progesterone (its precursor) levels in I-10 cells by activating adenylate cyclase via cAMP/PKA signaling, without altering phosphodiesterase activity. These findings highlight the potential benefits of GGOH as a therapeutic agent for low testosterone levels, such as late-onset hypogonadism in men. [ABSTRACT FROM AUTHOR]

Background Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is a complication of the bisphosphonate (BP) treatment and its pathopysiology is still not fully understood. The existing preventive and treatment options require updates and more attention. Geranylgeraniol (GGOH) so far demonstrated an increased activity and viability of the cells previously treated with zoledronic acid (ZA). The aim of this study was to evaluate the in vivo effects of GGOH on the development of BRONJ. Materials and methods A total of 30 male Wistar rats were included in the study, divided into three groups: two experimental groups (EG1 and EG2) and a control group (CG). Rats from EG1 and EG2 were treated with 0,06 mg/kg ZA ip weekly in a duration of five weeks, while CG received saline ip. On the third week all animals underwent extraction of the lower right first molars. The rats from EG2 received a local solution of GGOH in concentration of 5 mM in the socket every day after the tooth extraction. The analyses included clinical evaluation on the wound healing and pathohistological evaluation for presence and level of osteonecrosis. Results EG2 showed significantly improved wound healing and tissue proliferation, when compared to EG1. EG2 significantly differed from EG1 and CG (p<0,05) for the presence of microscopical osteonecrosis (80% vs 22,2% vs 0%). Regarding to the number of empty lacunes without osteocytes and the level of necrosis, all groups demonstrated significant differences. Conclusion Geranylgeraniol in a form of local solution may be a promising option for prevention and treatment of BRONJ. [ABSTRACT FROM AUTHOR]

Geranylgeraniol (GGOH), a natural isoprenoid found in plants, has anti-inflammatory effects via inhibiting the activation of nuclear factor-kappa B (NFκB). However, its detailed mechanism has not yet been elucidated. Recent studies have revealed that isoprenoids can modulate signaling molecules in innate immune responses. We found that GGOH decreased the expression of lipopolysaccharide (LPS)-induced inflammatory genes in human macrophage-like THP-1 cells. Furthermore, we observed that the suppression of NFκB signaling proteins, in particular interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), occurred in GGOH-treated cells prior to LPS stimulation, suggesting an immunomodulatory effect. These results indicate that GGOH may modulate and help prevent excessive NFκB activation that can lead to numerous diseases.

Annatto ( Bixa orellana L.) is a tropical crop indigenous to the Americas, probably Amazonia. Annatto is commercially valuable in the food and cosmetics industries as a natural dye used instead of synthetic dyes. In addition, annatto contains other important substances for human health, such as geranylgeraniol, tocotrienols and other carotenoids. The aim of the present study was to evaluate the genetic diversity of 63 accessions from the annatto germplasm bank at the Agronomic Institute (IAC), São Paulo, Brazil, using four phytochemical compounds and 16 microsatellite markers. Significant variation was observed for the phytochemical compounds, ranging from 2 to 7.31 g (100 g dry matter-DM) for bixin, 2.14-7.11 g (100 g DM) for lipids, 0.25-1.05 g (100 g DM) for tocotrienols, and 0.49-2.61 g (100 g DM) for geranylgeraniol content. A total of 73 alleles was observed in the molecular characterization with 16 microsatellite loci. We found higher expected heterozygosity than observed heterozygosity for all loci, indicating strong deficits of heterozygotes. For both molecular and phytochemical compounds, cluster and PCoA analyses tended to separate the accessions from Rondônia, northern Brazil, with only a few exceptions, from the Southwestern accessions. The same two groups were found in the Bayesian analysis with molecular data. Rondônia accessions showed higher values for all the phytochemical compounds and higher levels of genetic diversity. Some accessions presented bixin levels well above the average and are promising materials to be used in genetic improvement programs. [ABSTRACT FROM AUTHOR]

Objectives: Recent studies focused on angiogenesis in the pathophysiology of bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) and identified geranylgeraniol (GGOH) as a feasible option for BP-ONJ therapy. This study investigated the influence of GGOH on microvessel sprouting after BP-incubation in vitro. Materials and methods: Ten experimental set-ups were randomly designed in an in vitro 3D-angiogenesis assay. Two groups included HUVEC cell spheroids with and without (±) GGOH substitution as controls and eight groups pairwise contained either clodronate or the nitrogen-containing bisphosphonates (N-BP) ibandronate, pamidronate, and zoledronate ± GGOH. The size of the cell spheroids including the outbranching sprouts (SpS) as well as the density (SpD) and length of the sprouts (SpL) were analyzed by a grid system after 0, 24, 48, and 72 h. Results: For controls and NN-BP clodronate, no significant differences at any tested parameter and any point of measurement could be detected within the experimental set-ups ± GGOH ( p each ≥0.05). For N-BP ibandronate, the experimental set-ups +GGOH showed a significantly increased SpS, SpD, and SpL after 48 and 72 h ( p each ≤0.002) compared to the experimental set-ups −GGOH. For N-BPs pamidronate and zoledronate, the experimental set-ups + GGOH demonstrated a significantly increased SpS, SpD, and SpL after 24, 48, and 72 h ( p each ≤0.001) compared to the experimental set-ups −GGOH. Conclusions: The strong negative influence of N-BPs on microvessel sprouting could be significantly reversed by GGOH. Clinical relevance: Since supportive therapeutic options for BP-ONJ are lacking, GGOH might be a promising substitute for BP-ONJ prevention and therapy. [ABSTRACT FROM AUTHOR]

Optimization of supply and conversion efficiency of geranylgeranyl diphosphate (GGPP) is important for enhancing geranylgeraniol (GGOH) production in Saccharomyces cerevisiae . In this study, first, a strain producing 26.92 ± 1.59 mg/g of dry cell weight squalene was constructed with overexpression of all genes of the mevalonate (MVA) pathway, and an engineered strain producing 597.12 mg/L GGOH at the shake flask level was obtained. Second, through additional expression of PaGGPPs - ERG20 and PaGGPPs - DPP1 , and downregulating expression of ERG9 , the GGOH titer was increased to 1221.96 mg/L. Then, a NADH HMG-CoA reductase from Silicibacter pomeroyi ( SpHMGR ) was introduced to alleviate the high dependence of the strain upon NADPH, and the GGOH production was further increased to 1271.14 mg/L. Finally, the GGOH titer reached 6.33 g/L after optimizing the fed-batch fermentation method in a 5 L bioreactor, with a 24.9% improvement from the previous report. This study might accelerate the process of developing S. cerevisiae cell factories for diterpenoid and tetraterpenoid production.

Currently, leishmaniasis is difficult to manage owing to the limited choice and high toxicity of available drugs, and emergence of drug-resistant protozoa. Medicinal plants, which produce various bioactive molecules, can help counter this global shortage. In this study, we prepared Pterodon pubescens fruit extracts, which show antileishmanial activity, and developed a nanoemulsion of the optimized extract to improve its performance. The extracts were prepared using conventional methods and a supercritical fluid method and were tested for activity against Leishmania amazonensis promastigotes and amastigotes. The two most effective extracts were formulated as nanoemulsions. Although both extracts showed cytotoxicity, the supercritical extracts were more effective against L. amazonensis promastigotes and amastigotes than conventional extracts were. This was attributed to the high content of the geranylgeraniol derivative in the supercritical extracts. The nanoemulsions showed a better selectivity index and significantly improved activity against parasites (IC 50 : 2.7 μg/mL for nanoemulsion of hexane extract; IC 50 : 1.9 μg/mL for nanoemulsion of supercritical extract) compared to the Miltefosine standard (0.7 μg/mL). This could be due to the smaller droplets of the supercritical extracts, allowing better penetration. In conclusion, the extracts showed promise in in vitro tests, and could be used as a leishmaniasis treatment. [ABSTRACT FROM AUTHOR]

Introduction: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication of bisphosphonate therapy. The mechanism underlying BRONJ pathogenesis is poorly understood. Objectives: To determine the effects of zoledronic acid (ZA) and geranylgeraniol (GGOH) on the mevalonate pathway (MVP) in osteoblasts generated from the human mandibular alveolar bone in terms of cell viability/proliferation, migration, apoptosis and gene expression. Materials and methods: Primary human osteoblasts (HOBs) isolated from the mandibular alveolar bone were phenotyped. HOBs were cultured with or without ZA and GGOH for up to 72 h. Cellular behaviour was examined using a CellTiter-Blue® viability assay, an Ibidi culture-insert migration assay, an Apo-ONE® Homogeneous Caspase-3/7 apoptosis assay and transmission electron microscopy (TEM). Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to determine the simultaneous expression of 168 osteogenic and angiogenic genes modulated in the presence of ZA and GGOH. Results: ZA decreased cell viability and migration and induced apoptosis in HOBs. TEM revealed signs of apoptosis in ZA-treated HOBs. However, the co-addition of GGOH ameliorated the effect of ZA and partially restored the cells to the control state. Twenty-eight genes in the osteogenic array and 27 genes in the angiogenic array were significantly regulated in the presence of ZA compared with those in the controls at one or more time points. Conclusion: The cytotoxic effect of ZA on HOBs and its reversal by the addition of GGOH suggests that the effect of ZA on HOBs is mediated via the MVP. Clinical relevance: The results suggest that GGOH could be used as a possible therapeutic/preventive strategy for BRONJ. [ABSTRACT FROM AUTHOR]