Cannabis sativa L. are undoubtedly cannabidiol (CBD), a non-psychotropic compound, but with other pharmacological properties, and Δ9-tetrahydrocannabinol (Δ9-THC), which instead possesses psychotropic activity and is responsible for the recreative use of hemp. Recently, the homolog series of both CBDs and THCs has been expanded by the isolation in a medicinal cannabis variety of four new phytocannabinoids possessing on the resorcinyl moiety a butyl-(in CBDB and Δ9-THCB) and a heptyl-(in CBDP and Δ9-THCP) aliphatic chain. In this work we report a new series of phytocannabinoids that fills the gap between the pentyl and heptyl homologs of CBD and Δ9-THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and Δ9-tetrahydrocannabihexol (Δ9-THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and Δ9-THCH have been already identified and reported as monomethyl ether derivatives of the canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and Δ9-tetrahydrocannabinol monomethyl ether (Δ9-THCM). The unambiguously identification in cannabis extract of the n-hexyl homologues of CBD and Δ9-THC different from the corresponding methylated isomers  was achieved by comparison of the retention time, molecular ion, and fragmentation spectra with those of the authentic standards obtained via stereoselective synthesis, and a semi-quantification of these cannabinoids in the FM2 medical cannabis variety was provided. Conversely, no trace of Δ9-THCM was detected. Moreover, CBDH was isolated by semipreparative HPLC and its identity was confirmed by comparison with the spectroscopic data of the corresponding synthetic standard. Thus, the proper recognition of CBDH, CBDM and Δ9-THCH closes the loop and might serve in the future for researchers to distinguish between these phytocannabinoids isomers that show a very similar analytical behaviour. Lastly, CBDH was assessed for biological tests in vivo showing interesting analgesic activity at low doses in mice.The two most important and studied phytocannabinoids present in  In the academic research, new insights on cannabis chemistry have been disclosed thanks to the high-performing technological platforms for the identification of new compounds6. Although there is still much to do in the cannabis chemistry research, almost 150 phytocannabinoids can be count on the most updated inventory7. Our most recent works have disclosed the existence of new phytocannabinoids series besides those of the orcinoids, varinoids and olivetoids, belonging to the cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) type cannabinoids8. The new series of phytocannabinoids share the terpenophenolic core of CBD and Δ9-THC and differ for the length of the linear alkyl side chain; specifically, cannabidibutol (CBDB), and Δ9-tetrahydrocannabutol (Δ9-THCB) have a n-butyl side chain8, whereas cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) have a n-heptyl side chain4. The discovery of new phytocannabinoids, which were both directly isolated from the plant and synthetically prepared in the lab, has opened new gaps on their still unexplored biological activity, making us wondering about their pharmacological effects on humans.Cannabis research has made great progresses in the latest years in both clinical and academic field. For example, new cannabis based drugs, like Epidiolex, have been placed on the market for the treatment of severe forms of infant epilepsy not responding to conventional therapies9-THC, bearing a n-hexyl side chain on the resorcinyl moiety. At the best of our knowledge and according to the literature, no case of hexyl derivatives of cannabinoid has been reported so far. Conversely, cannabinoids with the same molecular formula and molecular weight have been classified as monomethyl ether derivatives of canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and Δ9-tetrahydrocannabinol monomethyl ether (Δ9-THCM)9. Whilst CBGM and CBDM have been already isolated and characterized11, Δ9-THCM has been detected in cannabis smoke12 and some authors reported that it is present in the plant, but they were not able to isolate it due to chromatographic issues13. Our findings on the presence of the hexyl homologs of CBD and Δ9-THC, which we named cannabidihexol (CBDH) and Δ9-tetrahydrocannabihexol (Δ9-THCH) respectively, were supported by the stereoselective synthesis of the corresponding pure standards that are found in the plant prior to decarboxylation.To further complicate the already intricate scenario, we report a new series of phytocannabinoids that fills the gap between the pentyl and heptyl homologs of CBD and Δ23H32O4, suggesting the presence of the carboxylic group. The analysis of the fragmentation spectra in negative ionization mode confirmed this hypothesis but showed three different fragmentation patterns  and the putative tetrahydrocannabinol monomethyl ether (THCM). However, we found three peaks corresponding to the same formula but different MS2 spectra. By a comparison with other CBD and THC homologs present in our spectral library, such as cannabidivarin (CBDV), cannabidibutol (CBDB), cannabidiphorol (CBDP), Δ9-tetrahydrocannabivarin (Δ9-THCV), Δ9-tetrahydrocannabutol (Δ9-THCB), and Δ9-tetrahydrocannabiphorol (Δ9-THCP), we were able to putatively identify two new homologs of CBD and THC with a hexyl side chain. As shown in Fig. 2 unit (14 amu) from the corresponding pentyl (CBD and Δ9-THC) and the recently identified heptyl homologs (CBDP and THCP), not only for the molecular ion [M+H]+ but also for all fragments. For both CBD  and tetrahydrocannabinolic acid monomethyl ether (THCMA). Similarly, cannabinoids with molecular formula Cd, a stereoselective synthesis of the putatively identified cannabinoids, which for sake of simplicity and consistency were called cannabidihexol (CBDH) and Δ9-tetrahydrocannabihexol (Δ9-THCH), and the monomethyl ether derivatives of CBD and Δ9-THC (CBDM and Δ9-THCM) was performed.In order to confirm the identity of these two new cannabinoids and unambiguously identify peak B9-THC, we hypothesized the presence in the FM2 variety of the hexyl homolog of CBG, which has a molecular formula C22H34O2 and [M+H]+ ion at m/z 331.2632. Only one peak resulted from the specific ion search, thus instilling the doubt about its identity as hexyl or methyl ether derivative. The fragmentation spectrum of its acidic precursor in the FM2 native extract showed a pattern different from that of cannabigerolic acid (CBGA), for which the analytical standard was available, and from those reported in the literature for the other series of CBG like cannabigerovarin (CBGV)14 and cannabigerobutol (CBGB)9. The match of retention time (20.98 min) and fragmentation spectrum with those of synthetic CBGM and the comparison with spectral data reported in the literature for the same cannabinoid confirmed that the additional methyl group was attached to the oxygen of the resorcinyl moiety and was not part of the alkyl side chain.Given the results obtained for CBD and Δ9-tetrahydrocannabihexol ((−)-trans-Δ9-THCH) was performed as previously reported for the synthesis of the corresponding homologs (−)-trans-CBDB, (−)-trans-CBDP, (−)-trans-Δ9-THCB and (−)-trans-Δ9-THCP8. The appropriate 5-hexylbenzene-1,3-diol (4) was prepared first as reported in Fig. 1) was easily prepared in quantitative yield by reaction of 1-(bromomethyl)-3,5-dimethoxybenzene with triphenylphosphine in refluxing toluene for 6 h. Wittig’s reaction between 1 and valeraldehyde in 0.1 M K2CO3 aqueous solution at reflux for 24 h gave 1-(hex-1-en-1-yl)-3,5-dimethoxybenzene (2) as a 55:45 E/Z mixture which was hydrogenated using the ThalesNano H-Cube flow reactor, to give the corresponding 1-hexyl-3,5-dimethoxybenzene (3) in 91% yield. The demethylation performed using BBr3 in anhydrous DCM, overnight at room temperature and under nitrogen atmosphere, gave 5-hexyl-resorcinol (4) in quantitative yield. In our previous works CBDB and CBDP were synthesized first by condensation of the appropriate resorcinol with -1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol, using pTSA as catalyst and stopping the reaction before CBDs evolve to THCs in the same conditions. Conversely, for the selective synthesis of (−)-trans-Δ9-THCB and (−)-trans-Δ9-THCP a longer procedure was adopted. The appropriate resorcinol was condensed with -1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol in the same condition described above for a longer reaction time . In this way, the CBDs were quantitatively converted into the corresponding Δ8-THCs. Hydrochlorination of the Δ8 double bond of (−)-trans-Δ8-THCs, allowed to obtain (−)-trans-HCl-THCs, which were successively converted to (−)-trans-Δ9-THCs by selective elimination on position 2 of the terpene moiety using potassium t-amylate as base. Although this procedure allowed to selectively prepare (−)-trans-Δ9-THCs, it has the inconvenience to be time consuming, and with low atom economy. Because the conversion of CBDs to Δ8-THCs passes through the formation of Δ9-THCs first, for the synthesis of (−)-trans-CBDH and (−)-trans-Δ9-THCH we evaluated the possibility to stop the reaction before Δ9-THCH starts to convert into Δ8-THCH. Therefore, 5-hexyl-resorcinol (4) was condensed with -1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol using pTSA as catalyst and the progression of the reaction was monitored every 15 min by HPLC–UV/Vis. After approximately 2 h, almost the 50% of (−)-trans-CBDH converted in (−)-trans-Δ9-THCH, but no traces of Δ8-THCH were detected. The reaction was therefore stopped and (−)-trans-CBDH and (−)-trans-Δ9-THCH were purified as reported in Material and Methods section. (−)-trans-CBDH and (−)-trans-Δ9-THCH were obtained in 17% and 20% yield, respectively. The total yield of the two phytocannabinoids was 37%, which is in line with the yield obtained for the sole synthesis of CBDB or CBDP using the same procedure, but quenching the reaction after the consumption of the starting materials and before that CBDs started to isomerize into THCs . Therefore, strictly monitoring the condensation of the appropriate resorcinol with -1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol it is possible to prepare in one-pot reaction both (−)-trans-CBDs and (−)-trans-Δ9-THCs, avoiding cumbersome and longer procedure to selectively prepare the (−)-trans-Δ9-THCs, without the awkward formation of their Δ8 isomers. The synthesis of the monomethyl ether derivatives of CBD, THC and CBG is reported in Fig. trans-CBDM and CBGM were easily prepared by methylation of the commercially available CBD and CBG by reaction with 0.5 equivalents of dimethylsulfate, in DMF at room temperature, using K2CO3 as base -trans-CBDM, through cyclization catalyzed by pTSA -trans-Δ9-THCH, (−)-trans-CBDM, (−)-trans-Δ9-THCM and CBGM, and their unambiguous 1H and 13C assignments were achieved by NMR spectroscopy -trans-Δ9-THCH, as already stated during the synthesis of (−)-trans-CBDB, (−)-trans-CBDP, (−)-trans-Δ9-THCB and (−)-trans-Δ9-THCP, and by comparison with the well-known homologs  no significant differences in the proton and carbon chemical shifts of the terpene and aromatic moieties were observed among CBD and Δ9-THC homologs. The sole exception observed regards the integration of the multiplet in the range 1.4–1.2 ppm in the 1H spectra and the number of carbon signals in the range 20–30 ppm of the 13C spectra, corresponding to the central methylene units of the alkyl chain on the resorcinyl moiety. The perfect match in the chemical shift of the terpene and aromatic moieties between the synthesized (−)-trans-CBDH and (−)-trans-Δ9-THCH and the respective homologues8, combined with the mass spectra and fragmentation pattern, allowed us to unambiguously confirm the chemical structures of the two new synthetic cannabinoids.The trans  configuration at the terpene moiety was confirmed by optical rotatory power. The new cannabinoids (−)-trans-CBDH, (−)-trans-Δ9-THCH, (−)-trans-CBDM and (−)-trans-Δ9-THCM showed an [α]D20 of − 146°, − 166°, − 113° and − 161°, respectively, in chloroform. The [α]D20 values were in line with those of the homologs15, suggesting a  configuration for the four phytocannabinoids. Lastly, the perfect superimposition between the 1H and 13C NMR spectra of both synthetic and extracted (−)-trans-CBDH was observed, confirming the identity of the new cannabinoids identified in the FM2 cannabis variety -trans-cannabidihexol ((−)-trans-CBDH) and (−)-trans-Δd could be assigned to CBDH and the second one Bd to CBDM . In particular, the hexyl homologs of CBD and Δ9-THC resulted 27 µg/g and 7 µg/g, while the methyl ether derivatives CBDM and CBGM were 50 µg/g and 102 µg/g. No Δ9-THCM was detected in the FM2.Thanks to the synthetically prepared analytical standards of CBDH, Δ = 17.32, P < 0.0001, time F = 67.80, P < 0.0001 and interaction F = 3.02, P < 0.0001); also, the dose of 2 mg/kg had a significant antinociceptive effect as compared to the vehicle group. The doses of 3 and 5 mg/kg had no effect on the formalin test  followed by a quiescent period, and then a second, prolonged phase (15–60 min) of tonic hyperalgesia Fig. C. In the8. The present work expanded the scope of cannabinoids identification completing the series of homologs with different alkyl side chain from three to seven methylene units. Up to now, only cannabinoids with an odd number of carbon atoms on the side chain have been reported and those with an even number of carbon atoms have been supposed to be artifacts derived from fungal ω-oxidation of their corresponding homologs7. The investigation of the origin of these species, such as those with a butyl and hexyl side chain, is beyond the scope of this work, but, although surprising, it is certain that such cannabinoids are actually present in a medicinal cannabis variety. The literature reports the existence of monomethyl ether derivatives of the canonical pentyl cannabinoids to justify the presence of compounds bearing an additional methyl group. Although on one side the structural identity of such derivatives was confirmed, our findings pointed out a new series of cannabinoids with the same molecular formula of the monomethyl ethers but with a different arrangement. Their origin, whether it is from the plant or from microorganisms, should be investigated as this might disclose new insights in the cannabis biochemistry. It is certainly important to underline that it is very easy to confuse CBDH and CBDM, as well as Δ9-THCH and Δ9-THCM. However, the match of the high-resolution fragmentation patterns with their pure synthetic standards was determinant to assign the respective chemical structure. This work might serve in the future for any researcher to distinguish between two species that show a very similar analytical behaviour. In a similar way, the methyl ether derivative of CBG was also identified (CBGM).The comprehensive characterization of the chemical profile of a cannabis variety is a rather arduous task as the analytical tools in the chemist’s hand are not able to cover such a broad range of compounds. However, the high sensitivity and selectivity of the high-resolution mass spectrometry, for example those achieved with the Orbitrap technology, can enable the identification of a reasonable number of molecules, even when present in very small traces. This approach allowed for the identification of new series of cannabinoids, CBD and THC homologs, with different lengths of the alkyl side chain, which were recently reported by our group9-THCM was detected in the FM2 variety. On the other hand, both CBDM and CBGM showed a high peak as well as their native precursors CBDMA and CBGMA. Achieved results are in accordance with what reported by Lumır Ondrej Hanus et al., which showed that the cannabigerol monomethyl ether is always presents in greater quantities than its products, THCMA and CBDMA16.It is worth noting that no Δ18.de Meijer et al. demonstrated that the cannabinoid acid synthases  show a different affinity for CBGA alkyl homologues. This concept would explain the achieved results. CBDAS could be competitively stronger than THCAS when the substrate is CBGMA9-THC, CBDP and Δ9-THCP were found in the FM2 at the concentration of 243 µg/g and 29 µg/g4; whereas, in the same cannabis variety, the butyl series of CBD and THC was found at the concentration of 500 µg/g and 400 µg/g for CBDB and Δ9-THCB respectively8. In this work, we found 27 µg/g and 7 µg/g for CBDH and Δ9-THCH respectively. However, this data should be verified considering a larger number of different varieties in order to provide a reliable statistical significance.The comparison of the results obtained for the concentrations of unorthodox cannabinoids in the FM2 variety suggested that there is no relationship between the different series as the same CBD to THC ratio is not respected. Considering our recent work on the heptyl derivatives of CBD and ΔThe CBDH could have pleiotropic mechanisms of action through which it can exert its pharmacological effect. We found that the doses of the 1 and 2 mg/Kg significantly reduced the late phase of the formalin-induced nocifensive behavior, whereas the higher doses 3 and 5 mg/Kg were uneffective. This could be due, at least in part, assuming that at these doses CBDH can activate receptor facilitating nociception such as TRPV1 or other channels. On the other hand, we can speculate that CBDH at the higher doses could block receptors involved in antinociception such as CB1 or CB2. However, further pharmacological studies are needed to better investigate the pharmacodynamics profile of this interesting compound.Cannabis Sativa L. cannabinoma has been added with this work. In particular, clarity has been made about the possible confusion between phytocannabinoids with a 6-term alkyl chain  and those with a methylated resorcinolic hydroxyl group . Furthermore, two new phytocannabinoids CBDH and THCH have been identified in the FM2 variety by comparison with their respective authentic synthesized compounds. In particular, CBDH has been isolated and its pharmacological activity has been evaluated in vivo in mice. At extremely low doses (1 mg/kg) it showed an interesting nocifensive activity. However, the CBDH concentration of 27 μg/g found in the FM2 variety is too low to exert the pharmacological effect but it is not excluded that other cannabis varieties may contain higher concentrations. More in-depth pharmacological studies are currently underway to clarify the mechanism of action of this new phytocannabinoid.Another piece of knowledge towards understanding 19 and analyzed by UHPLC-HESI-Orbitrap without further dilution. The remaining 9.5 g were treated according to the protocol of Pellati et al. with minor changes20. Briefly, freeze-dried plant material was sonicated with 400 mL of n-hexane for 15 min in an ice bath. After centrifugation for 10 min at 2000 × g the pellets were discarded. The same procedure was repeated twice more. The supernatants were then dried under reduced pressure and resuspended in 10 mL of acetonitrile, filtered and passed through a semi-preparative liquid chromatography for the isolation of the acidic species of the cannabinoids of interest.FM2 cannabis variety is produced from the strain CIN-RO bred by the Council for Agricultural Research and Economics (CREA) in Rovigo  and supplied to the Military Chemical Pharmaceutical Institute . Experiments on FM2 inflorescence (batch n. 6A32/1) were performed with the authorization of the Italian Ministry of Health (prot. n. SP/062). Two 5 g packs were finely grinded (< 2 mm particle size) and divided into two batches: 500 mg were extracted with 50 mL of ethanol 96% according to the procedure reported in the monograph of Cannabis Flos of the German Pharmacopoeia4. A Luna C18 with a fully porous silica stationary phase (Luna 5 µm C18(2) 100 Å, 250 × 10 mm)  was the column employed and a mixture of acetronitrile:0.1% aqueous formic acid 70:30 (v/v) was used as mobile phase at a constant flow rate of 5 mL/min. The fractions containing CBDHA (retention time 13.0 min) was isolated as reported in our previous work2. The fractions containing CBDHA (13.0 min) were analyzed by UHPLC-HESI-Orbitrap and dried on the rotavapor at 70 °C. The residue was placed in an oven at 120 °C for 2 h to achieve decarboxylation. An amount of about 0.3 mg of CBDH was obtained.A semi-preparative LC system  was used to separate the FM2 mixture into 80 fractions in a total run time of 80 min. The chromatographic conditions used are reported in the paper by Citti et al.4. Briefly, the capillary temperature was set at 320 °C, the vaporizer temperature at 280 °C, the electrospray voltage at 4.2 kV (for the positive ionization mode) and 3.8 kV (for the negative mode), the sheath gas and the auxiliary gas at 55 and 30 arbitrary units respectively, the RF level of the S lens at 45. Analyses were acquired in full scan data-dependent acquisition (FS-dd-MS2) in positive and negative mode with a resolving power of 70,000 FWHM and m/z of 200 using the Xcalibur 3.0 software . For the Orbitrap mass analyzer, a scan range of m/z 250–400, an AGC of 3e6, an injection time of 100 ms and an isolation window of m/z 0.7 were chosen as the optimal parameters. The collision energy for the fragmentation of the molecular ions was set at 20 eV. The exact masses of the [M+H]+ and [M−H]− molecular ions were extracted from the total ion chromatogram (TIC) of the FM2 extracts and matched with pure analytical standards for accuracy of the exact mass (5 ppm), retention time and MS/MS spectrum.Analyses on FM2 extracts were performed on a Thermo Fisher Scientific Ultimate 3000 provided with a vacuum degasser, a binary pump, a thermostated autosampler, a thermostated column compartment and interfaced to a heated electrospray ionization source and a Q-Exactive Orbitrap mass spectrometer (UHPLC-HESI-Orbitrap). The HESI and Orbitrap parameters were set following our previous work18 stationary phase  following the conditions employed for our previous work4.The chromatographic separation was carried out on a core shell C9-THC and CBD, their hexyl homologs CBDH and Δ9-THCH, and the methyl ether derivatives of CBD and CBG, CBDM and CBGM, was carried out using a calibration curve with the external standard method. A stock solution of CBD and Δ9-THC (1 mg/mL) was properly diluted to obtain five non-zero calibration points at the final concentrations of 50, 100, 250, 500 and 1000 ng/mL; a stock solution of CBDH, CBDM, Δ9-THCH, CBDM and CBGM was diluted to obtain the final concentrations of 5, 25, 50, 100 and 250 ng/mL. The linearity was assessed by the coefficient of determination (R2), which was greater than 0.992 for each analyte.A semi-quantitative analysis of Δ3); cyclohexane (CE); dichloromethane (DCM); diethyl ether (Et2O); dimethyl sulfoxide (DMSO); ethyl acetate (AcOEt). The reactions were monitored by thin-layer chromatography (TLC) using 60F-254 silica gel plates (from Merck) and inspected with UV lamp, or alkaline KMnO4 stain. Purification of the synthesized products was performed by flash chromatography on silica gel (40–63 μm). The mobile phase is specified in the respective following monographies. NMR spectra were recorded on a Bruker 400 (at 400.134 MHz for 1H and 100.62 MHz for 13C) or on a Bruker 600 (at 600.130 MHz for 1H and 150.902 MHz for 13C) spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) and referenced to the solvent residual peaks. Coupling constants are reported in hertz (Hz) and the splitting pattern is reported as: singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), quintet (quin), multiplet (m), broad signal (b). Monodimensional and bidimensional spectra were acquired using the same parameters previously reported8. Optical rotation (α) was acquired with a Polarimeter 240C from Perkin–Elmer , using a cell with a length of 100 mm, and a volume 1 mL.The reagents and the solvents used for the synthesis of the analytical standards were purchased from Sigma-Aldrich and VWR, respectively. In the synthetic procedures, the solvents were abbreviated as following: acetonitrile (ACN); chloroform  was added to a stirred solution of 1-(bromomethyl)-3,5-dimethoxybenzene , in 30 mL of toluene and refluxed for 6 h. After standing at room temperature overnight, the precipitate formed was collected by filtration, washed with diethyl ether and dried to give 10.4 g of a white solid (quant. yield).1H-NMR  δ 7.78–7.76 , 7.66–7.63 , 6.35 , 6.30 , 5.32 , 3.54 .1  in 20 mL of aqueous 0.1 M K2CO3. The mixture was refluxed overnight and chilled down at 0 °C. Cyclohexane (20 mL) was added, and the biphasic mixture was vigorously stirred in the same condition for two hours in order to precipitate triphenylphosphine oxide. The solid was removed by filtration and the organic phase separated. The aqueous layer was extracted two more times with cyclohexane. The combined organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated to give 540 mg (81% yield) of a yellow oil. The product was obtained as a 55:45 E/Z mixture of alkene, pure enough to be used in the next step without further purification.Valeraldehyde  was added to a stirred suspension of 1H NMR  δ 6.43 , 6.35 , 6.31 , 5.66 , 3.79 , 2.37–2.30 , 1.47–1.31 , 0.90 ; 1H NMR  δ 6.51 , 6.29 , 6.21 , 3.79 , 2.24–2.18 , 1.47–1.31 , 0.92 .2), solubilized in EtOH, was selectively reduced at the double bond by hydrogenation with the flux reactor H-Cube Mini Plus ThalesNano using the following conditions: temperature 30 °C, H2 20 psi, cartridge Pd/C, solvent EtOH, flow 1 mL/min. The solvent was evaporated obtaining 495 mg (91% yield) of a colourless liquid pure enough to be used in the next step without further purification.The mixture of (E/Z)-1-(hept-1-en-1-yl)-3,5-dimethoxybenzene  δ 6.37 , 6.32 , 3.80 , 2.57 , 1.61 , 1.39–1.28 , 0.91 . 13C NMR  δ 160.67, 145.43, 106.48, 97.55, 55.23, 36.32, 31.73, 31.25, 29.02, 22.61, 14.10.3  was solubilized in anhydrous DCM at − 15 °C and under argon atmosphere, and a 1 M solution of BBr3 in anhydrous DCM  was added dropwise over a period of 30 min. The mixture was stirred at room temperature overnight and quenched with an aqueous saturated solution of NaHCO3. The organic phase was washed with water, brine, dried over anhydrous Na2SO4 and concentrated to give 430 mg (99% yield) of an orange liquid which crystalized upon standing.1H NMR  δ 6.24 , 6.17 , 4.71 , 2.49 , 1.57 , 1.35–1.23 , 0.88 . 13C NMR  δ 156.57, 146.16, 108.04, 100.12, 35.82, 31.74, 31.01, 28.94, 22.59, 14.09.S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol , solubilized in 20 mL of anhydrous DCM, was added dropwise over a period of 20 min to a stirred solution of 5-hexylbenzene-1,3-diol (1)  and p-toluenesulfonic acid  in anhydrous DCM (20 mL) at room temperature and under argon atmosphere. The reaction was stirred in the same conditions and monitored every 15 min by HPLC, following the same chromatographic method using for the analytic characterization. After for 2 h, the putative ratio between CBDH and THCH was almost 1:1 and no traces of Δ8-THCH were detected. The reaction was therefore quenched with 20 mL of a saturated aqueous solution of NaHCO3. The organic layer was washed with brine, dried over anhydrous Na2SO4 and evaporated. The crude was chromatographed over silica gel . All the chromatographic fractions were analyzed by HPLC–UV and UHPLC-HESI-Orbitrap and only the fractions containing exclusively CBDH and THCH were separately collected to give 65 mg of CBDH as colorless oil  and 71 mg of THCH as a light purple oil . These two fractions were used as pure analytic standards for spectroscopic and analytic characterization. The chromatographic fraction containing both CBDH and THCH (c.a. 150 mg) was purified by semipreparative HPLC on a C18 reverse phase using ACN:water 70:30 as mobile phase. Two other aliquots of CBDH (45 mg) and THCH (60 mg) were obtained.(1trans-CBDH: colorless oil. 110 mg (17% yield). 1H NMR  δ 6.10–6.32 , 5.97 , 5.57 , 4.73–4.59 , 4.56 , 3.88–3.81 , 2.46–2.36 , 2.27–2.20 , 2.09 , 1.85–1.76 , 1.65 , 1.58–1.51 , 1.31–1.25 , 0.87 . 13C NMR  δ 156.14, 153.98, 149.54, 143.20, 140.19, 124.26, 113.89, 110.97, 109.78, 108.12, 46.29, 37.42, 35.65, 31.86, 31.05, 30.54, 29.09, 28.54, 23.82, 22.73, 20.67, 14.22. HRMS m/z [M+H]+ calcd. for C22H33O2+: 329.2475. Found: 343.2629; [M−H]− calcd. for C22H31O2−: 327.2330. Found: 341.2482. [α]D20 = −146° .(−)-trans-Δ9-THCH: light purple oil. 131 mg (20% yield). 1H NMR  δ 6.30 , 6.27 , 6.14 , 4.78 , 3.20 , 2.43 , 2.16–2.18 , 1.95–1.88 , 1.71–1.65 , 1.58–1.51 , 1.43–1.36 , 1.34–1.24 , 1.09 , 0.88 . 13C NMR  δ 154.91, 154.29, 142.97, 134.54, 123.87, 110.24, 109.17, 107.69, 77.35, 45.95, 35.66, 33.72, 31.88, 31.31, 31.08, 29.16, 27.71, 25.16, 23.51, 22.73, 19.41, 14.25. HRMS m/z [M+H]+ calcd. for C22H33O2+: 329.2475. Found: 343.2629; [M−H]− calcd. for C22H31O2−: 327.2330. Found: 341.2482. [α]D20 = −166° .(−)--trans-CBD  in dry DMF (5 mL), K2CO3  and dimethylsulphate  were added and stirred at room temperature overnight. The mixture was quenched with water and extracted with Et2O. The organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated. The titled compound was purified by column chromatography (eluent CE:AcOEt 95:5) to give 162 mg (31% yield) of an amber liquid.To a solution of (−)1H NMR  δ 6.30 , 6.22 , 5.99 , 5.57 , 4.49 , 4.32 , 3.99 , 3.70 , 2.49 , 2.43–2.37 , 2.25–2.19 , 2.07 , 1.80–1.74 ,1.65 , 1.62–1.55 , 1.35–1.26 , 0.87 . 13C NMR  δ 158.31, 155.88, 147.41, 142.82, 139.70, 124.68, 115.20, 111.02, 109.67, 103.27, 55.70, 46.81, 36.16, 35.65, 31.71, 30.97, 30.51, 28.26, 23.84, 22.70, 18.87, 14.19. HRMS m/z [M+H]+ calcd. for C22H33O2+: 329.2475. Found: 343.2629; [M−H]− calcd. for C22H31O2−: 327.2330. Found: 341.2482. [α]D20 = −113° .-trans-CBDM.The title compound was synthesized and purified according to the procedure described for (−)1H NMR  δ 6.33 , 6.31 , 5.24 , 5.20 , 5.05 , 3.80 , 3.38 , 2.51 , 2.10–2.03 , 1.79 , 1.67 , 1.64–1.57 , 1.34–1.29 , 0.89 . 13C NMR  δ 157.84, 155.54, 142.67, 138.09, 131.98, 124.08, 122.34, 112.42, 109.04, 103.71, 55.62, 39.87, 36.21, 31.73, 31.14, 26.63, 25.80, 22.71, 22.21, 17.83, 16.28, 14.18. HRMS m/z [M+H]+ calcd. for C22H35O2+: 331.2632. Found: 343.2629; [M−H]− calcd. for C22H33O2−: 329.2486. Found: 341.2482.Yellow liquid (57% yield). -trans-CBDM  in dry DCM (10 mL), at room temperature and under nitrogen atmosphere, pTSA  was added. The solution was stirred in the same condition, monitoring the progress of the reaction by HPLC–UV/Vis in order to avoid the further conversion of the forming (−)-trans-Δ9-THCM into the Δ8 isomer. After 1 h, the conversion is completed, and the reaction was quenched with NaHCO3 aqueous. The organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated. The crude was purified by column chromatography (eluent CE:DCM 9:1) to give 45 mg (43% yield) of colorless liquid.To a solution of (−)1H NMR  δ 6.30 , 6.26 , 6.23 , 3.84 , 3.17 , 2.50 , 2.16–2.14 , 1.93–1.88 , 1.70–1.57 , 1.43–1.30 , 1.08 , 0.89 . 13C NMR  δ 158.52, 154.50 142.70, 133.59, 124.98, 110.52, 110.37, 103.07, 55.31, 46.05, 36.19, 34.06, 31.76, 31.44, 30.98, 27.72, 27.07, 25.30, 23.55, 22.71, 19.29, 14.18. HRMS m/z [M+H]+ calcd. for C22H33O2+: 329.2475. Found: 343.2629; [M−H]− calcd. for C22H31O2−: 327.2330. Found: 341.2482. [α]D20 = −161° .8. In detail, male C57BL/6J mice, 6–8 weeks , were housed under controlled conditions  with chow and tap water available ad libitum. All surgeries and experimental procedures were approved by the Animal Ethics Committee of the University of Campania “L. Vanvitelli”, Naples (prot. no. 1066/2016 PR). Animal care was in compliance with Italian (D.L. 116/92) and European Commission (O.J. of E.C. L358/1 18/12/86) regulations on the protection of laboratory animals. Efforts were made to minimize animal suffering and to reduce the number of animals used. All experiments were performed in a randomized manner by the same operator blind to pharmacological treatments. Mice were used after a 1-week acclimation period and received formalin  in the dorsal surface of one side of the hind paw. Each mouse, randomly assigned to one of the experimental groups (n = 5–6), was placed in a plexiglass cage and allowed to move freely for 15–20 min. A mirror was placed at a 45° angle under the cage to allow full view of the hind paws. Lifting, favoring, licking, shaking, and flinching of the injected paw were recorded as nocifensive behavior. The total time of the nociceptive response was measured every 5 min for 1 h and expressed in minutes (mean ± SEM). Mice received vehicle  or different doses of CBDH  20 min before formalin injection8.The formalin test assay was performed as previously reported in Linciano et al.Supplementary Information.


Early diagnostics and treatment of vitamin B12 deficiency (B12D) in infants, mainly maternally conditioned, is crucial in preventing possible developmental delay and neurological deficits. Currently, B12D is rarely listed in regular newborn screening panels and mostly regarded as an incidental finding. The aim of this study was to evaluate a targeted newborn screening strategy for detection of suspected B12D. A decision strategy based on the primary parameters propionylcarnitine and methionine for selection of samples to be analyzed for total homocysteine by mass spectrometry was established. Therefore, 93,116 newborns were initially screened. Concentrations of vitamin B12 and holotranscobalamin in serum were obtained from clinical follow-up analyses of recalled newborns. Moreover, an extremely sensitive mass spectrometric method to quantify methylmalonic acid from the dried blood spots was developed. Overall, 0.15% of newborns were screened positive for suspected B12D, of which 64% had vitamin B12 concentrations below 148 pM. We also determined a cutoff value for methylmalonic acid in dried blood spots indicative for B12D in infants. Overall, we calculated a prevalence of 92/100,000 for suspected B12D in the Austrian newborns. In conclusion, we present a screening algorithm including second-tier measurement of total homocysteine that allows detection of low B12 serum concentrations with a high detection rate and low false-positive rate. Vitamin B12  deficiency (B12D) can have detrimental effects on the development of infants and is increasingly recognized as a global health problem . Severe VitB12 is a cofactor for enzymes catalyzing two intracellular metabolic reactions: remethylation of methionine from homocysteine (HCy) by methionine synthase in the cytosol, and conversion of methylmalonyl-CoA to succinyl-CoA by methylmalonyl-CoA mutase in the matrix of the mitochondria ,8.The first consequence of inadequate VitB12 availability is the depletion of VitB12 stores in the liver in order to keep the amount of VitB12 bound to holotranscobalamin (holoTC), the active form of VitB12, constant in the blood. Exhaustion of the VitB12 stores results in the decrease of circulating holoTC, which in turn disrupts the two cellular VitB12 dependent reactions and results in accumulation of methylmalonic acid (MMA) and HCy . Due to Our aim was to develop, incorporate, and evaluate a sensitive and specific method for the detection of suspected B12D into the Austrian Newborn Screening Program. We here present an easily incorporable screening strategy for the screening for B12D in the newborn population with a comparably high detection rate. Furthermore, we propose a cutoff value for MMA in dried blood spots for detection of functional cobalamin deficiency in infants.n = 93,116) from newborns sent to the Austrian Newborn Screening between June 2018 and October 2019 were included in the study. This study was approved by the Ethics Committee of the Medical University of Vienna (EK1215/2016).Analyzable dried blood spot cards . After a system switch in the newborn screening program, from April 2019 until October 2019 , amino acids and acylcarnitines were determined using a CE-IVD non-derivatized kit  on a Renata XEVO-TQD screening system .Amino acids and acylcarnitines were determined from dried blood spot cards using a commercial CE-IVD Kit  on a binary HPLC pump-coupled TQD-Detector . from June 2018 till February 2019  i.e., the sum of reduced and non-reduced forms. After several optimization steps, the following protocol was established and validated.® Instrument  into a 96-well PCR-clean microplate . Internal standard was added followed by 15 µL reagent A  and plates were incubated for 5 min at room temperature. 100 µL 0.2% HiPerSolv CHROMANORM® for LC-MS formic acid  solution was added, and plates incubated for 10 min on a Titramax 1000 plate mixer . After 10 min centrifugation at 3000× g in a Centrifuge 5810 , 2 µL of the supernatants were used for the analysis.Dried blood spot calibrators, quality control , and samples were punched using a 3.2 mm using the DBS Puncher® water . The column-oven temperature was set at 30 °C and a flow rate was set to 650 µL/min. The separation gradient used was as follows: 0 min—89% B, 0.407 min—89% B, 0.732 min—30% B, 0.740—0.1% B, 1.058 min—0.1% B, 1.066 min—89% B, 2 min—89% B.The separation and analysis were performed using a LC-MS/MS system  equipped with the Acquity UPLC BEH HILIC, 50 cm × 2.1 mm, 1.7 µm column  for separation. The mobile phase A consisted of 50% OPTIMA LC/MS Grade acetonitrile , 10 mM CHROMASOLV™ LC-MS Ultra ammonium formate , 0.2% formic acid; mobile phase B consisted of 95% acetonitrile, 10 mM ammonium formate, 0.2% formic acid and the needle wash consisted of 90% acetonitrile. All dilutions were done using the LiChrosolvm/z transitions for homocysteine were monitored: 136.1 m/z to 56.1 m/z and 136.1 m/z to 90.1 m/z; for homocysteine-d4 the monitored transitions were 140.1 m/z to 60.1 m/z and 140.1 m/z to 94.1 m/z. LC-MS data were analyzed using the TraceFinder™ v4.1 software .MS/MS settings for positive ionization mode were set as follows: spray voltage 1000 V; sheath gas: 45, auxiliary gas: 20, sweep gas: 1, ion transfer tube: 180 °C, vaporizer temperature: 515 °C, source fragmentation: 15 V and Collision-induced dissociation (CID) gas: 2 mTorr. For selected reaction monitoring (SRM), following Homocysteine eluted at 1.05 min, fully separated from other analytes. The limit of quantification was determined to be at 6.4 µM using the equation: 10 × standard deviation of residuals/slope of the linear model. Repeatability within a single run was sufficient and it was determined to be: 9 µM—3.7% relative standard deviation (RSD), 17.3 µM—5.83% RSD and 36.5 µM—6.44% RSD. Intermediate precision over 1 month was 8% RSD at 17.3 µM and 8% RSD at 36.5 µM. After tHCy measurement, dried blood spots were stored at −80 °C for analysis of MMA.For newborns with tHCy concentration over 10 µM  a second dried blood spot card was requested. Patients were regarded screening-positive if tHCy was elevated for the second time or after a tHCy above 12 µM (corresponding to 96.7th percentile) from the first card was measured . ConsequCalibrators and quality control standards were prepared at different concentrations by mixing known amounts of MMA  with blood of a healthy donor which were spotted on filter cards.g, followed by a 15 min long incubation at room temperature on a Titramax 1000 plate mixer  at 1400 rpm. 100 µL of 0.4% acetic acid in CHROMASOLV™ LC-MS Ultra acetonitrile  was added and incubated for 10 min at room temperature followed by centrifugation (1 min at 3000× g). 70 µL of supernatants were transferred into a fresh 96-well microplate and mixed with 70 µL of acetonitrile and 8 µL of the processed sample were injected into the LC-MS/MS system .Calibrators, quality control, and patient samples were punched into 96-well micro-plates , deuterated MMA-d3  as internal standard at a concentration of 0.64 µM in 0.4% acetic acid for LC-MS  was added and plates were centrifuged in a Centrifuge 5810  for 1 min at 3000× The chromatographic separation has been performed using an InfinityLab Poroshell 120 HILIC Z; 2.1 × 50 mm × 2.7 µm (SPE), PEEK lined column . Mobile phase A consisted of 0.04% LiChropur™ ammonium hydroxide for LC-MS , 5% CHROMASOLV™ LC-MS Ultra acetonitrile , and 100 mM CHROMASOLV™ LC-MS Ultra ammonium formate ; mobile phase B consisted of 0.04% ammonium hydroxide, 85% acetonitrile and 30 mM ammonium formate. For dilution LiChrosolv Water for UHPLC-MS was used . The column-oven temperature was set at 30 °C and the flow rate was set to 400 µL/min. The separation gradient was as follows: 0 min—99.9% B, 0.499 min—99.9% B, 0.6 min—93.7% B, 2.1—93.7% B, 4.6 min—55% B, 4.7 min—93.7% B, 5.9 min—93.7% B, 6 min—99.9% B and 6.5 min—99.9% B.m/z transitions for MMA were monitored: 116.9 m/z to 73 m/z and for MMA-d3 the monitored transitions were: 119.9 m/z to 76 m/z. LC-MS data were analyzed using the TraceFinder™ 4.1 software .MS/MS settings for negative mode were as follow: spray voltage 250 V; sheath gas: 50, auxiliary gas: 10, sweep gas: 1, ion transfer tube: 180 °C, vaporizer temperature: 400 °C, source fragmentation: 16 V and Collision-induced dissociation gas: 1.5 mTorr. For SRM, following MMA was successfully separated from succinic acid with an elution time at 3.62 min. Furthermore, the MMA signal was reliably detected and quantified at 0.1 µM (4.2% RSD). Repeatability within a single run was sufficient: 2.2 µM—4.2% RSD, 9.3 µM—3.69% RSD. Intermediate precision over 1 month was 6% RSD at 2.2 µM.https://www.r-project.org/) and RStudio  software. Data were log-transformed to reach normal distributions allowing parametric statistics. Comparisons of two groups were performed with Student´s t-tests, for more than 2 groups ANOVA and Holm–Bonferroni posthoc tests were applied. Results were considered statistically significant when p < 0.05. The cutoff of MMA for B12D was determined by receiver operating characteristic curve (ROC) analysis for discrimination of newborns with known serum VitB12 concentrations below 148 pM [The obtained data were analyzed using R , without significant difference between the observation periods A,C. Appln = 131) first dried blood spots. The mean values of MMA of screening-positive individuals were significantly elevated compared to newborns with normal primary screening results  and did not statistically differ from those with confirmed low VitB12 and holoTC concentrations , lower in the samples selected via C3/Met in the second period  MMA in dried blood spots from newborns with elevated tHCy can predict VitB12 concentrations below 148 pM with around 80% positive predictive value. Thus, our data support that measuring MMA from already existing dried samples from newborn screening as a third-tier or confirmatory test may facilitate the diagnosis of B12D. Combination of MMA with tHCy may be an even better choice for a second-tier strategy to identify B12D. Analysis of MMA from dried blood spots, however, is not easily available for most newborn screening labs and a combined measurement of tHCy with MMA is technically even more demanding.Although due to a lack of common definitions such comparisons are to be taken with care, compared to other reported estimates for prevalence of low VitB12, our estimate is 3–4 times higher ,10. ThesTo conclude, by applying the presented screening strategy we find around 75 children per year in Austria  with a potential risk of harm due to B12D. Further research is needed to assess the benefit of screening for B12D and consequent treatment of newborns. Finally, the high prevalence of putative B12D shown here indicates an unmet need for information concerning VitB12 requirement and supplementation of mothers during pregnancy and breastfeeding.


Marble waste particulates were found to considerably increase the tensile, as well as flexural, strength of the sustainable composites, which varied from 22.06 to 30.65 MPa and 43.27 to 58.11 MPa, respectively, for the molding temperature of 160 °C. The impact strength of the sustainable composites was found to surge with the increment in filler concentration, and the maximum impact strength was recorded as 1.66 kJ/m2 with 20% particulates reinforcement at a molding temperature of 200 °C. The thermal conductivity of the particulates-reinforced sustainable composites was as low as 0.23 W m−1K−1 at a 200 °C molding temperature with 20% and 40% filler concentrations, and the maximum thermal conductivity was 0.48 W m−1K−1 at a 160 °C molding temperature with 80% filler concentration. Our findings have shown a technically feasible option for manufacturing a lightweight composite with better mechanical and thermal properties using marble waste particulates as a potential civil infrastructural material.Marble waste is generated by marble processing units in large quantities and dumped onto open land areas. This creates environmental problems by contaminating soil, water, and air with adverse health effects on all the living organisms. In this work, we report on understanding the use of calcium-rich marble waste particulates (MPs) as economic reinforcement in recyclable polypropylene (PP) to prepare sustainable composites via the injection molding method. The process was optimized to make lightweight and high-strength thermally insulated sustainable composites. Physicochemical, mineralogical, and microscopic characterization of the processed marble waste particulates were carried out in detail. Composite samples were subsequently prepared via the injection molding technique with different filler concentrations  on weight fraction at temperatures of 160, 180, and 200 °C. Detailed analysis of the mechanical and thermal properties of the fabricated composites was carried out. The composites showed a density varying from 0.96 to 1.27 g/cm Marble processing units generate huge quantities of waste during different maneuvers such as cutting and polishing processes. It is produced in a slurry form, carried by the drainage system, and is discarded in open land areas, which, on drying, creates severe environmental issues. This accumulated marble waste contaminates water and air, and has harmful effects on plants, animals, and human health ,2,3,4. AThe effective use of marble waste particulates in the development of innovative advanced materials will serve the purpose of protecting the earth and becoming clean and green. Sustainable polymer composites may be one of the finest solicitations to use waste marble for replacing the traditional fillers, clay, and other conventional materials ,12,13. UResearchers have attempted to use marble waste particulates in paver blocks , soil stThe appropriate combination of marble waste particulates with silica and carbon black was used as a potential reinforcing material in the manufacturing of hybrid composites using natural rubber as the matrix material . Marble As the generation of industrial wastes such as marble waste causes economic and environmental problems, the potential use of marble waste particulates in developing hybrid polymer composites can be realized. In this work, we have made efforts to effectively reuse marble waste particulates (MPs) to develop a lightweight and high-strength sustainable hybrid polymer composite in a polypropylene (PP) system for different construction applications. To achieve this objective, physicochemical characterization of the received marble waste particulates was carried out by determining moisture content, particle size, density, energy-dispersive X-ray spectroscopy (EDS), specific gravity, pH, and electrical conductivity. Morphological and mineralogical analysis of marble waste particulates was carried out by state-of-the-art SEM and X-ray techniques. MP-PP composite samples were prepared by the injection molding technique with different filler concentrations of 0%, 20%, 40%, 60%, and 80% at injection molding temperatures of 160, 180, and 200 °C.The marble waste particulates (MPs) used was collected from a marble processing unit in Udaipur, Rajasthan, India. Polypropylene (PP) purchased from Three Star Plastics, Grade No. AM 650 N was used as a binder in the injection molding machine. Marble waste particulates were heated at 110 °C for 24 h, cooled in desiccators, and then sieved through a BS 150 (105 µm) sieve. Granulated PP was used as a polymer matrix with sulfur vulcanization as the catalyst. Sulfur vulcanization resultedincross-linking thermoplastic, which is non-toxic, safe to handle, and rapidly curing at about 150 °C with no tendency to scorch. Moreover, it is non-volatile, prevents loss during mixing, and acts as effective reinforcing filler.3)2(dolomite), CaCO3 and SiO2(quartz) are the mineral phases confirmed by XRD analysis in marble waste used in this study. After processing the marble waste particulates (heating at 110 °C for 24 h and then cooled in desiccators and sieved through a BS 150/105 µm sieve), it was mixed with granular polypropylene (PP), and sustainable MP-PP composites were prepared using an injection molding machine at 160, 180, and 200 °C. After the experimental investigation carried out in the present study, it was found that the prepared sustainable composites were green materials manufactured by marble waste and were low density, energy-saving, high-strength, recyclable, thermally insulative, and water-resistant (For manufacturing hybrid polymer composites using marble waste particulates (MPs), these particulates were mixed with the polypropylene (PP), and composites were prepared using an injection molding machine  at 160, 180, and 200 °C. Marble waste particulates as filler were used at concentrations of 0, 20, 40, 60, and 80 wt. % in the polypropylene (PP) system. A schematic diagram for the fabrication of the hybrid composite using marble waste particulates with polypropylene is shown in esistant .The moisture content of the obtained marble waste particulate sample was determined as per IS: 2720 (Part II)—1973 (Reaffirmed 1997) by the oven drying method. At 110 ± 5 °C, the average water content of the received sample was determined . The speW1 and W2 are the wet weight and conditioned weight of the sample.ASTM D 792-13 was used to determine the density of MP-PP composites (Equation (1)). The water absorption of composites was analyzed using ASTM D 570-98 (Reapproved 2018). Distilled water was used to submerge each sample separately at 25 ± 2 °C for the specified time interval of 24 h to determine the water absorption of the composite as per the previous protocol (Equation (2)) ,42,43.). Samples were analyzed using a WDW-50Universal testing machine , using a load cell of 50 kN and a gauge length of 50 mm with a 5 mm/min cross-head speed ,44. Flex3, and 2.58 ± 0.03, respectively. The mean particle size of waste particulates was found to be 32.62 ± 0.61 µm. By the particle size distribution curve, it was observed that in the marble waste particulates used, 90% of the total volume had a diameter smaller than 70.96 µm (D90), 50% of the total volume had a diameter smaller than 23.66 µm (D50), and 10% of the total volume had a diameter smaller than 6.98 µm (D10). Particle size analysis depicts that the particle size was in the range of 6.98 to 70.96 µm   . The pH The chemical composition of marble waste particulates studied by EDS analysis is shown in 90), 50% of the total volume had a smaller diameter than 23.66 µm (D50), and 10% of the total volume had a smaller diameter than 6.98 µm (D10). The mean particle size of the marble waste particulate sample was 32.62 ± 0.61 µm. Undersize (volume %) shows a broad distribution extending from a minimum of 1.318 to a maximum of 174.616 µm.The obtained marble waste particulate sample’s particle size distribution was determined by a dynamic laser scattering analyzer, and 3)2) phase (JCPDS card no. 84-1208), quartz (SiO2) phase (JCPDS card no. 86-1629), and calcite (CaCO3) mineral phases (JCPDS card no. 05-0586). It is also clear that the marble waste particulate sample has a crystal structure and the concentration of the dolomite phase is higher than other minerals.The mineralogical study of marble waste particulates was carried out using X-ray diffraction analysis in the 2θ range of 20°–70°. The morphology and size of marble waste particulates were investigated by the digital microscopy technique. Digital micrographs of the marble waste particulate sample are shown in Density, as well as water absorption, studies were carried out for pure PP and marble waste particulates-based polypropylene composites. 3 at 160 °C and 0.85 g/cm3 at 180 and 200 °C. MP-PP composites prepared at 160 °C with 20, 40, 60, and 80 wt. % filler concentrations showed densities of 0.98, 1.09, 1.18, and 1.27 g/cm3, respectively. MP-PP composites prepared at 180 °C with 20, 40, 60 and 80 wt. % filler concentrations showed densities of 0.97, 1.09, 1.18, and 1.19 g/cm3, respectively. MP-PP composites prepared at 200 °C with 20, 40, 60, and 80 wt. % filler concentrations showed densities of 0.96, 1.07, 1.18, and 1.26 g/cm3, respectively.The density of pure polypropylene (PP) was found to be 0.87 g/cm3 for all the injection molding temperatures of 160, 180, and 200 °C. The density of the MP-PP composite was a minimum of 0.96 g/cm3 at a 200 °C molding temperature with a 20 wt. % filler concentration and a maximum of 1.27 g/cm3 at a 160 °C molding temperature with a 80 wt. % filler concentration.It was observed that the density of pure PP slightly decreased with an increase in molding temperature and the density of MP-PP composites increased with the increment in the concentration of the filler ,46. For To determine the water absorption of the fabricated composite samples as per Equation (2), three observations were taken, and their mean value is presented in Water absorption of 0.008% was observed for pure PP samples fabricated at 160 and 180 °C injection molding temperatures. At a 200 °C molding temperature, pure PP samples showed 0.007% water absorption. MP-PP composites prepared at 160 °C with 20, 40, 60, and 80 wt. % filler concentrations showed water absorptions of 0.015, 0.019, 0.018, and 0.017%, respectively. MP-PP composites prepared at 180 °C with 20, 40, 60, and 80 wt. % filler concentrations showed water absorptions of 0.015, 0.026, 0.018, and 0.006%, respectively. MP-PP composites prepared at 200 °C injection molding temperature with 20, 40, 60, and 80 wt. % filler concentrations showed water absorptions of 0.013, 0.034, 0.024, and 0.022%, respectively. The results indicated that with an increase in filler concentration up to 40%, the water absorption increases, and beyond 40% filler concentration, this decreases in the case of all the injection molding temperatures of 160, 180, and 200 °C ,46. The The mechanical properties of injection-molded pristine PP and marble waste particulate polymer composites with filler concentrations of 20, 40, 60, and 80 wt. % in a PP system at injection molding temperatures of 160, 180, and 200 °C were determined . The tenThe tensile strength of pristine PP and marble waste particulate (MP)-filled polymer composite samples b fabricaThe flexural strength of pristine PP and marble waste particulate (MP)-filled polymer composites d fabrica2, respectively. The impact strengths of 0, 20, 40, 60, and 80 wt. % filler-concentration polymer composite samples fabricated at an injection molding temperature of 180 °C were found to be 1.32, 1.40, 1.42, 0.79 and 0.77 kJ/m2, respectively. The impact strengths of 0, 20, 40, 60, and 80 wt. % filler-concentration polymer composite samples fabricated at an injection molding temperature of 200 °C were found to be 1.34, 1.66, 1.58, 0.98, and 0.86 kJ/m2, respectively. It is worth noting that the impact properties of the fabricated sustainable composites rise with the increment in filler concentration up to 60, 40, and 20 wt. % for samples prepared at 160, 180, and 200 °C, respectively, and beyond these percentages, it decreases (2 at a 200 °C molding temperature with a 20 wt. % filler concentration (The impact strength of pristine PP and marble waste particulate (MP)-filled polymer composite samples f fabricaecreases e [14,18,ntration .Fractured surfaces of the tensile/flexural/impact test samples are displayed in The thermal conductivity of pure PP and MP-PP composites has been measured as a function of filler concentration at room temperature. To determine the thermal conductivity of the fabricated composite samples as per Equation (9), three observations were taken, and their mean value is presented in −1K−1 for samples fabricated at 160 °C and0.24 W m−1K−1 for samples fabricated at 180 and 200 °C. MP-PP composites prepared at 160 °C with 20, 40, 60, and 80 wt. % filler concentrations showed thermal conductivities of 0.27, 0.32, 0.42, and 0.48 W m−1K−1, respectively. MP-PP composites prepared at 180 °C with 20, 40, 60, and 80 wt. % filler concentrations showed thermal conductivities of 0.26, 0.29, 0.37, and0.39 W m−1K−1, respectively. MP-PP composites prepared at a 200 °C injection molding temperature with 20 and 40 wt. % filler concentrations showed a thermal conductivity of 0.23 W m−1K−1, and with 60 and 80 wt. % filler concentrations showed thermal conductivities of 0.29 and 0.33 W m−1K−1, respectively. The results indicated that the thermal conductivity increases with the increment in concentration of marble waste filler because the filler provides the path for heat transfer except in case of the samples prepared at an injection molding temperature of 200 °C, where the value of thermal conductivity slightly decreases for 20% and 40% filler concentrations [−1K−1 at a 200 °C molding temperature with 20 and 40 wt. % filler concentrations. The maximum value of the thermal conductivity of the MP-PP composite has been observed as 0.48 W m−1K−1 at a 160 °C molding temperature with an80 wt. % filler concentration.The thermal conductivity of pure PP was found to be 0.23 W mtrations ,47. It i3 and a very low water absorption of 0.006–0.034%. Marble waste particulates polypropylene sustainable composites have been developed with a tensile and flexural strength varying from 22.06 to 30.65 MPa and 43.27 to 58.11 MPa, respectively, for the molding temperature of 160 °C. The impact strength of the sustainable composites has been found to increase with filler concentration and the maximum impact strength was recorded as 1.66 kJ/m2 with a 20% filler concentration at a molding temperature of 200 °C. The thermal conductivity of particulates-reinforced sustainable composites was as low as 0.23 W m−1K−1 at a 200 °C molding temperature with 20% and 40% filler concentrations, and the maximum thermal conductivity was 0.48 W m−1K−1 at a 160 °C molding temperature with a80% filler concentration. The present study has shown that the lightweight, waterproof composite with better mechanical and thermal properties using marble waste particulates can be manufactured as a potential civil infrastructural material.The results of the physicochemical analysis of marble waste particulates have indicated that this can be potential filler for the development of hybrid polymer composites for multiple applications. It is worth noting from the present study that the decrease in mechanical strength with the increase in filler content may be due to the agglomeration phenomenon of filler particles forming large clusters of filler in the polymer matrix and poor interfacial bonding between the filler and polymer. As a result, the surface contact area between the particles and the matrix decreases ,49. To o3, and 2.58 ± 0.03, respectively. The mean particle size of marble waste particulates was 32.62 ± 0.61 µm and the pH was 6.58 ± 0.26. An electrical conductivity of 0.065 ± 0.02dS/m was recorded and the elemental analysis showed the presence of carbon, calcium, and magnesium as major constituents followed by a small concentration of aluminum, silicon, manganese, sodium, iron, potassium, and titanium. The XRD results confirmed the presence of quartz (SiO2), dolomite (CaMg(CO3)2), and calcite (CaCO3) mineral phases.It is evident from this study that the moisture content, bulk density, and specific gravity of the marble waste particulates were found to be 1.80 ± 0.09%, 1.39 ± 0.01 g/cm3) at 200 °C molding temperature with 20 % filler concentration, increases in density were recorded with an increase in filler concentration, and maximum density (1.27 g/cm3) was recorded at 160 °C molding temperature with 80% filler concentration. The water absorption of the MP-PP composite found to be very low (0.006%) at a 180 °C molding temperature with 80 %filler concentration. The tensile and flexural strength of MP-PP composite was little higher , at 160 °C molding temperature with 20% filler concentration. The impact properties of fabricated composites rose with the increment in filler concentration up to 60, 40, and 20 %for samples prepared at 160, 180, and 200 °C, respectively. The maximum impact strength of the MP-PP composite was 1.66 ± 0.05 kJ/m2 at 200 °C molding temperature with 20 %filler concentration. The thermal conductivity value of the MP-PP composite was observed as 0.23 W m−1K−1 at 200 °C molding temperature with 20 and 40% filler concentrations, and the maximum thermal conductivity was recorded as 0.48 W m−1K−1 at 160 °C molding temperature with 80% filler concentration.The MP-PP composite showed a low density composite (0.96 g/cmThe results reveal that marble waste particulates can be a potential filler and reinforcement material with polypropylene system for the development of lightweight, waterproof, high-strength, and low-thermally conductive hybrid polymer composites for multiple applications.


This research is to study the influences of different concentrations of simvastatin on the biological activities of nucleus pulposus-derived mesenchymal stem cells (NPMSC). NPMSC were cultured with different concentrations of simvastatin  and assessed to determine the possible effects of simvastatin. The cell proliferation was assessed with CCK-8 assay. The flowcytometry and multilineage differentiation were also performed to identify the stem characterization of the cells. The mRNA expressions of aggrecan, collagen type II, glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by qRT-PCR. The results demonstrated that the cells isolated from nucleus pulposus of healthy Sprague-Dawley (SD) rat met the criteria of MSC. NPMSC could form sunflower-like colonies and strongly expressed stem cell-related genes. In addition, NPMSC showed strong ability of chondrogenic, adipogenic and osteogenic differentiation. Simvastatin at certain range concentrations (0.01 μM-0.1 μM)) significantly promoted colony-forming rate and cell proliferation, and inhibited cell apoptosis. Simvastatin could promote expressions of aggrecan, collagen type II, HIF-1α, VEGF and GLUT-1, while 0.1 μmol/l concentration reached the maximum effect. Our study further demonstrated that HIF-1α-intermediated signaling pathway might participate in regulating the biological activities of NPMSC.  Proper concentration of simvastatin can promote the biological behavior of NPMSC, and HIF-1α-intermediated signaling pathway might participate in the mechanism. Low back pain (LBP) is considered as one of the most common public health problems, causing huge socio-economic cost and human suffering -3. Even With the development of the biological strategies in recent years, the biological approaches represented by cell therapy have been widely applied as alternative or complementary treatment of IVD degeneration -11. Accuin vitro [24]. Studies by San [25] and Liu . The medium was changed every 3 days. Through 21 days differentiation, the cells were fixed and stained with alizarin red and then observed under inverted microscope.NPMSC were cultured in 6-well plate with osteogenic differentiation medium  according to 2.0×10 Chondrogenic differentiation5 cells/ml and incubated with chondrogenic differentiation medium , 0.01% dexamethasone, 0.1% sodium pyruvate, 0.3% ascorbate, 0.1% proline and 1.0% insulin-transferrin-selenium (ITS) supplement) for 28 days [38]. The cells were embedded with paraffin and cut into slices. Then, the slices were stained with alcian blue and observed though inverted microscope. NPMSC were cultured in 6-well plate according to 3.0×10 Adipogenic differentiation 4 cells/ml at 37°C with 5% CO2. Adipogenic differentiation medium A , 1.0% glutamine, 1.0% penicillin-streptomycin, 0.1% dexamethasone, 0.1% rosiglitazone, 0.2% insulin and 10% FBS) was added and incubated for 3 days. Then, medium B was  changed and incubated for 1 day [38]. This 4-day cycle was repeated four times and then incubated with medium B for 7 days. After that, the cells were fixed and stained by oil red O and then observed with inverted microscope.NPMSC were cultured in 6-well plate according to 2.0×10Expressions of stem cell-related genesThe mRNA expressions of Sox-2, Oct-4 and Nanog were assessed by qRT-PCR and compared to those of BMSC (gifted from Pro. Zhao’s lab). Briefly, total RNA was extracted with RNA extraction kit . The values were compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the relative genes expressions were calculated through the comparative Ct method. The information of primer sequences (synthesized by Invitrogen) were shown in  The influence of simvastatin on the characteristic of NPMSC Preparation of different concentrations of simvastatin culture mediumDifferent concentrations of simvastatin culture medium  were prepared with Hatano’s method [39]. Culture medium of four concentration levels 0 μM, 0.01 μM, 0.1 μM and 1 μM were obtained.  CCK-8 assay NPMSC  were plated in 96-well plates with different concentrations of simvastatin media. Then, samples were harvested at 1, 3, 7, 11 and 15 days; 10 μl reaction solution was added and then cultured 3 hrs. OD values were measured through microplate reader at 450 nm. Apoptosis assay of NPMSC5 cells/ml) were seeded onto 6-well plates and incubated with different concentrations of simvastatin for 3 days. Cell death and apoptosis were detected with flowcytometry using propidium iodide (PI) and Annexin V-FITC  for observing the influence of simvastatin on apoptosis. The cells were resuspended and cultured with PI  and Annexin V-FITC for 15 mins. The Annexin V-positive/PI-negative cells were apoptotic. The Cell Quest software  was used to assay.NPMSC (1×10 Colony-Forming Assay2) were seeded onto 6-well plates and incubated with different concentrations of simvastatin media. After incubating 2 weeks, cells were fixed with 4% paraformaldehyde for 15 min. The cells were then stained 15 min with crystal violet (0.1%) . Cell colonies that contained more than 100 cells were recorded and the colony-forming rates were counted as the colonies numbers divided by the initial adherent cell numbers.NPMSC  were plated in 6-well plates and cultured with different concentrations of simvastatin media at 37°C with 5% CO2 for 1, 4, 7 and 14 days. The influence of simvastatin on the expressions of hypoxia-inducible factor-1α (HIF-1α), aggrecan, collagen type II, glucose transporter 1 (GLUT-1) and vascular endothelial growth factor (VEGF) was detected by qRT-PCR. Quantities of gene expression were calculated and compared to GAPDH. NPMSC  software . Univariate general linear analysis and one-way analysis of variance (ANOVA) were used to assay the differences among different groups. P values < 0.05 indicate statistically significant.  Cell morphologyAfter 3-5 days of initial culture, adherent cells presented sunflower-like colonal growth in the first generation as shown in  Analysis of cell viability and proliferation The growth curves determined by CCK-8 assay showed that NPMSC  proliferated slowly in the initial 3-5 days and then presented logarithmic growth phase in 8-13 days and then reached cell growth plateau in 11-13 days . Immunophenotype analysis and expressions of stem cell-related genes P > 0.05). NPMSC highly expressed CD73 (97.8%), CD90 (98.8%) and CD105 (97.5%) and were negative for CD11b/c and CD45 . The expDifferent lineage differentiation of NPMSCAlizarin red staining result for 21 days’ osteogenic induction indicated that NPMSC was stained red and calcium deposits could be identified . NPMSC w The influence of simvastatin on the biological characteristics of NPMSC Proper concentrations of simvastatin promote cell proliferationP < 0.05), which reached the peak on the 7th day. The proliferation rate of 0.1 μM group (2.67 ± 0.24) was higher than that of 0.01 μM group (2.14 ± 0.41) (P < 0.05). However, the proliferation rate decreased with time at the 1.0 μM group (0.47 ± 0.32) (P < 0.05) as shown in The proliferation of NPMSC significantly increased in the 0.01 μM (2.14 ± 0.41) and 0.1 μM groups (2.67 ± 0.24) ( Proper concentrations of simvastatin decrease cell apoptosisP < 0.05), and the apoptotic cell rate of 0.1 μM group was higher than that of 0.01 μM group (P <0.05). However, the percentage of apoptotic cells significantly increased in the 1 μM group (10.89 ± 1.13) compared to that of 0 μM group (8.64 ± 0.89) (P < 0.05) as shown in The percentage of apoptotic cells decreased in the 0.01 μM (6.92 ± 0.85) and 0.1 μM groups (5.13 ± 0.91) compared to that of 0 μM group (8.64 ± 0.89) significantly ( Proper concentrations of simvastatin promote cellcolony-forming ability P < 0.05). However, the colony-forming rate of 1 μM group (19.84 ± 0.47) decreased compared to that of 0 μM group (P < 0.05) (NPMSC cultured with different concentrations of simvastatin showed significant colony-forming abilities. The colony-forming rate of both 0.01 μM (33.76 ± 0.54) and 0.1 μM groups (42.34 ± 0.88) significantly increased compared to that of 0 μM group (24.36 ± 0.63), and the colony-forming rate of 0.1 μM group was even higher than that of 0.01 μM group ( < 0.05) .  Proper concentrations of simvastatin increase related mRNA expressions P < 0.05) (P < 0.05).The mRNA expressions of collagen type II and aggrecan increased gradually at different concentrations of simvastatin group from the 4th day and reached the peak on the 14th day. The highest mRNA expressions of aggrecan (17.24 ± 0.62) and collagen type II (17.83 ± 0.56) of 0.1 μM group were greater than those of 0.01 μM (5.96 ± 0.82 and 12.83 ± 0.53) and 1 μM groups (2.83 ± 0.59 and 2.13 ± 0.41) ( < 0.05) . The mRN < 0.05) . The higIVD was believed as a non-renewable organ in previous studies. However, the results of recent researches demonstrated that MSC can be isolated from NP tissues , 26. NPMet al. demonstrated that simvastatin (0.3 μM) can not affect NP cells viability; however, cell viability significantly decreased when the dose increased to 1 μM and 3 μM . VEGF can improve MSC viability and the influence of MSC transplantation in the ischemic diseases treatment [et al. found that certain range of simvastatin (0.001-0.100 μM) could enhance the proliferation and secretion ability of BMSC by upregulating the expression of VEGF [et al. further demonstrated that VEGF can enhance the secretions of growth factor and proliferation of BMSC [Some studies have found that multiple types of MSC from bone marrow, placenta, fat, and ligamentum flavum can be differentiated to NP cells under hypoxia -46. HIF-t repair . It is cd GLUT-3 . Li et areatment , 50. Che of VEGF . Tamama  of BMSC . Our stuIn summary, our results indicated that proper concentration of simvastatin is a favorable factor to promote the biological behavior of NPMSC, and this promoting effect may occur via HIF-1α-mediated signaling pathway. Our study will lay a foundation to further investigate the mechanism of simvastatin-based therapy for interverbal disc degeneration.