Y. lipolytica biomass as a whole-cell catalyst in the synthesis of lipophilic antioxidants, with the example of esterification of five phenolic acids with 1-butanol.The aim of the study was to evaluate the possibility of using Y. lipolytica biomass was successfully applied as a biocatalyst in the synthesis of esters of phenylpropanoic acid derivatives with 75–98% conversion. However, in the case of phenylacetic acid derivatives, results below 10% were obtained. The biological activity of phenolic acid esters was strongly associated with their chemical structures. Butyl 3-(4-hydroxyphenyl)propanoate showed an IC50 value of 19 mg/ml (95 mM) and TEAC value of 0.427. Among the compounds tested, butyl esters of 3-(4-hydroxyphenyl)propanoic and 4-hydroxyphenylacetic acids exhibited the highest antifungal activity.Freeze-dried Lipophilization of phenolic acids achieved by enzymatic esterification creates prospects for using these compounds as food additives with antioxidant properties in lipid-rich food matrices. Free radicals are atoms, ions, or molecules with one or more unpaired electrons capable of independent existence. High levels of instability and reactivity increase their ability to cause damage in various molecular structures, and the overproduction of reactive oxygen species (ROS) leads to oxidative stress, a deleterious process contributing to the development of many civilization diseases , butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT)  of Institute of Immunology and Experimental Therapy Polish Academy of Sciences . C. cylindracea was purchased from Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures. R. mucilaginosa and S. cerevisiae were isolated and identified in Department of Chemistry . Prior to testing yeasts and bacterial strains were stored at − 20 °C using Protect Microorganism Preservation System .In the study following yeast and bacterial strains were used: Candida antarctica Lipase B  was purchased from Sigma-Aldrich . Chemicals were purchased from Sigma-Aldrich and Avantor Performance Materials . Culture media components were purchased from BTL Sp. z o. o. , and extra virgin olive oil , which was used as a carbon source in the cultivation of Y. lipolytica was purchased from the local supermarket in Warsaw (Poland).p-nitrophenyl laurate  and freeze-dried Y. lipolytica biomass (2 g) were used as biocatalysts. Reactions were carried out in Erlenmeyer glass flasks (100 ml) in 10 ml of a mixture of isooctane and methyl-tert-butyl ether ) using an orbital shaker (200 rpm) at 28 °C. Liquid samples were withdrawn after 1, 2 and 24 h from the reaction mixtures and analyzed by gas chromatography (GC).Following phenolic acids and their analogs were used: phenylacetic acid, 3-phenylpropionic acid, 4-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, and 3-(4-methoxyphenyl)propionic acid. 1-Butanol was chosen as a second reaction substrate. Reaction scheme of enzymatic esterification of phenolic acids with 1-butanol was presented in Fig. N,O-bis(trimethylsilyl)trifluoroacetamide) + 1% TMCS (trimethylchlorosilane) added in equal volumes and heated for 30 min at 70 °C. Then samples were analyzed by gas chromatography equipped with a flame ionization detector (GC-FID). Analysis was carried out using Agilent Technologies 7820A with HP-5 column . Nitrogen was used as carrier gas at a flow rate of 1.5 ml/min. The temperature program was as follow: 70 °C for 3 min, 70 °C to 150 °C (3 °C/min), 150 °C to 300 °C (40 °C/min), and 300 °C for 10 min. Injector temperature: 250 °C and detector temperature: 290 °C, injection volume: 1 µl. Percentage conversion was calculated based on the area under peaks of acid and ester.Samples were derivatized with pyridine and BSTFA  mixture was applied for the purification of the remaining esters). The 1H NMR : δ 0.94 , 1.37 , 1.63 , 3.64 , 4.12 , 7.15–7.30 .Butyl phenylacetate: 1H NMR : δ 0.94 , 1.37 , 1.63 , 2.65 , 2.98 , 4.12 , 7.16–7.38 .Butyl 3-phenylpropanoate: 1H NMR : δ 0.94 , 1.37 , 1.63 , 3.64 , 4.12 , 5.60 , 6.70–6.81 , 7.08–7.19 .Butyl 4-hydroxyphenylacetate: 1H NMR : δ 0.94 , 1.37 , 1.63 , 2.65 , 2.98 , 4.09 , 4.99 , 6.71–6.82 , 7.03–7.14 .Butyl 3-(4-hydroxyphenyl)propanoate: 1H NMR : δ 0.94 , 1.37 , 1.63 , 2.65 , 2.98 , 3.81 , 4.09 , 6.74–6.85 , 7.09–7.20 .Butyl 3-(4-methoxyphenyl)propanoate: Antimicrobial activity of synthesized esters was evaluated using the disk diffusion method with Mueller–Hinton agar  or Sabouraud dextrose agar  plates. Blank 6 mm discs  were soaked with 10 µL of the compound. Bacterial or yeasts suspensions in 0.9% NaCl solution at a density of 0.5 McFarland were spread over the surface of the agar plates. Then discs were placed on the agar and plates were incubated for 16–18 h at 37 °C (bacteria) or 48 h at 28 °C (in case of yeasts). After incubation, inhibition zone diameters were measured.50 parameter  was determined. Additionally, CUPRAC (cupric reducing antioxidant capacity) method was carried out following Apak et al.  method following Zanetti et al. . The perk et al.  with neoTo calculate physicochemical properties of the compounds Osiris DataWarrior was used . Following parameters were calculated: partition coefficient  and solubility in water .Analyses were performed using Statistica 13.3 software . One-way analysis of variance (ANOVA) was used to measure the significance of the main effects. Tukey’s test was used to determine significant differences among means (p < 0.05).Y. lipolytica was used in the esterification of five different phenolic acids with 1-butanol. C. antarctica lipase B was used as a comparison. Conversion of phenolic acids to butyl esters is presented in Fig. Freeze-dried biomass of Y. lipolytica biomass), and in the case of 4-hydroxyphenylacetic acid, maximum conversion was 46% (about 10% with freeze-dried Y. lipolytica biomass). 3-phenylpropionic acid was less reactive than in the previous case, and 75% conversion was achieved after 24 h. For the other two substituted 3-phenylpropionic acids, the esterification yield did not exceed 25%.Using the CALB as a catalyst, an opposite trend in reactivity of tested phenolic acids was observed. The highest conversion (93% after 24 h) was achieved in a reaction with phenylacetic acid  and are responsible for the hydrolysis of ester bonds in triacylglycerol molecules. In 1985, Zaks and Klibanov described that lipases can act in nearly anhydrous environments and catalyze reactions inverse to hydrolysis, such as esterification, transesterification, aminolysis, etc.  describeC. antarctica lipase B, as a representative of lipases, has a substrate binding pocket in the shape of an elliptical, steep funnel and shows higher activity for short- and medium-chain fatty acids than long-chain fatty acids. Reducing the size of the binding site can lead to steric conflicts with the substrate, but on the other hand, its larger size leads to suboptimal binding of the substrate due to the presence of free space , as well as α-substituted phenolic acids are not suitable substrates for CALB esterification  with linear alcohol were studied, and similar observations to Otto et al. . Thus, describing substrate specificity of Y. lipolytica intracellular enzymes using whole-cell biocatalyst is not easy. The authors considered the involvement, in the catalytic process, of some intracellular lipases of Y. lipolytica found in the cytosol or associated with cell wall structures, but there is still little knowledge about them and their substrate specificity propionic acid did not significantly affect the antioxidant activity, unlike 4-hydroxyphenylacetic acid and its ester, where the ester was much less active. The obtained results of antioxidant activity with the DPPH radical were comparable to the CUPRAC method. Compounds were characterized by the highest TEAC values in the following order: 3-(4-hydroxyphenyl)propionic acid > 4-hydroxyphenylacetic acid > butyl 3-(4-hydroxyphenyl)propanoate >  > butyl 4-hydroxyphenylacetate >  >  >  > the remaining compounds. The following TEAC values were obtained for the three most active compounds: 0.579, 0.504, and 0.427, respectively.Compounds obtained in esterification reactions and the substrates used for their synthesis were tested for antioxidant activity in DPPH and CUPRAC methods. Compounds containing a hydroxy group in the phenyl ring showed the highest antioxidant activity. The most effective DPPH radical scavengers were 3-(4-hydroxyphenyl)propionic acid and its butyl ester, with antioxidant activities around 50%. Also, 4-hydroxyphenylacetic acid was an effective compound, with about 42% activity, and its butyl ester activity was 1.5-fold lower. For other compounds, antioxidant activity did not exceed 10%. ICortho or para positions to the carboxylic group showed better antioxidant properties, due to their hydrogen donating capacity against radicals  were more potent than hydroxybenzoic acid and its derivatives, due to the better stabilization of radical structures in view of the presence of a double bond conjugated with the aromatic ring . In the case of E. coli PCM 2057 and B. subtilis PCM 486, butyl 3-(4-hydroxyphenyl)propanoate was ineffective. A similar trend was observed for compounds that acted on yeast. Butyl 3-(4-methoxyphenyl)propanoate did not show any antimicrobial activity. Butyl esters of phenylacetic and 3-phenylpropionic acids weakly inhibited yeast growth. Yeasts were more sensitive than bacteria, and C. cylindracea DSM-2031 and R. mucilaginosa were the most sensitive tested microorganisms, where inhibition zone diameters for esters of 4-hydroxypheynlacetic and 3-(4-hydroxyphenyl)propionic acids exceeded 20 mm.Antimicrobial activity of the obtained esters was also determined due to the rising problem of foodborne pathogens and their drug resistance. Dual-functioning compounds  may become useful as multi-task food additives in the near future. Esters without any group or with a methoxy group in phenyl ring did not possess antibacterial activity, and inhibition zone diameters were 8 mm maximum Table . Only esE. coli and Listeria monocytogenes growth. Researchers showed that the examined ester was able to lyse cells, disrupt cell membranes, and also, affect the protein expression system, causing changes in membrane protein conformations and contents  are a broadly studied issue, investigated most frequently for cinnamic acid and its phenolic derivatives. Shi et al. , 2019 obd Guzman . Accordid Guzman . Thus, tE. coli than phenolics with hydroxy and methoxy substituents. On the other hand, one comparable observation has been made in the current study—compounds with shorter side chain length (phenylacetic > phenylpropionic) have higher antimicrobial activity.Contrary to hydroxycinnamates, esters of phenylacetic, 3-phenylpropionic acids and their hydroxy derivatives are less studied. Cueva et al.  examinedY. lipolytica KKP 379 was confirmed to be capable of producing intracellular lipolytic enzymes that catalyzed esterification reactions in organic solvents. Furthermore, these enzymes have significant substrate specificity—freeze-dried Y. lipolytica biomass can be used as a biocatalyst in the synthesis of esters of selected phenolic acids. In particular, esters of phenylpropanoic acid derivatives were synthesized with 75–98% yield. However, in the case of phenylacetic acid, results below 10% were obtained.In the current study, 50 value  to its precursor—3-(4-hydroxyphenyl)propanoic acid  and TEAC values of 0.427 and 0.579, respectively. Considering its better solubility in lipid substances, this compound has prospects for use as a food additive with antioxidant properties. Moreover, butyl esters of 3-(4-hydroxyphenyl)propanoic and 4-hydroxyphenylacetic acids exhibited high antifungal activity, and butyl 4-hydroxyphenylacetate was more active against bacteria than butyl 3-(4-hydroxyphenyl)propanoate, as determined by the larger zones of growth inhibition.The biological activity of phenolic acid esters is strongly associated with their chemical structure, i.e. the presence of substituents in the benzene ring. The hydroxy group is responsible for the antioxidant and antimicrobial activities, and among the obtained compounds, butyl 3-(4-hydroxyphenyl)propanoate showed a similar ICFurther research relating to the enzymatic synthesis of phenolic esters may allow the development of alternative methods for the production of food additives, which is economical and environmentally friendly.


T and RB56T, were determined using a polyphasic approach. Both were isolated from the fungus-farming termite Macrotermes natalensis. Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860T (98.9 %) and Nocardia nova DSM44481T (98.5 %) for RB20T and Nocardia takedensis DSM 44801T (98.3 %), Nocardia pseudobrasiliensis DSM 44290T (98.3 %) and Nocardia rayongensis JCM 19832T (98.2 %) for RB56T. Digital DNA–DNA hybridization (DDH) between RB20T and N. miyunensis JCM12860T and N. nova DSM 44481T resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56T and N. takedensis DSM44801T and N. pseudobrasiliensis DSM44290T showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56T and N. rayongensis JCM19832T resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia, such as the presence of meso-diaminopimelic acid (A2pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids , and the predominant menaquinone MK-8 . The main fatty acids for both strains were hexadecanoic acid (C16 : 0), 10-methyloctadecanoic acid (10-methyl C18 : 0) and cis-9-octadecenoic acid (C18 : 1 ω9c). We propose two novel species within the genus Nocardia: Nocardia macrotermitis sp. nov. with the type strain RB20T (=VKM Ac-2841T=NRRL B65541T) and Nocardia aurantia sp. nov. with the type strain RB56T (=VKM Ac-2842T=NRRL B65542T).The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20 Nocardia are characterized as Gram-positive, non-motile, aerobic bacteria that form a branched mycelium which is easily fragmented forming rod to coccoid-like structures [Actinobacteria together with Corynebacteriaceae and Mycobacteriaceae due to the presence of mycolic acids in the cell membrane [Members of the genus ructures . The genructures . They fomembrane .Nocardia have been mostly recognized as pathogens of humans, plants and animals [Nocardia species harbour an enormous biosynthetic potential to produce structurally unique natural products with antiviral, antifungal, antibacterial and immunosuppressive functions [Like members of these two families, strains of  animals . Nonethe animals  and as s animals . In lighunctions .Nocardia species isolated from the gut of fungus-growing termite Macrotermes natalensis.We have recently focused on the phylogenetic and chemical characterization of Actinobacteria associated with fungus-growing termites , which aMacrotermes natalensis were collected from a termite colony Mn160  and actinobacterial strains RB20T and RB56T were isolated from termite guts as previously described [−1 cycloheximide were incubated aerobically for 21 days at 30 °C and checked daily for the appearance of colonies. Single colonies were transferred onto International Streptomyces Project (ISP) 2 medium. The isolated pure cultures of RB20T and RB56T were maintained on ISP2 at 30 °C and as glycerol suspensions  at −80 °C.In February 2015, termite workers of the genus T and RB56T were carried out as previously described [T and RB56T were extracted from whole genome data  using Artemis [blastn analysis was determined using the NCBI database and the results indicated that strains RB20T and RB56T were members of the genus Nocardia. The 16S rRNA gene sequences of selected Nocardia reference strains were downloaded from the LPSN database (date of access: 2 March 2020) [et al. [sina sequence alignment service was used to generate 16S rRNA gene sequence alignments [mega version 7.0.26 [+G) and the rate variation model allowed for some sites to be evolutionarily invariable (+I). For the NJ algorithm, rate variation among sites was modelled with a gamma distribution. The reliability of the tree topology was evaluated by bootstrap analysis with 1000 resamplings [Genomic DNA extraction, genome sequencing, PCR amplification and sequencing of the 16S rRNA genes of RB20 Artemis . blastn ch 2020)  and pair [et al.  on the G [et al. . The sinignments . Phylogen 7.0.26  using thn 7.0.26  and maxin 7.0.26  algorithn 7.0.26  was usedamplings .T shared highest 16S rRNA gene similarity with Nocardia miyunensis 117T  [Nocardia nova DSM44481T  [Nocardia niigatensis IFM330T  and Nocardia pseudobrasiliensis DSM44290T  [T shared highest 16S rRNA similarity with Nocardia takedensis DSM44801T  [Nocardia pseudobrasiliensis DSM44290T  and Nocardia rayongensis JCM19832T  [Nocardia species .Strain RB20 98.9 %) , Nocard 98.5 %) , Nocard 98.3 %) . Strain  98.3 %) , Nocard 98.2 %) . Lower lT formed a cluster with a larger clade containing N. nova JCM6044T. Strain RB56T clustered with N. rayongensis RY45-3T and N. pseudobrasiliensis DSM44290T  [The DNA G+C content of the genomic DNA was determined from the whole genome sequences . DNA–DNAet al.  and the  [et al.  and Huss [et al. . Further and S4) .Nocardia . For strain RB56T and the closest relatives N. takedensis DSM44801T and N. pseudobrasiliensis DSM44290T the dDDH values were 20.7 and 22.3 %, respectively (Table S4). Wet-lab DDH was performed for RB56T and N. rayongensis JCM19832T due to the lack of whole genome sequence data for N. rayongensis JCM19832T and resulted in a DDH value of 14.5 % (10.2 %). In both cases, the obtained values are below the threshold value of 70 % for the definition of bacterial species recommended by Wayne et al. [It was recommended by Stackebrandt and Ebers  that a 1CM12860T , N. novSM44481T , N. takSM44801T , N. pseSM44290T , N. rayCM19832T  and our e et al. .T and RB56T showed that both strains had a similar genome size of approximately 8.6 Mb with 60 contigs for RB20T and 67 contigs for RB56T (Table S5) N50 size of RB20T is 425 626 bp and for RB56T 451 059 bp. Total CDS were 7454 and 7605 for RB20T and RB56T and the genomes had a completeness of 98.9 and 99.7 %. The G+C content was 67.2 % for RB20T and 69.4 % for RB56T, which is typical for this genus (64–72 %) [Genome analysis of RB20et al. [et al. [4, ω-cyclo) type strains of N. asterioides (IMET 7547T) [N. carnea (IMET 7504T) producing this menaquinone were analyzed in parallel. Polar lipids were extracted by the method described by Minnikin [Rhodococcus rhodochrous IMET 7374T containing glycolyl and Nocardoides albus IMET 7807T containing acetyl muramic acid. Gram-staining was performed as described by Kamlage et.al. [et al. [For chemotaxonomic analyses, freeze-dried cells were obtained from culture grown in ISP2 for 3 days at 28 °C on a rotary shaker at 180 r.p.m. The diagnostic diamino acid of the cell wall was determined in whole-cell hydrolysates by paper chromatography according to Hasegawa . Whole-cet al.  and iden [et al. . To veriT 7547T)  and N. Minnikin  and idenMinnikin . ExtractMinnikin . The glyMinnikin . The refe et.al. . Decompo [et al. . Antibio [et al. . Antibio−1 agar) at 30 °C using light microscope  and a field emission scanning electron microscope. Scanning electron microscopy was performed as described by Groth et al. [et al. [−1). Sodium chloride tolerance was tested on ISP2 by changing sodium chloride concentrations from 1–15 %. The pH tolerance (pH range 4–10) was tested in ISP2 broth using a buffer system described by Xu et al. [Morphological characteristics of the strains were determined on cultures grown for 5–14 days on ISP2 agar  and small amounts of menaquinone MK-9(H2) were detected in both strains  and two glycolipids . Strain RB20T contained additional two unpolar lipids  and more polar lipid L3, which were not present in RB56T. In contrast, RB56T revealed a third phospholipid (PL3) as well as two polar lipids , which is different from the lipids of RB20T .Strains RB20T and RB56T were consistent with those of the genus Nocardia (T was composed of the major fatty acids C16 : 0 (39.6 %), C18 : 0 10-methyl (19.0 %), C18 : 1ω9c (13.4 %) and C18 : 0 (9.9 %). The closest-related species, N. miyunensis JCM12860T and N. nova DSM44481T, had similar fatty acid profiles and contained predominant amounts of C16 : 0 (39.5 and 38.3 %), C18 : 0 10-methyl (18.8 and 14.7 %) and C18 : 1ω9c (13.9 and 16.6 %).The overall cellular fatty acid profiles of RB20ocardia  and S7. T exhibited the major fatty acids C16 : 0 (42.8 %), C18 : 1ω9c (16.1 %) and C18 : 0 10-methyl (12.6 %) and minor amounts of C14 : 0 (6.9 %). The closest relative N. takedensis DSM44801T was characterized by a relatively high amount of C18 : 1ω9c (27.4 %) and the presence of C18 : 0 (4.3 %) and C20 : 1ω9c (4.4 %)   and S7.T and RB56T, respectively  and at 0–3 % (w/v) NaCl . Strain RB20T tolerated a temperature range from 15 to 37 °C, but with only weak growth at 15 and 37 °C, and an optimal growth temperature of 28 °C. RB20T showed fragmenting hyphae into short rod or coccoid forms . Overall, strain RB20T exhibited different phenotypic characteristics compared to the reference strains N. miyunensis JCM12860T, N. nova DSM44481T and N. pseudobrasiliensis DSM44290T  with the following major differences: While RB20T showed good growth on ISP2, ISP6 and Suter medium (+Tyr) and white-beige aerial and substrate mycelium, the closest relative N. miyuensis JCM 12860T showed only very weak growth and ochre to orange aerial and substrate mycelium on ISP2 and ISP6, and good growth but ochre aerial and substrate mycelium on Suter medium (+Tyr). Similarly, RB20T exhibited white-beige aerial and substrate mycelium on ISP7, while N. nova DSM 44481T produced orange aerial and substrate mycelium.Strain RB20T grew at pH range 5–7  and at 0–1 % (w/v) NaCl . Strain RB56T tolerated a temperature range of 15–37 °C, with only weak growth at 15, 37 and 45 °C. The optimal growth temperature was 28 °C. On ISP2 medium, strain RB56T formed short, round and ellipsoidal cells . The substrate mycelium was orange on ISP1, ISP2, ISP6, ISP7 and Suter medium, white on ISP3, yellowish-white on ISP4 and orange-yellow on ISP5. White aerial mycelium developed on ISP3–5, white yellowish aerial mycelium on ISP7 and very poor orange aerial mycelium on ISP1 and ISP2. A soluble reddish pigment was observed on ISP7.Strain RB56T exhibited different phenotypic characteristics to the reference strains N. takedensis DSM44801T, N. pseudobrasiliensis DSM44290T and N. rayongensis JCM19832T with the following major differences: While RB56T showed good growth on ISP3 and white aerial and substrate mycelium, N. takedensis DSM 44801T showed only weak growth and orange yellow substrate mycelium. Similarly, N. pseudobrasiliensis DSM 44290T and N. rayongensis JCM 19832T showed good growth on ISP4 with white aerial and substrate mycelium, whilst RB56T and N. takedensis DSM 44801T grew only moderately to weakly with yellowish to orange substrate mycelium. Finally, growth of RB56T was only weak on Suter medium (with/without tyrosine), whilst all reference strains grew well showing orange to brown soluble pigmentation.Overall, strain RB56T and RB56T, were resistant to oxytetracycline, azlocillin, lincomycin, trimethoprim, carbenicillin, piperacillin, cefoxitin, mezlocillin, penicillin G, cephalothin and chlortetracycline.Both strains, RB20T was furthermore resistant against tetracycline and novobiocin, whereas RB56T exhibited resistance against polymyxin B and erythromycin (Table S8).Strain RB20T and RB56T as two novel species of the genus Nocardia.The morphological, physiological, genetic and chemotaxonomic data support the delineation of RB20Nocardia macrotermitis .Cells are Gram-stain-positive, aerobic and acid-fast. Colonies form branched vegetative mycelium that fragment into short rod and coccoid forms. Good growth occurs on all media tested within 12 days. Aerial mycelium is formed on ISP2, ISP3, ISP5, ISP7 and Suter and poorly on ISP1, ISP4 and ISP6.The pH range for growth is pH 5–7. Growth temperature range is between 15–45 °C with optimum growth at 28 °C. The maximum concentration of NaCl for growth is 3 % (w/v). The strain grows on the following sole carbon sources: glucose, arabinose, xylose, inositol, mannitol, fructose and rhamnose; but does not grow with raffinose and cellulose.The strain is able to utilize the organic acids propionate, malate and decomposes pyruvate and hypoxanthine weakly. Resistant to oxytetracycline, azlocillin, lincomycin, tetracycline, chlortetracycline, trimethoprim, carbenicillin, piperacillin, cefoxitin, mezlocillin, penicillin G, cephalothin and novobiocin.meso-diaminopimelic acid (meso-A2pm). Mycolic acids and N-glycosylmuramic acid in the glycan part of the peptidoglycan are present.The diagnostic diamino acid of the cell wall is 4, ω-cyclo). The major fatty acids are hexadecanoic acid (C16 : 0), 10-methyl octadecanoic acid (10-methyl C18 : 0), cis-9-octadecenoic acid (C18 : 1 ω9c) and octadecanoic acid (C18 : 0). The DNA G+C content of strain RB20T is 67.2 mol%.The sugars in whole-cell hydrolysates are arabinose, galactose and traces of glucose. Major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, two phospholipids  and two glycolipids . Furthermore, two unpolar lipids  and a polar lipid (L3) are present. The predominant menaquinone is MK-8(HT (=VKM Ac-2841T=NRRL-B65541T), was isolated from the gut of the termite Macrotermes natalensis (major worker). The strain has been deposited in the All-Russian Collection of Microorganisms (=VKM Ac-2841T) and the Agricultural Research Service Culture Collection (=NRRL B65541T). The GenBank/EMBL accession number for the partial 16S rRNA gene sequence is KY558706.2. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession WEGK00000000 . The version described in this paper is version WEGK01000000.The type strain, RB20Nocardia aurantia .Cells are Gram-stain-positive, aerobic and acid-fast. Colonies form branched vegetative mycelium that fragment into short rod and coccoid forms. Good growth occurs on ISP2, ISP5 and ISP7, moderate growth on ISP1, ISP3 and ISP4, and poor growth on ISP6 and Suter medium. Aerial mycelium is formed on ISP3, ISP4 and ISP7 media, very poorly on ISP1, ISP2 and ISP5, but not at all on ISP6 or Suter medium. Short, round, ellipsoidal spores are formed. A reddish pigment is produced on ISP7.The pH range for growth is pH 5–7. The growth temperature range is 15–37 °C with optimal growth at 28 °C. The maximum concentration of NaCl for growth is 1 % (w/v). The strain grows on the following sole carbon sources: glucose, arabinose, xylose and rhamnose; but not with raffinose, cellulose, inositol or mannitol.The strain is able to weakly utilize the organic acids acetate, propionate, pyruvate and hypoxanthine, and decomposes malate. Resistant to oxytetracycline, azlocillin, lincomycin, trimethoprim, carbenicillin, piperacillin, cefoxitin, mezlocillin penicillin G, cephalothin, chlortetracycline, polymyxin B and erythromycin.meso-diaminopimelic acid (meso-A2pm). Mycolic acids and N-glycosylmuramic acid in the glycan part of the peptidoglycan are present.The diagnostic diamino acid of the cell wall is 4, ω-cyclo). The major fatty acids are hexadecaonic acid (C16 : 0), cis-9-octadecenoic acid (C18 : 1cis-9) and 10-methyl octadecanoic acid (10-methyl C18 : 0). The DNA G+C content of strain RB56T is 69.4 mol%.Whole-cell hydrolysates contain arabinose, galactose and traces of glucose. Major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, three unidentified phospholipids , two unknown lipids and two glycolipids . The predominant menaquinone is MK-8(HT (=VKM Ac-2842T=NRRL-B65542T), was isolated from the gut of the termite Macrotermes natalensis (major worker). The strain has been deposited in the All-Russian Collection of Microorganisms (=VKM Ac-2842T) and the Agricultural Research Service Culture Collection (=NRRL B65542T). The GenBank/EMBL accession number for the partial 16S rRNA gene sequence is KY558730.2. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession WEGI00000000 . The version described in this paper is version WEGI01000000.The type strain, RB56Click here for additional data file.


Anlotinib is a novel oral multi-targeted receptor tyrosine kinase inhibitor, which selectively inhibits VEGFR2/3, FGFR1-4, PDGFR α/β, c-kit, and Ret. It shows antitumor effect in patients with advanced refractory solid tumors. The detailed absorption, metabolism, and excretion pathways of anlotinib have not yet been fully investigated.14C]-anlotinib hydrochloride. Group B (containing four patients) received 14.15 mg/120 µCi/subject [14C]-anlotinib hydrochloride. The blood, urine, and feces of all the six patients after orally administration of [14C]-anlotinib were collected. The absorption, metabolism, and excretion of [14C]-anlotinib were investigated, and the efficacy and safety of anlotinib were evaluated.Six male patients were enrolled and divided into two groups. Group A (containing two patients) received 14.15 mg/80 µCi/subject [Tmax) of total radioactivity was 4.42 h and the average peak concentration (Cmax) of total radioactivity was 18.80 ng Eq./g. The average values of AUC0-last, AUC0-∞, and MRT0-t were 4071 h.ng Eq./g, 13,555 h.ng Eq./g, and 125 h, respectively. The average recovery of total radioactivity (TRA) in urine and feces was 62.03%, accounting for 48.52% and 13.51% in feces and urine of the total dosage, respectively. The parent drug, a carboxylic metabolite (M30), and mono-oxidation products (M46/M66) were major drug-related components in human plasma. Oxidative metabolism played the major role in drug clearance in human. The major metabolic pathways include oxidative deamination to M2, mono-oxidation to M1, and the formation of M30. Adverse events occurred in five patients and severe adverse events (SAE) occurred in one. Tumor response were evaluated as stable disease (SD) in three, partial response (PR) in one, and progressive disease (PD) in one of the patients, respectively.In plasma, the average time to peak concentration  contains supplementary material, which is available to authorized users. Plasma, urine, and feces were collected followed by total radioactivity analysis, radioactivity profiling, and metabolite structural characterization. Consequently, exposures to drug-related components, mass balance, and metabolic clearance pathways in human were determined.The main objective of this phase I clinical study was to investigate the absorption, metabolism, and excretion of ; (5) adequate renal function [serum creatinine < 1.5 × ULN or creatinine clearance rate (CCR) > 30 mL/min]; (6) adequate cardiac function [left ventricular ejection fraction (LVEF) > 50%].Patients who provided written consents aged between 18 and 70 years with histologically or cytologically confirmed advanced refractory solid tumors , progressed from or relapsed after the first- or second-line treatment, were eligible. Other inclusion criteria included: (1) Eastern Cooperative Oncology Group (ECOG) Performance Score 0–1; (2) an estimated life expectancy > 3 months; (3) adequate bone marrow function  > 1.5 × 10The exclusion criteria included: (1) uncontrolled hypertension ; (2) urinary protein (+ +) and 24-h urinary protein quantification (> 1.0 g) was confirmed; (3) exposure to any drug that inhibits or induces drug metabolic enzymes within 30 days before study entry; (4) major surgical treatment, incision biopsy, or significant traumatic injury within 28 days; (5) arteriovenous thrombosis events within 6 months, such as cerebrovascular accident (including temporary ischemic attack), and deep vein thrombosis and pulmonary embolism; (6) large doses of radiation within 1 year, or radioactivity pharmacokinetic studies of human carbon 14 within 1 year.The basic characteristics of patients are shown in Table 14C]-anlotinib hydrochloride suspension was prepared by XBL-China, Inc.  The suspension containing anlotinib hydrochloride and [14C]-anlotinib hydrochloride which was prepared for orally administration of the patients contained 234 mg of excipients . Patients in Group A administered a single oral dose containing 14.15 mg/80 µCi [14C]-anlotinib hydrochloride . Patients in Group B administered a single oral dose containing 14.15 mg/120 µCi [14C]-anlotinib hydrochloride . anlotinib hydrochloride-related radioactivity for oral administration of [14C] anlotinib hydrochloride. Total radiocarbon concentrations in organ/tissue distribution and excretion data obtained after oral administration of [14C] anlotinib hydrochloride to male rats were utilized for this analysis.A dosimetry analysis was conducted to estimate the human absorbed radiation dose of  anlotinib hydrochloride would predict an effective radiation absorbed dose at or below 1 mSv . In conclusion, a total dose of up to 81 μCi in humans is expected to result in a radiation exposure that is within the 100 mrem (1 mSv) acceptable absorbed dose limit in humans. 80 µCi/ subject was used for Group A, due to long half-life of anlotinib in humans, radioactivity in collected samples post-dose was near to background, and 120 µCi/ subject was used for Group B to obtain more precise data.For a human mass balance study involving radioactivity, the Effective Dose (ED) is typically kept below 1 mSv (Risk Category IIa or below) if possible. Based on the analysis presented herein, a single oral administration of 81 μCi or lower of -anlotinib hydrochloride in male patients. The Tmax of total radioactivity (TRA) in plasma is 4.42 h in average and the Cmax of TRA is 18.80 ng Eq./g in average. The average values of AUClast and AUC0-∞ are 4071 h∙ng Eq./g, 13,555 h∙ng Eq./g respectively, and the average value of MRT0-t is 145 h. The plasma concentration–time curve of anlotinib in plasma samples from six patients is shown in Fig. Individual differences are significant after a single oral administration of 14.15 mg/80 μCi/human or 14.15 mg/120 μCi/human [14C]-anlotinib. Recovery of total radioactivity of the dose was 62.03%, of which 13.51% was from urine and 48.52% was from feces; feces was the major excretion route. The details are listed in Table 14C] anlotinib hydrochloride are shown in Fig. For all of six patients, urine and fecal samples were collected during the 2648 h after administration of [14C]-anlotinib, which were designed as M1, M2, M9, M17, M18, M23, M28, M29, M30, and M46/M66  PlasmaThe parent drug, M30, and M46/M66 were major drug-related component in human plasma . In addition, M1, M2, M9, M17, M18, M28, and M29 were detected in plasma. The identities of M1, M2, M9, M17, M18, M30, and M46/M66 were determined using LC–MS/MS analysis and the comparison with those in urine and feces. However, due to matrix interference and low concentration, M28 and M29 in plasma were not detected by LC–MS/MS. Their identities were determined by comparing their retention time with those in urine and feces. In addition, metabolites with high polarity  were detected in plasma samples, but their structures remained to be determined.(2) UrineThe parent drug was one of the major drug-related components in urine , accounting for 0.73% of the dose. M2, M9, M28, and M30 were the major metabolites in urine, accounting for 0.32% ~ 0.88% of the dose. M1, M17, M18, M23, M29, and M46/M66 were minor metabolites in urine, accounting for 0.07% ~ 0.25% of the dosage. In addition, highly polar metabolites  were detected in urine samples, but their structures remained to be determined. Other metabolites were less than 0.40%.(3) FecesThe parent drug was one of the major drug-related components, accounting for 5.64% of the dose . M9, M28, and M30 were major metabolites in feces, accounting for 2.97% ~ 5.91% of the dosage; M1, M2, M17, M18, M23, M29, and M46/M66 were minor metabolites in feces, accounting for 0.20% ~ 2.57% of the dosage. Other metabolites accounted for less than 1.20% of the dose.(4) Metabolic pathwaysThe major metabolic routes of anlotinib in human include: (1) oxidative deamination to M28, (2) mono-oxidation to M1, followed by further oxidation to M2 and sulfate conjugation to M9, and 3) oxidation to carboxylic acid product (M30). Minor metabolic routes include: (1) mono-oxidation to M23, M46/M66, followed by glucuronide conjugation to M17 and (2) O-dealkylation to M18. The metabolic pathways of anlotinib in human are proposed in Fig.  oxidatioAfter a single dosage, adverse events occurred in five patients, while SAE occurred in one during the follow-up period. Treatment-related grade 3 adverse events occurred in the course of drug use. The frequency of adverse events was sorted as following: increased lipase, increased bilirubin, increased alanine aminotransferase, hypophosphatemia, increased gamma-glutamyltransferase, increased aspartate aminotransferase, and liver function damage. Grade 1/2 adverse events included increased lipase, hypoalbuminemia, increased bilirubin, constipation, proteinuria, etc. The details are listed in Table 14C]-anlotinib hydrochloride suspension was administered followed by anlotinib (12 mg for 2 consecutive weeks followed by 1 week discontinuation) after 432 h (504 h for Subject 04). As a result, [14C]-anlotinib hydrochloride and its metabolites were diluted and redistributed after the large quantity of unlabeled drugs entered circulation, reducing the excretion rate of labeled drugs and their metabolites -anlotinib hydrochloride administration, and the individual difference was significant.The plasma concentration of anlotinib hydrochloride was analyzed by LC–MS/MS. All of the data were in accordance with the standard operating procedures of Nanjing Mesino Pharmaceutical Technology Co., Ltd. The results showed that TRA concentration in plasma and blood was low after [14C]-anlotinib hydrochloride. The main clearance method of anlotinib hydrochloride was considered to be metabolized by phase I enzyme and then excreted from liver to feces. Besides, the direct excretion of anlotinib from the kidney is also another important way of clearance. According to metabolite identification of [14C]-anlotinib hydrochloride, the main metabolic pathways of anlotinib hydrochloride include: (1) amino oxidation (amino to hydroxyl) to form M28; (2) mono-oxidation to form M1, and further to oxidative metabolite (M2) and sulfuric acid conjugate (M9); (3) carboxylation to form M30. Additionally, the secondary metabolic pathways of anlotinib hydrochloride in the patients including: (1) mono-oxidation to form M23 and/or M46/M66 and further convert to glucuronic acid-binding product (M17); (2) O-dealkylation to form M18 [The liver was the main excretory organ and the kidney was the secondary excretory organ after a single dosage of [form M18 .14C]-anlotinib hydrochloride suspension during the observational period, and most of them presented with grade 1/2 adverse events. It is suggested that the safety of patients could be guaranteed with a single dosage of [14C]-anlotinib hydrochloride suspension (14.15 mg/80 µCi or 14.15 mg/120 µCi).Patients were well tolerated to single dosage of 14.15 mg/80 µCi/human or 14.15 mg/120 µCi/human [In conclusion, anlotinib showed a pharmacokinetic profile of rapid absorption, long half-life, and extensive hepatic metabolism. The efficacy was promising with a favorable toxicity profile as expected, demonstrating that anlotinib could be further investigated in clinic.Supplementary file1 (DOCX 297 kb)Below is the link to the electronic supplementary material.


Cisplatin and oxaliplatin are treatment options for a variety of cancer types. While highly efficient in killing cancer cells, both chemotherapeutics cause severe side effects, e.g., peripheral neuropathies. Using a cell viability assay, a mitochondrial stress assay, and live-cell imaging, the effects of cis- or oxaliplatin on the mitochondrial function, reactive oxygen species (ROS) production, and mitochondrial and cytosolic calcium concentration of transient receptor potential ankyrin 1 (TRPA1)- or vanilloid 1 (TRPV1)-positive dorsal root ganglion (DRG) neurons of adult Wistar rats were determined. Mitochondrial functions were impaired after exposure to cis- or oxaliplatin by mitochondrial respiratory chain complex I-III inhibition. The basal respiration, spare respiratory capacity, and the adenosine triphosphate (ATP)-linked respiration were decreased after exposure to 10 µM cis- or oxaliplatin. The ROS production showed an immediate increase, and after reaching the peak, ROS production dropped. Calcium imaging showed an increase in the cytosolic calcium concentration during exposure to 10 µM cis- or oxaliplatin in TRPA1- or TRPV1-positive DRG neurons while the mitochondrial calcium concentration continuously decreased. Our data demonstrate a significant effect of cis- and oxaliplatin on mitochondrial function as an early event of platinum-based drug exposure, suggesting mitochondria as a potential target for preventing chemotherapy-induced neuropathy. Cytostatic drugs as cis- and oxaliplatin are still common in tumor therapy today. While cisplatin is used for the treatment of lung, testicular, or ovarian cancer, oxaliplatin is majorly used for colorectal cancer ,2,3,4. B1) receptors are discussed as playing a critical role in the induction of polyneuropathies or neuropathic pain in different models  stimuli. Therefore, these neurons express a specialized setup of receptors and channels that can be activated by mechanical, thermal, or chemical stimuli and contribute to typical symptoms of CIPN as hyperalgesia, allodynia, or numbness  activates at high temperatures (>43 °C) by capsaicin and an acidic pH . TRPV1 aNext to damage in the deoxyribonucleic acid (DNA) of DRG neurons, mitochondrial and dysfunctional changes in the calcium homeostasis and increased reactive oxygen species (ROS) have been suggested to cause CIPN ,28,29,30Dysfunction and changes in expression of voltage-gated calcium channels (VGCC) in DRG neurons have been shown to contribute to calcium-dyshomeostasis after cis- or- oxaliplatin exposure ,17,18,31Using a mitochondrial stress assay, live-cell antibody staining, and imaging, the influence of cisplatin or oxaliplatin on the mitochondrial function of DRG neurons was examined.A luminescence viability assay was performed to examine the influence of cis- or oxaliplatin on DRG neurons’ viability. DRG neurons were cultured and exposed to 0.1 µM, 1 µM, 10 µM, or 100 µM for 24 h. As a positive control, neurons were exposed to 0.01% Triton X-100 to induce cell death after 24 h.p < 0.001) , compared to untreated control cells (135700 ± 1603.12). Exposure to 100 µM led to a decrease of the neuronal viability  A.p < 0.001). Furthermore, exposure to 0.01% Triton-X100 led to a decreased viability of cultured DRG neurons  . In contrast to these findings, exposure to 100 µM oxaliplatin resulted in a decreased viability  A.To measure the influence of chemotherapeutics on ROS production in DRG neurons, cell were cultured as described above and exposed to 0.1 µM, 1 µM, 10 µM, or 100 µM cis- or oxaliplatin for 24 h and stained with CellRox B.p < 0.01; 1 µM: 1.36 ± 0.03, *** p < 0.001; 10 µM: 1.86 ± 0.04, *** p < 0.001; 100 µM: 2.24 ± 0.02, *** p < 0.001) compared to untreated control neurons (1 ± 0.03). A concentration-dependent effect was observed for a concentration range of 1 µM to 100 µM   C.p < 0.01; 1 µM: 1.41 ± 0.02; *** p < 0.001; 10 µM: 1.87 ± 0.04, *** p < 0.001; 100 µM: 2.30 ± 0.01; *** p < 0.001). A concentration-depend effect was shown for the range of 0.1 µM to 100 µM   D.A mitochondrial stress assay was conducted to determine the different respiration rates during the addition of Oligomycin, carbonyl cyanide-4 (trifluoromethoxy)-phenylhydrazone (FCCP), and Rotenone and Antimycin A. Cells were exposed either 10 µM cis- or oxaliplatin overnight.p < 0.05)  A,B.p < 0.001) and after oxaliplatin exposure to 14.68 ± 0.98 pmol/min (*** p < 0.001) compared to the control at 67.09 ± 2.43 pmol/min. Cisplatin exposure had a more substantial effect on the BR than oxaliplatin (###p < 0.001) (p < 0.001) and after an oxaliplatin exposure to 17.857 ± 2.594 compared to the control cells at 179.52 ± 6.96 pmol/min (*** p < 0.001). Oxaliplatin exposure had a more substantial effect on the MRC than cisplatin (###p < 0.001) (p < 0.01) and was reduced to 3.92 ± 1.74 pmol/min after an oxaliplatin exposure (** p < 0.01). There was no difference in PL between cis-or oxaliplatin (p > 0.05)  rate decreased after cisplatin exposure to 5.85 ± 0.71 pmol/min (*** < 0.001) C. As wel< 0.001) D. Under  > 0.05) E.p < 0.001) and to 10.76 ± 0.76 pmol/min (** p < 0.01) after exposing the cells to oxaliplatin. Cisplatin exposure had a more substantial effect on the ATPR than oxaliplatin (###p < 0.001) (p < 0.01) compared to control conditions (112.43 ± 4.57 pmol/min). Exposure with oxaliplatin for 24 h reduced the SRC to 3.17 ± 1.97 pmol/min (*** p < 0.001). Oxaliplatin exposure had a more substantial effect on the SRC than cisplatin (###p < 0.001) (The adenosine triphosphate-linked respiration (ATPR) is 31.52 ± 3.50 pmol/min at the control level, whereas it was decreased after exposure to cisplatin to 1.61 ± 0.40 pmol/min (*** < 0.001) F. The sp< 0.001) G.p < 0.01) and oxaliplatin: 2.78 ± 1.63 pmol/min (** p < 0.01)). Oxaliplatin exposure had a stronger effect on the non-mitochondrial respiration than cisplatin (#p < 0.05)  was 11.67 ± 0.91 pmol/min. Here, an exposure with cis- or oxaliplatin for 24 h decreased the non-mitochondrial respiration (cisplatin: 7.29 ± 0.43 pmol/min (**  < 0.05) H.p < 0.01) at 70 min. The mitochondrial calcium concentration dropped below the control level after 10 min (* p < 0.05) and continuously decreased to 0.56 ± 0.05 (** p < 0.01) after 70 min (p < 0.001) after 15 min with a maximum at 1.24 ± 0.07 (*** p < 0.001) after 70 min. The intramitochondrial calcium concentration continuously decreased to a minimum at 70 min   C.p < 0.05, ** p < 0.01). The mitochondrial calcium concentration also had a stronger decrease in TRPV1-positive neurons after 5 min   compared to TRPA1-positive neurons (*  < 0.01) D,E.p < 0.001) and a maximum for TRPV1-positive neurons at 1.48 ± 0.03 (*** p < 0.001) after 70 min (p < 0.001) for TRPA1 and to 0.72 ± 0.07 (** p < 0.01) for TRPV1-positive neurons after 70 min (p > 0.05)  D,E.p > 0.05)  A–C.p < 0.05, ** p < 0.01)  D.p < 0.001) after 30 min. Afterward, it continuously decreased and reached the control level after 45 min . The relative ROS production further decreased to 0.370 ± 0.035 (*** p < 0.001) at 70 min  was applied directly while measuring and was observed for 70 min A. The ROt 70 min B.p < 0.001) 5 min after exposure to cisplatin. The ROS production decreased after 35 min and dropped below the control level after 50 min  with a minimum at 0.75 ± 0.07 (** p < 0.01) after 70 min (The ROS production of TRPV1-positive neurons continuously increased to a maximum of 1.23 ± 0.07 (*** r 70 min C.p < 0.01). It continuously increased with a peak at 15 min . Afterward, the ROS production decreased towards control levels   was applied directly while measuring and was observed over 70 min A. The RO > 0.05) B.p < 0.05) and decreased to the control condition. After 30 min, the ROS production was decreased  with a minimum at 70 min  (The ROS production of TRPV1-positive neurons increased after 5 min at 1.08 ± 0.01 (* < 0.001) C.p < 0.05). After 45 min until 70 min, ROS production was decreased after cisplatin exposure compared to oxaliplatin exposure (* p < 0.05)  A.p < 0.05, ** p < 0.01)  B.We here show that the platinum-based drugs cis- and oxaliplatin induce mitochondrial dysfunction of TRPA1- or TRPV1-positive DRG neurons. Here we observed changes in ROS production, mitochondrial, and cytosolic calcium concentration.Both cisplatin and oxaliplatin are known to contribute to induction of apoptotic processes in different cell types. When cultured DRG neurons were treated with different concentrations of these chemotherapeutics, no change in cell viability was observed when cells were treated with 0.1 µM, 1 µM, or 10 µM after 24 h. Viability was only decreased by cis- or oxaliplatin in a concentration of 100 µM. These findings are in line with other cell culture studies, in which reduced cell viability was only observed with concentrations of cis- or oxaliplatin higher than 10 µM ,33. In oThe mitochondrial stress assay showed that DRG neurons demonstrate altered mitochondrial respiration after long-term exposure to cis- or oxaliplatin for 24 h. The lower ATPR and BR possibly determine a diminished ATP generation. The BR indicates an endogenous ATP generation. Consequently, a low BR after exposure to cis- or oxaliplatin indicates a lower ATP generation during the resting state of the DRG neurons, leading to the suggestion that DRG neurons exposed to cis oxaliplatin have a lower ATP generation and ATP supply. However, cisplatin-exposed DRG neurons had lower ATPR and consequently were more affected than oxaliplatin-exposed DRG neurons. Similar results have been shown for the influence of cisplatin on ampullary tissue or cerebellar cortex of guinea pigs . In rat SRC reflects the ability of neurons to react to different energetic states. Oxaliplatin-exposed DRG neurons demonstrated a lower SRC than cisplatin-exposed DRG neurons, indicating an insufficient energy supply during stressful situations as a contributing mechanism for sensory neuron malfunction or neuronal degeneration.After platin exposure, a lower MRC and a direct effect on the SRC was observed. Oxaliplatin-exposed DRG neurons showed a lower MRC compared to cisplatin-exposed DRG neurons. These findings pinpoint an inhibitory effect of platin on the mitochondrial respiratory chain complexes I-III as evidenced by a markedly diminished electron flux, which could be chemically restored. The PL of cis- or oxaliplatin-exposed DRG neurons was also reduced. Generally, the PL generates heat and can be influenced by a couple of factors, e.g., the composition of the inner mitochondrial membrane and ROS production. As shown here, both cis- and oxaliplatin exposure increased ROS production, possibly due to changes in the PL, MR, and SRC. In other studies, an increased ROS production after cis- or oxaliplatin exposure has also been observed in Caco-2 and a prostate cancer cell line ,37.ROS production of DRG neurons exposed to cisplatin or oxaliplatin showed alterations during long-term and short-term exposure. Cultured DRG neurons treated with cis- or oxaliplatin showed an increased ROS production after 24 h. In recent studies, an increased ROS level of DRG neurons treated with 50 µM cisplatin for 24 h was described . Other sIn contrast, we found increased ROS production after exposure with cis- or oxaliplatin, but a decline to or below control level after a short period of time. Possible cyclic regulation of ROS explains the absence of increased ROS production after exposure with 10 µM oxaliplatin after 24 h. The abrupt and significant decline may occur due to antioxidant defenses within the neuron. In a model of diabetic neuropathy, activation of the transcription factor nuclear factor erythroid 2-related factor (Nrf2) in DRG neurons results in an expression of antioxidant enzymes and thus, an immediate decrease in the ROS production and preventing hyperglycemia-induced damage to the neuron . TRPV1-pMitochondrial dysfunction caused by platinum-based chemotherapeutics are well-described for different types of cancer cells. In prostate cancer cell line, exposure with cisplatin for 24 h led to an increase in ROS production . ExposurThe increase in cytosolic calcium concentration and the simultaneous decrease in mitochondrial calcium concentration indicate a discharge of calcium out of the mitochondria into the DRG neurons’ cytosol. In HeLa-S3 cells, exposure with cisplatin led to an increased intracellular calcium concentration. The increase in intracellular calcium was dependent on extracellular calcium concentration. Using an IP3 receptor blocker, the cisplatin-mediated increase of intracellular calcium was diminished . In gastThe increased cytosolic calcium concentration during short-term exposure of cisplatin or oxaliplatin contributes to diminished VGCC currents, as observed in a former study . Here, aAdult Wistar rats of both sexes at the age of approximately three weeks (60–80 g) were used to cultivate DRG neurons. The animals were kept at the animal research lab at the University Duisburg-Essen in cages with up to 4 animals. The animals had free access to food and water (ad libitum) and were kept in a 14/10 h day and night rhythm.All experiments were conducted under the animal welfare guidelines of the University Duisburg-Essen.2. Afterward, DRGs were washed and incubated in 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA)  solution for 8 min at 37° C and 5% CO2. The enzyme reaction was stopped with 1 mL of fetal bovine serum  and washed three times in DMEM/F12. Afterward, the ganglia were titrated in 1 mL of DMEM/F12 until a cell suspension was formed. To facilitate neurons’ adhesion to coverslips, 24-well dishes were placed in an incubator for 2 h. Following the adhesion, 500 µL of DRG-culture medium containing DMEM/F12, 10% fetal bovine serum , 1% penicillin/streptomycin , 1% neuronal growth factor , and 1% N2  was placed in each well. After 24 h, the medium was replaced with neuronal feeding medium with 10 µM cytosine arabinose  for 72 h. Seventy-two hours later, the neurons were prepared for further experiments.For the isolation of the DRG neurons, the rats were narcotized with Isoflurane, and the DRGs were removed as previously described . BrieflyDRG neurons were counted using a Neubauer Counting Chamber and plated in a density of 1500 neurons/well for all experiments.Oxaliplatin  and cisplatin  were diluted with Aqua bidest to a concentration of 10 mM for oxaliplatin and 5 mM for cisplatin as a stock solution. DRG neurons were exposed to 0.1 µM, 1 µM, 10 µM, or 100 µM cisplatin or oxaliplatin for 24 h for measuring viability and ROS production. All further experiments were performed with 10 µM cisplatin or oxaliplatin. For measurement of short-term effect on mitochondrial and cytosolic calcium concentration and ROS production, chemotherapeutics were directly applied to cells during measurements for 70 min. The concentrations used were based on viability experiments and previous cisplatin and oxaliplatin studies ,18,32,33For examining the influence of cisplatin or oxaliplatin on the viability of cultured DRG neurons, cells were cultured as described above, treated with 0.1 µM, 1 µM, 10 µM, or 100 µM cisplatin or oxaliplatin and a luminescence RealTime-Glo MT Cell Viability Assay  was performed according to the manufacture’s protocol.To determine the effects of the chemotherapeutics on the TRPA1- or TRPV1-positive subpopulations, cell cultures were prepared as described above. The cells were first stained with extracellular TRPA1 or TRPV1 antibodies .The cell culture medium was removed from each of the wells and wells were washed twice with cold and sterile blocking solution  in phosphate-buffered saline (PBS)).For already tagged primary antibodies , cells wIf a secondary antibody  was needTo analyze the long-term effects of cisplatin and oxaliplatin on ROS production in DRG neurons or the short-term effect on TRPA1- or TRPV1-positive subtypes of DRG neurons, cells were cultured and prepared as described above. For the study of short-term effects, DRG neurons were tagged with primary antibodies .For the study of long-term effects, DRG neurons were treated with 0.1 µM, 1 µM, 10 µM, or 100 µM cisplatin or oxaliplatin for 24 h. Afterward, 1 µL of the CellRox  solution was added to 500 µL of the cell culture medium and incubated for 30 min in the incubator. The cells were washed twice with sterile PBS and transferred to the microscopy chamber, and 2 mL of the live-cell imaging solution was added.For short-term effects, naïve cultured DRG neurons were treated with 10 µM cisplatin or oxaliplatin after CellRox incubation, and images were obtained every 5 min until a 70 min cut-off.For the determination of the cytosolic calcium concentration of treated TRPA1- or TRPV1-positive DRG neurons, the cells were cultured and stained as described above . Cells wThe cells were incubated for 15 min with 0.5 µL Fluo-4  in 500 µL serum-free culture medium. Afterward, the cells were washed with sterile PBS, 2 mL live-cell imaging solution was added, and cells were treated with 10 µM cisplatin or oxaliplatin. Images were obtained every 5 min until a cut-off at 70 min.To determine the effects of cis- and oxaliplatin on the intramitochondrial calcium concentration, cells were cultured as described above. DRG neurons were stained for TRPA1 or TRPV1, as described above .The cells were incubated for 15 min with 0.5 µL Rhod-2  in 500 µL serum-free culture medium. Afterward, the cells were washed with sterile PBS, and 2 mL of the live-cell imaging solution was added. The cells were treated with 10 µM cisplatin or oxaliplatin, and images were obtained every 5 min until a 70 min cut-off.The mitochondrial stress assay  targets specific complexes of the electron transport chain (ETC) and inhibits them from determining the oxygen consumption rate (OCR). The first inhibitor added is oligomycin inhibits the complex V, the ATP-synthase, of the ETC. The next inhibitor added is carbonyl cyanide-4 (trifluoromethoxy)-phenylhydrazone , which collapses the proton gradient and disrupts the membrane potential. The last injection contains a mixture of rotenone, a complex I inhibitor, and Antimycin A, an inhibitor of complex III.Dorsal root ganglion neurons were cultured as described above and treated with 10 µM cisplatin or oxaliplatin for 24 h.2 incubator at 37 °C overnight.On the day before the assay, 200 µL of calibrant solution  was added to each well, and the sensor cartridge was lowered until the sensors were submerged into the solution. The sensor cartridge was put in a non-COOn the day of the assay, the cell culture medium was removed and washed once with assay medium before adding 180 µL of assay medium to each well. The injections of the inhibitors were prepared and loaded into the disposable injection ports.After creating an appropriate assay template with “Wave” software , the sensor cartridge with the hydrated probes was loaded into the Seahorse Analyzer to allow calibration. Afterward, the well-plate containing the cells was put into the Analyzer. Further instructions were followed, as indicated by the apparatus. For analysis of data, we calculated basal respiration (BR), maximal respiratory capacity (MRC), proton leak (PL), ATP-linked respiration (ATPR), spare respiration capacity (SRC), and non-mitochondrial respiration (NMR).In summary, our results show a drug-specific effect of cisplatin or oxaliplatin on mitochondrial function in DRG neuron subpopulations. Possibly, mitochondria can be a potential target for future prevention strategies of CIPN.


BackgroundObesity is one of the main risk factors of coronary heart disease (CHD). Although a range of anthropometric measures are available to evaluate obesity, which measure is the most precise to predict the risk of CHD is still controversial. Therefore, we assess the prevalence of obesity among patients with CHD by using various anthropometric techniques to find out the most efficient method to predict the risk of CHD. MethodsIn this cross-sectional study, we included 300 CHD patients and 100 age and sex-matched healthy controls, aged 45-70 years. Various anthropometric measurements  were taken to assess the prevalence of obesity among the selected population.Resultsp < 0.001) and 97.8±12.1 vs. 86.9±5.3 (p < 0.001) respectively. The average waist-hip ratio among patients of both genders was significantly higher than controls 1.0±.06 vs. 0.92±.04 (p < 0.001) among males and 0.96±.07 vs. 0.88±.04 (p < 0.001) among females, respectively. The average body mass index (BMI) was not different among male patients (24.6±4.0) compared to controls (24.3±2.3); however, the frequency distribution of BMI among male patients and controls was significantly different (p < 0.05), whereas female patients had significantly higher BMI compared to controls 27.7±4.9 and 25.1±2.4, (p < 0.001). Similarly, body fat percentage and visceral fat percentage were elevated among female cases vs. female controls, but no significant difference was observed in the body fat percentage of male cases vs. controls 28.0±5.0 vs. 28.1±2.7; (p > 0.05). However, visceral fat percentage was significantly elevated among male cases vs. controls 11.6±5.7 vs. 9.6±2.6 (p < 0.05).Average waist circumference among male and female patients was significantly higher than the controls; 94.0±13.2 vs. 86.4±4.4 , waist-hip ratio (WHR), and visceral fat percentage were uniformly present in patients of both sexes and are stronger predictors of risk of CHD relative to the BMI. Coronary heart disease (CHD) is one form of cardiac disease. The pathogenesis is attributed to atherosclerosis, resulting in the formation of plaques which further lead to a compromise in the availability of oxygenated blood to the cardiac muscles . AtherosObesity is a major risk factor for cardiovascular disease (CVD) including CHD, heart failure (HF), hypertension (HT), and arrhythmias. Increased body weight is also associated with increased coronary events and mortality among heart disease patients -8. GlobaA cross-sectional study was carried out on newly diagnosed CHD patients based upon symptoms, finding of non-invasive tests, and angiographic evidence of CAD residing in the northwest region of India. Data were collected from 300 symptomatic CHD patients who visited the Cardiology OPD and Cardiac Care Unit of Government Medical College and Hospital, Chandigarh, India. Age and gender-matched asymptomatic subjects with no clinical evidence of CHD were recruited as controls (n-100). Patients with other co-morbidities including fever, infections, autoimmune disorders, and respiratory and gastrointestinal distress were excluded. Ethical clearance for collecting data from human subjects was obtained from the Institutional Ethics Committee (IEC), Panjab University, Chandigarh. Furthermore, ethical approval from the IEC of GMCH was also obtained to conduct fieldwork. The patient consent form was read to the participants in vernacular language  and their duly signed consent was taken before participation. It was clearly explained to the subjects that their identity will be kept confidential.2) and classification for Asians was used in the study [X2) was applied to observe the frequency distribution of BMI among cases and controls.Selected subjects were interviewed to obtain primary data for the study. Anthropometric measurements such as BMI, WC, WHR, and bioelectrical impedance analysis (BIA) were collected. Bodyweight (kg) was recorded with the help of a portable electric scale (Omron H-301 Korea). Total standing height was measured from the top of the head (Vertex) till foot-sole, using a portable anthropometric rod  . The waihe study . The bodAs shown in Table 2) was observed in 28 (16.6%) male patients, 45 (34.4%) female patients, and 6 (12%) female controls, whereas obesity class-I was not present among the male control group. According to the results, obesity class-II (BMI: 32.5-37.5 kg/m2) was reported among 5 (3.0%) male patients and 19 (14.5%) female patients, similarly, obesity class-III (BMI>37.5+ kg/m2) was observed among 1 (0.6%) male patients and 4 (3.1%) female patients but none among control groups. Pearson’s chi-square test (X2) was applied to the frequency distribution of BMI among cases and controls. The frequency distribution of BMI was significantly different among male cases and controls (p < 0.05) and obesity (BMI: 27.5-37.5 kg/m2) was prevalent among 34 (20.2%) male patients. In contrast, obesity (BMI: 27.5-37.5 kg/m2) was even more prevalent among 68 (52%) female cases in comparison to 6 (12%) female controls, and frequency distribution of BMI was highly significant (p < 0.001) among them.A comparison between CHD patients and controls regarding the frequency distribution of BMI according to Asian classification is shown in Table p < 0.001) and 97.8±12.1 vs. 86.9±5.3 (p < 0.001), respectively. Similarly, the average WHR among patients of both genders was significantly higher than controls, 1.0±.06 vs. 0.92±.04; (p < 0.001) among males and 0.96±.07 vs. 0.88±.04 (p < 0.001) among females, respectively. The average BMI was not different among male patients 24.6±4.0 compared to male controls 24.3±2.3 (p > 0.05), whereas, the female patients had a significantly higher average BMI compared to female controls, 27.7±4.9 and 25.1±2.4, respectively (p < 0.001). The average BF % was identical among male patients 28.0±5.0 compared to male controls 28.1±2.7 (p > 0.05), however, the female patients had significantly higher average BF % compared to female controls, 38.3±5.0 and 32.8±2.3, respectively (p < 0.001). Similarly, the average VF % among patients of both genders were significantly higher than controls 11.6±5.7 vs. 9.6±2.6 (p < 0.05) among males and 11.9±5.9 vs. 7.9±2.8 (p < 0.001) among females, respectively.The descriptive analysis of anthropometric measurements, as well as body composition indices of CHD patients and controls, is presented in Table 2) was prevalent only among 34 (20.2%) male patients, however, 135 (79.9%) male patients were below obesity range (BMI <27.5 kg/m2) according to Asian BMI classification [2) was even more prevalent among 68 (52%) female patients in contrast to 6 (12%) female controls. Furthermore, BMI >30 kg/m2 has been reported as an independent risk factor for the occurrence of coronary diseases [2 have also been linked with increased CVD mortality among the East Asian population [In our study, we attempted to determine the prevalence of obesity by using various anthropometric measurements among CHD patients, to identify the risk of CHD. Our results show that obesity from class I-III . In addition, CHD patients’ WHR was also reported above WHO reference range  , 19. MorBased on our study, female patients had significantly higher BF % than female controls however, no major difference was observed in BF % of male patients and male controls. Additionally, raised body fat % has been correlated with an increased probability of a cardiac event, while higher fat-free mass has been correlated with the inferior chance of a cardiac event in CHD patients. Therefore, BF %, not BMI is linked with “major adverse cardiovascular events” . In our study, male as well as female patients had significantly higher VF % than controls. Hypertension, ischemic heart disease (IHD), diabetic cardiomyopathy, and elevated VF have been associated with coronary events. The VF stores, including fat build-up in epicardial linings, are linked with higher CVD risk. This results from the effect of increased lipid emission, pro-inflammatory factors, and adipokines from adipose tissues . InteresResults of the present study indicate that WC, WHR, and VF % measurements were uniformly elevated among CHD patients than controls in both genders. Whereas, average BMI was similar among male cases and controls and showed no signification difference, conversely, the frequency distribution of BMI was significantly different among male cases and controls, thus the outcomes were found to be ambiguous to apply for CHD risk assessment. People with identical bodyweight may have immeasurable diversity in their body composition as BMI does not differentiate within the lean mass, fat mass, and fat-free mass . TherefoOur study has some limitations. First, in this cross-sectional research, we did not have the opportunity to examine the cause and effect association between obesity and CHD. Besides, the sample size of the control group can be taken as a restricting factor. The changed body water distribution among people is one of the constraining factors to utilize BIA in the assessment of body fat and obesity. The results of this research may be inadequate for the diverse cultures, demographic groups, and metropolitan communities in India. The anthropometric measurements could not be evaluated for sensitivity, specificity, positive predictive value, and negative predictive values to examine the precision of these measurements. However, the anthropometric measurements were taken by a trained anthropometrist (NG) by following all the standard procedures and protocols. Therefore, further researches are required in line to explore a greater understanding of the accuracy and precision of these methods.Anthropometric measurements are a simple and inexpensive tool for assessing adiposity and risk of CHD. Our results also show that there are wide variations between populations, in the effect of BMI, WC, WHR and, VF% on predicting the risk of CHD. While BMI is a useful tool to assess obesity in the mass population, it should be used in conjunction with other markers of central adiposity  for better prediction of CHD risk. It is therefore important to conduct population-specific studies and develop country-wise or even province-specific references for predicting CHD risk using these anthropometric measurements of predicting central adiposity.


Ziziphus jujuba. Since the pharmacological effects and associated mechanisms of UNA are not well-known, in this work, we attempt to introduce the therapeutic potential of UNA with a comparison to ursolic acid (ULA), a well-known secondary metabolite, for beneficial effects. UNA has a keto group at the C-3 position, which may provide a critical difference for the varied biological activities between UNA and ULA. Several studies previously showed that UNA exerts pharmaceutical effects similar to, or stronger than, ULA, with UNA significantly decreasing the survival and proliferation of various types of cancer cells. UNA has potential to exert inhibitory effects in parasitic protozoa that cause several tropical diseases. UNA also exerts other potential effects, including antihyperglycemic, anti-inflammatory, antiviral, and antioxidant activities. Of note, a recent study highlighted the suppressive potential of UNA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular modifications of UNA may enhance bioavailability, which is crucial for in vivo and clinical studies. In conclusion, UNA has promising potential to be developed in anticancer and antiprotozoan pharmaceuticals. In-depth investigations may increase the possibility of UNA being developed as a novel reagent for chemotherapy.Plants have been used as drugs to treat human disease for centuries. Ursonic acid (UNA) is a naturally occurring pentacyclic triterpenoid extracted from certain medicinal herbs such as  Historically, plants have long been used to create or obtain medicines to treat a variety of diseases . MedicinAmong triterpenoids, ursolic acid (ULA) is a widespread secondary metabolite found in pomace, cork, flower, sprout, leaf, and the bark of various plants . ULA is While the therapeutic effects and underlying mechanisms of ULA are well understood, as documented in various scientific studies and reviews, those of UNA are relatively less studied and elucidated. Although ULA and UNA seem quite similar in terms of chemical structure, they are definitely distinct compounds and can exert different biological activities. In this review, we focus on the sanative effects and related cellular mechanisms of UNA. With a comparison between UNA and ULA, we attempt to emphasize the pharmaceutical potential of UNA as a novel drug candidate.To search the articles regarding UNA and its therapeutic potential, we used PubMed and Google Scholar as databases. The name of UNA and its synonyms were used for keywords to search the literature. We used terms “Ursonic acid”, “3-oxo-urs-12-en-28-oic acid”, “3-oxours-12-en-28-oic acid”, “3-oxo-ursolic acid”, “3-keto ursolic acid”, “UA derivatives” plus “ketone”, “UA derivatives” plus “3-oxo”, “UA acid derivatives” plus “3-keto”, “Ursolic acid derivatives” plus “Ketone”, “Ursolic acid derivatives” plus “3-oxo”, and “Ursolic acid derivatives” plus “3-keto” to search papers relevant to our topic. We included papers from between 1980 and 2020, and there was no restriction on language or type of publication. In total, 22 articles were retrieved from PubMed and 980 publications were found using Google Scholar. We selected papers which conducted experiments regarding extraction or synthesis or the pharmacological potential of UNA. Duplicates and unrelated articles were excluded, and 41 papers were selected for references in regard to UNA.ULA (3β-hydroxy-urs-12-en-28-oic-acid) is a pentacyclic triterpenoid which has C30H48O3 as its molecular formula and a molecular mass of 456.7 g/mol A 14]. U. U14]. UNocardia species and Bacillus megaterium CGMCC 1.1741 was found to biotransform ULA to generate UNA [Aspergillus flavus resulted in isolation of UNA [Triterpenoids can be synthesized by plants through complicated biochemical pathways ,23. In prate UNA ,22. Alson of UNA . Moreoven of UNA ,25,26,27Crataegus Pinnatifida and Malus baccata [Ziziphus jujuba, a main ingredient of traditional Chinese medicinal formula PHY906, which is used for adjuvant therapy of patients with cancer [Pistacia atlantica, Bursera delpechiana, and trees of the Dipterocarpaceae family contain triterpenoids, including UNA [Toona sinensis, Piper betle, and Ficus microcarpa [Lantana camara, which was traditionally used for treatment of eczema, ulcers, rheumatism, and malaria, also resulted in isolation of UNA [Lantana tiliaefolia and Rauvolfia vomitoria [Catharanthus Roseus and Dracocephalum komarovi, which are widely used as folk medicines in Asia, Europe, and African countries [UNA can be found in several parts of various plants used in traditional herbal medicines. According to previous studies, UNA can be extracted from fruits of  baccata ,29. In ph cancer ,30,31. Iding UNA ,33,34. Ucrocarpa ,35,36. En of UNA . It is komitoria ,39. Furtountries ,41. This50 = 7.7 μM), SK-OV-3 ovarian cancer cells (IC50 = 32.1 μM), SK-MEL-28 melanoma cells (IC50 = 22.2 μM), XF498 glioblastoma cells (IC50 = 25.9 μM), and HCT15 colon cancer cell lines (IC50 = 4.6 μM) [50 values of 5.2 μM, 4.0 μM, and 6.3 μM, respectively [50 = 23.6 μM) and H460 (IC50 = 17.6 μM) lung cancer, and Jurkat (IC50 = 23.9 μM), K562 (IC50 = 12 μM), HL60 (IC50 = 12.8 μM), and K562-Lucena 1(IC50 = 12.6 μM) leukemia cells in vitro [50 values of ULA in same cancer cells, which were 8.8 μM, 8.2 μM, and 4.7 μM, respectively [50 = 19.8 μM), L1210 murine leukemia (IC50 = 6.9 μM), SK-MEL-2 human melanoma (IC50 = 7.6 μM), and HL60 human leukemia cells (IC50 = 54.9 μM) [50 = 5.5 mM), one of targets for cancer treatment [Toona sinensis reduced the viability of several cancer cell lines and induced apoptosis of MGC-803 gastric cancer cells and PC3 prostate cancer cells [50 > 50 μM), whereas the IC50 of UNA on cancer cells ranged from 13.6 μM to 26.5 μM, suggesting that UNA can selectively reduce the survival of cancer cells while minimizing side effects against normal cells. UNA also repressed human cathepsin B for 49% at 20 μM [50 > 20 μM) and the well-known anticancer agent Cisplatin (IC50 = 10.52 μM) [Cancer is a deadly disease, representing the second leading cause of death in the United States . Major h 4.6 μM) . In otheectively . UNA alsin vitro . These rectively . Cytotox54.9 μM) ,48,49. Uer cells . Noticeaat 20 μM . Since cat 20 μM . In studat 20 μM ,54. Of nMetastasis occurs when cancer cells from primary tumors spread to other organs. Metastasis is often fatal to cancer patients, as it is responsible for approximately 66.7% of cancer deaths . The extLeishmania species, a protozoan that causes the tropical disease leishmaniasis [Leishmania mexicana through a partially noncompetitive inhibition [50 values of 3.78 µM, superior to the effect exerted by ULA (IC50 = 8.1 µM) [Trypanosoma cruzi, a parasitic protozoan known to cause Chagas’ disease [Trypanosoma cruzi was 50 µM, while the concentration of ULA was 100 µM. UNA also exhibited amoebicidal activities against Acanthamoeba Castellanii Neff (IC50 = 34.9 µM) and Acanthamoeba Griffini (IC50 = 11.1 µM), which can cause Acanthamoeba keratitis and granulomatous amebic encephalitis [50 > 100 µM) compared to ULA (IC50 = 17.7 µM) and the antiseptic agent chlorhexidine (IC50 = 7.4 µM), indicating the possibility of UNA to selectively suppresses Acanthamoeba spp. without affecting mammalian cells [Trichomonas vaginalis, which is responsible for trichomoniasis, a sexually transmitted disease (55% inhibition at a dose of 100 µM) [Plasmodium falciparum in a micromolar dosage (IC50 = 41 µM), which was slightly more active than the effect from ULA (IC50 = 53 µM) [50 values with micromolar concentrations.UNA also has the potential to treat various human diseases other than cancer . Protozomaniasis . UNA selhibition . UNA dem 8.1 µM) ,66. It w disease . A minimphalitis . This stan cells . Moreove 100 µM) . In addi= 53 µM) . It is n50 = 101 µM) more effectively than the FDA-approved antidiabetic drug acarbose (IC50 = 236.3 µM) [50 = 57 µM) [50 = 2.47 µM), and this suppression was significantly stronger than that by ULA (IC50 = 5.08 µM) and acarbose (IC50 = 573.5 µM) [50 = 8.4 µM) more strongly than UNA (IC50 = 101 µM) in vitro [Hyperglycemia, which is highly associated with diabetes mellitus and acute stress, is a condition in which cellular glucose uptake fails and blood glucose levels aberrantly increase ,71. Incr36.3 µM) . UNA inh= 57 µM) ,76. In a73.5 µM) . It was 73.5 µM) . ULA is 73.5 µM) . In somein vitro . Also, din vitro ,78. Howein vitro . In prep50 = 4.94 µM), whereas ULA showed no repressive action [50 = 0.26 mM) and exerted an anti-inflammatory effect in rat paw edema induced by carrageenan (53% of inhibition at dose of 50 mg/kg) [50 = 4.41 µM) and aspirin (28–71% of inhibition at 150–300 mg/kg), which are well-known anti-inflammatory molecules [While inflammation is responsible for numerous diseases, including arthritis, diabetes, multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease, it is also highly associated with cancer progression ,79. Antie action . Notably0 mg/kg) . These iolecules ,36. Altholecules , indicat50 = 17.6 µM) against herpes simplex virus type I was stronger than the antiviral effect of ULA (IC50 = 175.1 µM) [It was reported that several types of triterpenoids can exhibit antiviral activities ,82,83. E75.1 µM) . Another75.1 µM) . While U75.1 µM) . This re75.1 µM) . There w75.1 µM) . Moreove75.1 µM) ,88. Mole50 = 55 µM), although this effect was weaker than the effect of ULA (IC50 = 26.2 µM) [Oxidative stress, which is induced by reactive oxygen species (ROS), can affect various cellular events such as cancer proliferation, inflammation, and aging . Some st26.2 µM) . Another26.2 µM) . However26.2 µM) . These c50 = 0.037 µM), and this effect was more powerful than the activity of ULA (IC50 = 0.24 µM) [Human carboxylesterase 1 (hCE1), a serine hydrolase distributed in liver and adipocytes, is highly associated with type 2 diabetes and hypertriglyceridemia . It was 0.24 µM) .Staphylococcus aureus (82% inhibition at 100 µM), although it did not exert antibacterial activity compared to ULA [50 > 100 µM), whereas ULA significantly reduced cell viability (IC50 < 5 µM), indicating that UNA may be developed as a safer antibiofilm reagent [Biofilm is one of major factors that lowers the efficacy of antibacterial drugs. In a previous study, UNA showed antibiofilm activity of d to ULA . UNA has reagent .50 = 1.23 μM) and attenuated NO production in RAW264.7 macrophages (IC50 = 4.94 µM) [Most cellular activities involve complex signaling pathways, including apoptotic pathways and mitogen-activated protein kinase (MAPK) pathways. In intrinsic apoptotic signaling pathways, tumor suppressors such as p53 induce apoptosis via regulation of proapoptotic proteins  and antiapoptotic proteins  . MAPKs a4.94 µM) ,49. Furt4.94 µM) . This im4.94 µM) .50 = 3.6 µM), and induced cellular apoptosis, mitochondria depolarization, and cell cycle arrest. Also, 2α/2β-bromo UNA showed high solubility in water and stability in human plasma, indicating high bioavailability for in vivo tests [Despite the strong pharmacological potential of UNA, there is a disadvantage that triterpenoids, including UNA, have poor water solubility, which is one of important parameters for bioavailability parameters, such as absorption, permeation, first effects, and elimination of drugs . As UNA vo tests .50 ranging from 0.94 to 15 µM [50 from 0.71 to 10.32 µM), while the IC50 values of ULA and UNA were 5.08 and 2.47 µM, respectively [Modifications of UNA could be conducted to improve its pharmacological potential. In another work, derivatives of UNA generated by reaction with oxalyl chloride and various phenyl amines inhibited tumor necrosis factor-α (TNF-α)-induced nuclear factor-kappa B (NF-κB) activation of H460 human lung cancer cells, with values of ICto 15 µM . Some UNectively .Oher derivatives of UNA, which are synthesized by reaction with organic compounds such as potassium thiocyanate, alkyl ammonium acetate, and o-aminobenzaldehyde, are well documented ,103. HowLeishmania Mexicana, and hCE1, studies of the interactions between UNA and various proteins may facilitate understanding of the pharmacodynamics of UNA as a drug candidate. Other attempts, including molecular modifications and cotreatment with other well-known compounds, may be conducted to enhance the bioactivities of UNA.Results from previous publications demonstrated that UNA could be a strong drug candidate to treat human diseases. However, there is a limitation in that most of experiments regarding UNA were preliminary and lacked profound assays, while the pharmaceutical effects of ULA were observed from various in vivo tests and its complex mechanisms of action were well explained . Since tIn conclusion, UNA, a phytochemical from medicinal herbs, has therapeutic potentials that are similar to, or stronger than, ULA in some aspects. In particular, UNA showed strong potential anticancer, antiproliferative, and antiprotozoan effects. However, in contrast to ULA, scientific studies regarding UNA’s therapeutic activities are still lacking. Since most assays concerning UNA were conducted in vitro, additional in vivo experiments may be required to confirm these potential pharmaceutical effects. Moreover, detailed studies to elucidate the molecular and cellular mechanisms are needed to help understanding of activities exerted by UNA. Further analyses and applications may facilitate the possibility of UNA being developed in new medications to treat various human diseases.