Lentinus squarrosulus is markedly unknown. Here, nutritional profile, digestion and colonic fermentation of L. squarrosulus powder (LP) were determined. The powder contained mainly carbohydrate and protein. SEM and F-TIR analysis of the resistant hydrolysate (RH) revealed that the structure and ratio of carbohydrate and protein components were altered, and released known immunomodulation agents; beta-glucans and mannose. Both LP and RH promoted selected probiotic bacteria, especially Bifidobacterium strains. Using fecal microbiota of five volunteers , RH stimulated the microbiota of all used volunteers, via decreasing the ratio of Firmicutes/Bacteroidetes ranging from 1.3 to 8.2 times. Also, RH increased the relative abundance of vital immunomodulators; Bacteroides, Bifidobacterium, Clostridium cluster XIVa and IV, and Sutterella. Additionally, RH fermentation enriched the content of branch-chain fatty acids (BCFA) and short-chain fatty acids (SCFA), indicating protein and carbohydrate usage. Notably, propionic and butyric acids were abundant in V1, V2 and V3, while in V4 and V5, acetic and butyric acids were most enriched. Suggesting L. squarrosulus as functional mushroom to improve health and prevent diseases by enhancing gut health.Humans have long-used mushrooms as food and medicine, but digestion and colonic fermentation of most mushrooms, including  It has been shown that food matrix determines food stability during gastric and intestinal digestion, and the proportion that reach the colon or that will potentially be absorbed2. Nowadays, concerns about GI health problems including inflammatory bowel disease (IBD)3, diabetes4, cancer5, and obesity6 and liver disease7 have increased. Meanwhile, functional food components as potential therapeutic agents are being highlighted8. Edible mushrooms are rich in functional ingredients, and have since been consumed and are valued for their low calorific value, and rich compounds such as carbohydrate and protein that are indigestible but can be utilized by gut microbiota9.The gastrointestinal (GI) tract play important role in human health10. Variation in the microbiota among individuals takes place in response to diseases and environmental factors11, whereas substantial changes have been documented in response to mushroom active compounds12. Previous reports have indicated that different edible mushrooms and their functional components affect host intestinal flora and mechanisms, prospect to improve health and prevent diseases. Edible mushrooms  were reported to have a positive role in gastrointestinal tract health by producing SCFAs and regulating the intestinal microbiota12. Chang et al.13 found that Ganoderma lucidum polysaccharides produced microbiota-modulating effects and could be used as prebiotics agents to prevent obesity. While it is understood that mushrooms contain mainly carbohydrate and protein, which may synergistically affect microbiome composition, however, most reports focused on the impact of the carbohydrate fraction but not the protein. Since, people generally consumed whole mushroom14, it is important to understand the effect of gastrointestinal condition on whole mushroom matrix and the impact of the indigestible fraction on human gut microbiota.The human microbiota is dynamic and complex, and its composition is shown to differ widely across healthy individuals. The gut microbes co-evolve with individuals that provide the microbes with stable environment, while the microbes participate in a series of metabolism including digestion and fermentation of foodLentinus squarrosulus is an edible mushroom belonging to Polyporales, which is mostly consumed in Thailand, and other parts of Asia and central Africa15. This mushroom has recently been highlighted due to a wide range of bioactive properties including immunomodulation16, antimicrobial17, antiproliferative18 and antiulcer19 studied in vitro. Our recent report indicated that L. squarrosulus contains various polysaccharides with medicinal value20. Obviously, due to the rich source of functional components such as carbohydrate, dietary fiber, protein and low fat19. Notably, most of the biological activities of L. squarrosulus are linked to its carbohydrate and protein fractions. Despite the fact that many of the biological properties of this mushroom have been reported, there is no information about its digestion and colonic fermentation. Carbohydrate and protein metabolisms were considered as markers for the study. Therefore, we evaluated simulated digestion profile of L. squarrosulus powder, and the effect of its resistant hydrolysate on the fecal microbiota of different individuals. Specifically, our findings outlined the comprehensive and beneficial effects of L. squarrosulus on human gut health.L. squarrosulus are presented in Table Pleurotus; P. flabellatus (57.4%) and P. florida (63.0%) , but higher than that of Lentinula edodes (30.2%). Also, protein content of L. squarrosulus was higher than that of Lentinula edodes (17.0%), but similar to the amount reported in P. citrinopileatus (30.0%) and P. djamor var. roseus (35.5%) . L. squarrosulus exhibited fat content slightly higher than L. edodes (1.9%)21. Carbohydrate was the major component in L. squarrosulus, and crude fiber fraction was 14.81 g. This was equivalent to oyster mushrooms, which range from 11.2 to 15.0%22, and was relatively low compared with L. edodes (39.4%)21. The detergent fiber profile was analysis, which consisted of 37.26 g neutral detergent fiber (NDF), 15.62 acid detergent fiber (ADF) and 1.03 g acid detergent lignin (ADL). Cellulose content was 14.59 g and the value of hemicellulose was found to be 21.65 g. The concentration of β--glucan and α-glucan were 29.49 g and 0.55 g, respectively. Lastly, the energy value was 349.315 kcal/100 g dw or 19.5 kcal/100 g fw, which is lower compare to Agaricus bisporus , Pleurotus spp. , Lentinula edodes  and Flammulina velutipes 23. The results indicated that LP consist of essential carbohydrates and substantial amount of protein, and low fat, which can be consider as a low-calorie food. Also, the high fiber composition may greatly influence it digestion and impact on gut microbiota of human.The results of the nutritional value g/100 g) obtained for 00 g obta24. Therefore, SEM image analysis was used to observe the effect of simulated GI condition on the hydrolysate of LP particles. As evidenced by SEM images in Fig. 24. Interestingly, most of the particles retained their shapes, possibly as a result of resistant to the low pH and the enzymes activities.During digestion, the structure of food particles are altered due to disruption of aggregates and breakdown of linkages-1 , recorded in the range of 4000 to 400 cm-1 Fig. A. It was28. Also, mannose-rich exopolysaccharide (EPS) from Tremella mesenterica was reported to induce the immune system through macrophage receptors29. Thus, simulated GI conditions affected the components of L. squarrosulus powder (LP) along with an increase of potential immunomodulatory agents; β-glucan and mannose. We posit a potential stimulation of intestinal immunity during digestion; however, further study is required to affirm this claim.Moreover, principal component analysis (PCA) Fig. C showed S) and lower molecular weight proteins. Before (0 h) digestion, the reducing sugars (RS) content was 0.19 ± 0.03 mg/mL, while after digestion in the gastric juice (stomach condition), RS was 0.75 ± 0.01 mg/mL, showing 23.44% hydrolysis and 76.56% resistant of carbohydrate  in RS in the gastric juice, but slight increase (P > 0.05) in RS in the small intestine fluids. This means that glycoside bonds were destroyed in the gastric juice, possibly due to the low pH. However, pancreatic α- amylase had a limited effect on carbohydrate in the mushroom powder after passing through the gastric juice. In addition, the protein content (%) prior (0 h) to digestion was 30.84 ± 0.81, whereas after digestion, protein (12.24 ± 0.70) substantial (P < 0.05) reduced in gastric juice, and a slight reduction but no significant (P > 0.05) of protein (11.91 ± 0.68) recorded in the small intestine fluid. This indicates 58.59% hydrolysis and 41.41% resistant in the gastric juice, and 59.79% hydrolysis 40.21% resistant in the intestine fluids , and showed different growth characteristics in dependence on sample and strain specificity. As presented in Fig. L. crispatus JCM 5810 was reached at 4 h, similarly toward LP and RH but greatly reduced thereafter, especially with RH. Whereas, L. reuteri KUB-AC5 was continually enhanced by LP and RH from 4 to 18 h in a comparable trend, but better with LP. Moreover, B. bifidum TISTR 2129 showed a negative growth enhancement, while B. animalis TISTR 2194 was positively promoted after 4 h by LP and RH as shown in Fig. L. reuteri KUB-AC5, B. bifidum TISTR 2129 and B. animalis TISTR 2194 were more sensitive toward LP than RH. Also, RH promoted Bifidobacterium better than Lactobacilli. Thus, indigestible carbohydrates and proteins are important energy sources for the human gut microbiota. Hence, this information may serve as a basis to evaluate how RH can stimulate the human gut microbiota.The ability of LP and RH to enhance the growth of lli Fig. A and Bifium Fig. B was detL. squarrosulus powder in simulated human upper gut  on gut microbiota by high throughput sequencing analysis. S. chao 1 revealed the richness of the different microbiota. In Fig. p > 0.05) and lastly, V4 and V5, which seems to be comparable. Also, the richness of bacterial between the control (C) and RH treated (S) groups for each individual microbiota during 24 h fermentation varied (p < 0.05). RH advanced bacterial richness in V1, V2 and V4 at 12 h, and also, in V5 at 12 h and 24 h. Whereas, in V3, RH slightly improved bacterial richness at 24 h. The supplement of RH might support the growth of the least populated bacterial group, which could improve bacterial richness of individual gut microbiota. The Shannon and Simpson indexes signified the diversity of the various microbiota. As shown in Fig. 31. Among individuals, V2 exhibited the most rich and diverse bacterial composition and afterwards, V3 displayed the most bacterial richness and diversity. V1 showed more bacterial richness but low diversity when compared to V4 and V5. According to previous report, a healthy human colon microbiota yields a relatively high rich and diverse abundance of microbes31. In this study, V2 and V3 may have better or more resilience gut microbiota than V1, V4 and V5. Interestingly, RH improved bacterial richness in all the individuals but reduced bacterial diversity in V1, V2 and V3. Indicating that RH may have a significantly impact on both weak and resilient gut microbiota.The study investigated the effects of resistant hydrolysate (RH); sample (S) obtained after digesting Also, the microbiota structure and composition between the control and RH groups were analyzed by NMDS-Jaccard distance and NMDS- wunifrac distance. As shown in Fig. Proteobacteria (p = 0.00), Bacteroidetes (p = 0.001), Firmicutes (p = 0.00), Actinobacteria (p = 0.00) and Fusobacteria (p = 0.208) with significant differences among the five volunteers  and Firmicutes (18.04%). V2 was abundant in Firmicutes (60.08%) and Bacteroidetes (18.84%). V3 composed of mainly Firmicutes (70.25%) and Bacteroidetes (13.13%). V4 consisted of Firmicutes (48.68%) and Bacteroidetes (42.32%). V5 was made-up of comparable abundant of Firmicutes (32.95%), Bacteroidetes (30.28%) and Proteobacteria (31.22%). Actinobacteria was significantly different (p = 0.000), which was abundant in V2 (3.98%) and V1 (2.51%) but significantly lower and similar among V3, V4 and V5. Whereas, Fusobacteria abundant detected was not significantly different (p = 0.208) among the volunteers. Additionally, Firmicutes/Bacteroidetes ratio for V1, V2, V3, V4 and V5 was 4.7, 3.2, 5.3, 1.1 and 0.9, respectively, which were significantly different (p = 0.00). This means that V1, V2 and V3 may have more risk of obesity than V4 and V5.The structures of the gut microbiota of five volunteers  were investigated to compare the effects of resistant hydrolysate (RH) on the intestinal microbes. Figure Bacteroidetes in the gut microbiota of V1 and V2 (p < 0.05)  but showed a trend of decreasing in RH treated groups  reduced the ratio of Firmicutes/Bacteroidetes (F/B)  in gut microbiota of all volunteers   Proteobacteria abundance in V3 and V5 when compared to the control after 24 h. according to previous reports, the Firmicutes/Bacteroidetes ratio is linked with obesity, and was positively associated with a reduced energy harvest. However, a reduction of Firmicutes/Bacteroidetes signified intestinal health and weight loss32. Also, in another report, increasing the abundance of species of Proteobacteria was recognized as a potential microbiological characteristic of diseases, such as inflammatory bowel disease (IBD), metabolic disorders and lung diseases33. Indicating that RH improved the gut microbiota of all the volunteers by decreasing F/B ratio and Proteobacteria abundance. Hence, L. squarrosulus powder (LP) has a potential to stimulate the gut microbiota of human and may reduce the risk of obesity and control gut related diseases.In comparison with the control during fermentation, RH addition significantly increased the relative abundance of 2 p < 0.0  the relative abundance of Phascolarctobacterium and Prevotella in the gut microbiota of all volunteers  compared to the control. Phascolarctobacterium reduction occurred at all time points in V1, V2, V3 and V5, but at 12 h and 24 h in V4. Whereas, Prevotella decreased after 24 h in V1, V3 and V5 but at 12 h and 24 h in V4. Also, the relative abundance of Coprococcus and Catenibacterium significantly decreased in RH group of V1, V2, V3 and V5. While, Gemmiger significantly decreased in V1, V2, V3 and V4. Again, the relative abundance of Faecalibacterium and Megamonas significantly declined in RH groups. Faecalibacterium decreased in V2, V3 and V5, while Megamonas reduced in V2, V3 and V4.A total of 92 kinds of genera Fig. A were idsis Fig. B. As it p > 0.05) increased in RH treatment groups combined, compared to the control groups. The relative abundance of Bacteroides, Bifidobacterium, Clostridium cluster XIVa and IV, and Sutterella were significantly (P > 0.05) improved in the gut microbiota of all volunteers but at different time points. Bacteroides increase occurred at 6 h and 12 h of fermentation in V1, 6 h and 24 h in V2, 12 h in V3, and 6 h in V4 and V5. Bifidobacterium was improved only at 6 h in V2 microbiota, and 24 h in the gut microbiota of V1, V3, V4 and V5. Clostridium cluster XIVa and IV were enriched at 6 h and 24 h fermentation in V2 and V5, 12 h and 24 h in V1, V3 and V4. High abundance of Sutterella was observed at all time points in V1, V3, V4, but at 12 h in V2 and at 12 h and 24 h in V5. Also, the relative abundance of Dialister were significantly enriched (p < 0.05) in four volunteers at different time points. Dialister was significantly enriched at 12 h fermentation of RH in V1 and at 24 h in V2, V3 and V5. Again, RH significantly increased the abundance of Ruminococcus, Desulfovibrio, Collinsella and Citrobacter in three of the volunteers. While Ruminococcus and Lachnospira were improved in V1, V4 and V5, Citrobacter in V1, V2 and V3, Desulfovibrio in V1, V2 and V5, Lactococcus after 24 h in V2 and V4. Moreover, RH significantly enriched abundance of Streptococcus in V3 and V4, Collinsella in V4 and V5, Oscillospira in V1 and V4. Fucobacterium 24 h fermentation of RH in V2 and 12 h in V4. Furthermore, RH significantly improved Megamonas and Mitsuokella in V1 and V5, Enterococcus in V2, Roseburia, Blautia, Lactobacillus, Catenibacterium and Eubacterium in V4, and Veillonella in V5. In particular, the relative abundance of Bacteroides  in murine gut, correlated negatively with SARS-CoV-2 load in fecal samples taken from patients35.Bifidobacterium as an acid-producing bacteria, is a commonly used probiotic that contribute importantly to host health36. Bifidobacterium has a potential to inhibit pathogenic microorganisms in infants37, protect the host from inflammatory diseases38, and improve disease symptoms in mice of the T-cell transfer colitis mode39. Also, in recent report, Bifidobacterium has known immunomodulatory bacterial remained low in COVID-19 patients, even after disease resolution40. RH enriching Bifidobacterium in the gut microbiota of all volunteers may release metabolites, activate immunomodulatory pathway, and finally reduce the risk of diseases.Bifidobacterium initiate oligofructose degradation, with a relation production of acetate, while some of the Clostridium cluster XIVa and IV bacteria in the colon are capable of degrading simultaneously oligofructose and associated consumption of acetate, resulting in the production of butyrate. Also, Clostridium cluster XIVa are capable of converting lactate to butyrate. In this work, Clostridium cluster XIVa and IV were enhanced in the gut microbiota of all volunteers when RH was added. This signified cross-feeding, which indicates direct stimulation of butyrate-producing colon bacteria in these hosts by RH.It was found that Dialister, together with Coprococcus were reported depleted in people with important indicators of depression41. We observed RH improved the abundance of Dialister in the microbiota of four volunteers, V1, V2, V3 and V5. Suggesting that RH may potentially help to condition human gut microbiota to fight depression. However, further research is needed to confirm this claim.Ruminococcus, Desulfovibrio, Collinsella, Citrobacter, Streptococcus and Mitsuokella in at 3 volunteers, while Lactococcus, Oscillospira, Collinsella, Fucobacterium, Eubacterium were improved in at least 2 volunteers. Other genara, Megamonas, Roseburia, Blautia, Lactobacillus and Catenibacterium were enhanced in at least 1 volunteer. Some of these groups such as Roseburia, Lactobacillus, Megamonas42, Oscillospira43 and Lactococcus44 have been reported to contribute to host health. Streptococcus45 and Eubacterium46 were indicated to confer beneficial effects on obesity and inflammation. The results showed that RH produced positive changes in the gut microbiota, but these depend on the initial composition of a volunteer’s gut microbiota. Notably, RH enhanced the relative abundance of known immunoregulatory bacteria, such as Bacteroides, Bifidobacterium and Clostridium cluster XIVa and IV. This signifies a potential application to stimulate an altered gut microbiota to reduce gut related diseases.In addition, RH promoted Bacteroidetes, Proteobacteria, Firmicutes and Actinobacteria  effect size (LEfSe) was performed to illustrate the differences in the microbial profiles of the gut microbiota of volunteers between control (A) and RH treated group (B). All the volunteer microbiota in the control and RH group were mainly consisted of ria Fig. . Based op < 0.05) increased by the addition of RH at different time points compared to the control. Hence, RH influenced the production and richness of SCFAs and BCFAs metabolites. However, the increased in richness of lactic acids, SCFAs and BCFAs were dependent on volunteer and time . After fermentation, lactic acid richness did not vary significantly (p > 0.05) among volunteers at various time points. Lactic acid concentration (mM/mL) fluctuated between 0.059–0.387, −0.295–0.267, −0.448–0.228, 0.014–0.145 and −0.173–0.344 for V1, V2, V3, V4 and V5, respectively. The increased lactic acid during RH fermentation was stable in V1 and V4 than the others. Meanwhile, the richness of SCFAs including acetic, propionic and butyric acids during RH fermentation differed significantly among volunteers and at various time points for each volunteer. The concentration (mM/mL) of SCFAs ranged between 5.6–24.01, 0.25–33.17, 4.56–39.60, 3.45–32.84 and 0.71–15.58 for V1, V2, V3, V4 and V5, accordingly. Notably, propionic was the most increased SCFA in V1, V2 and V3, while acetic acid was the most enhanced in V4 and V5. The proteolytic fermentation produces less SCFAs, but also branched-chain fatty acids BCFAs, including isovaleric and iso-butyric acids47. In this study, the richness of BCFAs, consisting isovaleric and iso-butyric acids did not differ significantly among volunteers during fermentation of RH. The BCFA concentration (Mm/mL) ranged between 1.28–2.38, 0.16–1.99, 0.38–1.69, −1.15–1.59, −0.02–4.61 for V1, V2, V3, V4 and V5, respectively.Lactic acid, short-chain fatty acids (SCFA) and branch-chain fatty acids (BCFA) concentrations during 24 h fermentation of resistant hydrolysate (RH) of fecal inocula from five healthy volunteers were evaluated. The RH was fermentable in the gut microbiota of all volunteers. Propionic, acetic, lactic, butyric, isovaleric and iso-butyric acids were detected in RH and control treatments Fig. A. IntereMoreover, in order to better visualize the influence of RH on production of SCFAs in gut microbiota of volunteers, PCA-Biplot analysis was employed. The biplot for first and second components showed the impact of RH on individual acids in the excreta of five volunteers at 3 h, 6 h, 9 h, 12 h, 18 h and 24 h fermentation  group was abundance in Clostridium, Megamonas and Bacteroides in V1S6h, Clostridium, Dialister and Bacteroides in V1S12h, and Bacteroides and Clostridium in V1S24h. Volunteer 2 (V2) group was abundance in Clostridium, Bifidobacterium, Escherichia and Bacteroides in V2S6h, V2S12h and V2S24h. Volunteer 3 (V3) group was abundance in Clostridium, Escherichia and Bacteroides in V3S6h, V3S12h and V3S24h. Volunteer 4 (V4) group was abundance in Clostridium, Escherichia and Bacteroides in V4S6h, Clostridium, Bifidobacterium, Escherichia and Bacteroides in V4S12h, and Clostridium and Bacteroides in V4S24h. Volunteer 5 (V5) group was abundance in Clostridium and Bacteroides in V5S6h, V5S12h and V5S24h. Notably, V1S6h and V1S12h showed a positive correlation with acetic and propionic acids, while V1S24h also showed a very strong positive relation with these acids. In V2 group, V2S6h showed a strong positive correlation with acetic and propionic acids, while V2S12h showed acetic, propionic and butyric acids. V2S24h had a strong positive correlation with acetic acid but an extremely strong correlation with propionic acid. In V3 group, V3S6h positively correlated with acetic and propionic acids, likewise V3S12h positively linked with acetic acid but with an extreme correlation with propionic acid. V3S24h showed a very strong correlation with acetic and propionic acids. In V4 group, V4S6h had a strong positive relation with acetic and propionic acids, while V4S12h showed a very strong correlation with acetic and butyric acids. V4S24h had an extreme positive correlation with acetic and with a strong positive correlation with propionic and butyric acids. Moreover, V5S6h had a strong positive correlation with acetic acid and a very strong positive correlation with butyric and propionic acids, while V5S12h showed a very strong positive correlation with acetic, isovaleric and propionic acids. Whereas, V5S24h only showed a very strong positive correlation with acetic acid. Notably, high abundance of Clostridium in V1, V2 and V3 from 12 to 24 h correlated strongly with propionic acid richness. Whereas, its less abundance in V4 and V5, correlated strongly with acetic acid. This means Clostridium might use acetic acid as precursor to produce other acids, which is in line with gut microbiota composition analysis. In general, RH enhanced the growth of beneficial bacteria along with the production of SCFAs and BCFAs known to ensure a balanced gut microbiota, but influenced by the baseline microbiota of the individuals.As clearly shown in Fig. L. reuteri KUB-AC5, B. bifidum TISTR 2129 and B. animalis TISTR 2194 were significantly enhanced but were better with LP. RH was fermented by the fecal microbiota of five volunteers. RH significantly decreased Firmicutes/Bacteroidetes ratio in V1, V2, V3, V4 and V5 by 2.9, 8.2, 5.4, 2.3 and 1.3 times, respectively. Also, the relative abundance of Bacteroides, Bifidobacterium, Clostridium cluster XIVa and IV, and Sutterella were enriched by RH, which was dependent on time. Moreover, RH fermentation enriched Lactic acid, SCFAs and BCFAs concentrations, indicating that carbohydrate and protein were utilized. The results showed that Lactic acid and BCFA enrichments were comparable among volunteers. However, propionic, acetic and butyric acids as major SCFAs produced varied significantly among volunteers. The gut microbiota of V1, V2 and V3 used RH to produce more of propionic acid and less butyric acid, while V4 and V5 fermentation resulted in more of acetic and butyric acids. In addition, the abundance of Bacteroides and Clostridium in the microbiota of V1, V2 and V3 revealed strongly correlations with propionic and acetic acids. Whereas, in the microbial ecology of V4 and V5, the abundance of Bacteroides, Clostridium, Bifidobacterium showed significant correlation with the production of propionic, butyric and acetic acids. The results indicated that most of the LP could escape GI digestion and reach the colon to regulate the composition of gut microbiota and SCFAs of different individuals depending on their baseline microbiota.GI conditions altered the structure and ratio of micronutrients in LP, and 71.67% carbohydrate and 40.41% protein of RH could reach the colon safely. With fermentation by probiotic bacteria, L. squarrosulus powder (LP) that passed through a 40-mesh sieve.Whole mushroom fruiting bodies were washed thoroughly to free them from mud, ferns and other extraneous material. White and gray fruiting bodies were dried in an oven at 60 °C and then pulverized into powder using a laboratory blender to produce Moisture, fat and ash content were determined by the procedure of AOAC method 925.45A, 922.06 and 920.153, respectively (AOAC 2016). Carbohydrate composition was measured according to In-house method TEC-CH-169 based on Compendium of Methods for Food Analysis, Thailand, 1st Edition, 2003. Protein was analyzed by In-house Method TE-CH-042 based on AOAC (2016) 981.10. Neutral detergent fiber (%NDF), Acid detergent fiber (%ADF), Acid detergent Lignin (%ADL), cellulose (%C), hemicellulose (%HC) and crude fiber (%CF) were obtained using the method by Van Soest, (1963). Energy was estimated using the following equation: Energy  = 4 (g protein) + 3.75 (g carbohydrate) + 9 (g fat).24. Mushroom powder was hydrolyzed sequentially with pepsin and pancreatic α-amylase  by simulating gastric and small intestine conditions. Approximately 1 g LP was combined with 50 mL of HCl buffer (pH 1) containing; (g/L); NaCl, 8.00; NaH2PO4, 14.35; KCl, 0.20; CaCl2, 0.10; MgCl2.6H2O, 0.18; NaHPO4.2H2O, 8.25. The pH was adjusted using 5 M HCl and pepsin was added to obtained final concentration of 20 units per mL and incubated for 4 h. An aliquot, 5 mL was taken to new tube at 0 and 240 min. The pH was adjusted to 6.9 using 1 M NaOH following the addition of porcine pancreatic α- amylase solution to obtained final concentration of 1units/mL and further incubated for 6 h. An aliquot of 5 mL was taken to new tubes at 6 h. Total sugar and reducing sugar contents were determined using phenol sulfuric acid and DNS methods, respectively56. Percentage hydrolyzed (H%) was estimated by the ratio of reducing sugar released to total sugar content based on the equation below57. Protein content before and after digestion were also determined by Kjeldahl Method58.In vitro enzymatic hydrolysis of LP was performed using the method described byThe hydrolysates were transferred into a dialysis tube , and dialyzed against water at 4 °C and then freeze-dried to obtain resistant hydrolysate (RH).L. squarrosulus powder (LP) and the resistant hydrolysate (RH) were visualized. A High Resolution (HR) SEM  was used as previously reported24. Samples were placed on double adhesive tape fixed on a metallic stub, coated with gold and viewed at 1200 × resolution.The morphological characteristics of 20. The measurements were made by synchrotron radiation (SR)-FTIR micro-spectroscopy  at beamline 4.1 at the Synchrotron Light Research Institute (SLRI), Nakhon Ratchasima 30,000, Thailand. The Samples were completely dried and grounded. Spectra collection were performed using OPUS8.0 software . Spectra acquisition were obtained with 36X objective lens using the transmission mode. All spectra were record from 4000 to 400 cm−1 with diamond compression cell at an aperture size of 20 × 20 µm; spectral resolution of 4 cm−1; 64 scans for background and sample.Spectro-microscopic measurements of carbohydrate, protein and fat fingerprint regions of LP and RH were achieved by a recently used procedureLactobacillus ssp. (L. reuteri KUB-AC5 and L. crispatus JCM 5810), Bifidobacterium ssp.  following a reported method 59. Lactobacillus strains were cultivation in MRS medium at 37 °C under aerobic condition. Whereas, Bifidobacterium strains were grown in MRS-0.005 L-cysteine HCL broth at 37 °C anaerobically. Overnight cultures, adjusted to 0.5–0.6 OD absorbance at 600 nm were used to inoculate (1%) sterilized 10 mL appropriate broth containing 1% LP or RH or no sample (control). Enhanced activity (%) of Lactobacillus strains were evaluated at 4, 8 and 16 h, while Bifidobacterium strains were assessed at 6, 12 and 24 h using spread plating method on MRS agar plate.LP and RH were tested on selected probiotic bacteria; Fresh fecal samples were collected from five healthy volunteers  who had not taken antibiotics at least for 3 months. All volunteers provided written informed consent prior to specimen collection. Immediately following collection, each fecal sample was stored in an anaerobic culture swab and used within 2 h. The study was performed and approved in accordance with the guidelines of Kasetsart University Ethics Committee on Human Research. All methods in this study were in accordance with relevant guidelines and regulations of the Declaration of Kasetsart University Ethics Committee.2HPO4, 40 mg KH2PO4, 10 mg MgSO4, 10 mg CaCl2, 2 g NaHCO3, 25 mg hemin, 0.5 g cysteine-HCl, 0.5 g bile salt, 4 mL resazurin; 0.05 mg/mL stock, 2 mL Tween 80 and 20 μL vitamin K1 into 1 L distilled water and adjusted the pH to 7.0 by using 0.1 mol/L HCl solution. After the sterilization, the media was bubbled with CO2 to remove the O2 in solution and cool down to 3 7 ºC before use. This was performed in accordance with the reported method60 with minor modifications.Fecal samples were homogenized with sterilized 0.1 M phosphate buffered saline, pH 7.0 to obtain 10% (w/v) suspension. The basal nutrient medium was prepared by adding 1.2 g peptone, 1.2 g yeast extract, 100 mg NaCl, 40 mg KWater-jacketed fermenter containing basal medium was used as control group, and fermenter in other vessel supplemented with 1% resistant hydrolysate (RH) was used as treatment group. Then, the vessels were magnetically stirred with temperature control at 37 °C and pH maintained at pH 6.65–6.95. Anaerobic conditions were kept by sparing the vessels with oxygen-free nitrogen gas at 15 mL/min. The fermented samples for further analysis were collected at 0, 3, 6, 9, 12, 18 and 24 h fermentation.2SO4 in HPLC-grade water, and the flow rate was 0.6 mL/min. Quantification of metabolites in the samples was carried out using calibration curves of lactate, acetate, propionate, iso-valerate, iso-butyrate and butyrate at concentrations of 1.0, 10, 20, 40, 80 and 100 mM. Tartaric acid  at a final concentration of 20 mM was used as the internal standard. The results were express as mmol/mL.The samples collected during fermentation were centrifuged and the supernatants were filtered using a 0.22-μm filter unit . Twenty microliters were then injected into an HPLC system  equipped with a UV detector (Waters 2489 UV visible detector). An ion-exclusion Aminex HPX-87H column , set at 50 °C was employed. The mobile phase was 8 mM H47. The bacterial pellets were collected by centrifugation and transferred into 2 mL lysate tubes containing zirconium beads and inhibitEx was adding prior to agitation. The cell suspension was centrifuged and 400 µL of the supernatant was combined with 400 µL of buffer AL and, 17 µL of proteinase K in a new tube. The content was vortexed and incubated at 70 °C for 10 min, and allowed to cool down at room temperature. The lysate was added with 400 µL of ethanol and mixed by vortex. A 600 µL of the content was repeatedly applied to QIAamp spin column until all lysate was loaded. And then 500 µL of buffers, AW1 and AW2 were respectively added and centrifuged. After that 200 µL buffer ATE (elution buffer) was pipetted directly onto the QIAamp membrane. The DNA concentration was determined using a NanoDrop 1000 .The bacterial DNA was isolated using QIAamp DNA Stool Mini Kit https://github.com/lh3/seqtk). The processed pair of sequences were denoised, then merged into amplicon sequence variants (ASVs) using DADA2 pipeline (v.1.10) with pseudo-pooling mode. We assigned a taxonomy label to each ASV with QIIME2’s sklearn classifier (v.2020.2) and Greengenes (v.13.8)61. We exclude ASV that either has no phylum label and a singleton from the analysis. Alpha diversity and Beta diversity were computed with vegan (v.2.5–6)62 and GUniFrac (v.1.1)63.The microbiota was analysed by MiSeq sequencing. DNA integrity was detected by the agarose gel electrophoresis. The genomic DNA was used as the template to amplify the V3–V4 regions of 16S rDNA with the forward primer (Illumina adapter sequence 1 + CCTACGGGNGGCWGCAG) and the reverse primer (Illumina adapter sequence 2 + GACTACHVGGGTATCTAATCC) and then specific index sequence was added. The PCR products were purified for library construction. High throughput sequencing was performed on the Illumina MiSeq platform  to generate 2 × 250 bp paired-end reads. The reverse reads were right truncated from the right to 240 bps as a quality control measure. After that the primers at the 5’ of reads were trimmed using seqtk  and treatment (S/RH) of five fecal samples taken at six periods  for metabolites (SCFA and BCFA) concentrations, and at four periods  for bacterial populations. Differences between bacterial groups were computed using T-test and among individuals were achieved using one-way ANOVA for continuous data in SPSS. Shannon, Simpson and S. chao 1 index were used to estimate the community diversity. A Bray–Curtis and Unifrac similarity matrix was derived and a permutational analysis of variance (PERMANOVA) pair wise comparison was conducted to compare all community samples. A


Moringa oleifera seeds-removed ripened pods (SRRP) were used for papersheet production and for the extraction of bioactive compounds. Fibers were characterized by SEM–EDX patterns, while the phytoconstituents in ethanol extract was analyzed by HPLC. The inhibition percentage of fungal mycelial growth (IFMG) of the treated Melia azedarach wood with M. oleifera SRRP extract at the concentrations of 10,000, 20,000, and 30,000 µg/mL against the growth of Rhizoctonia solani and Fusarium culmorum was calculated and compared with fluconazole (25 µg). The produced papersheet was treated with the ethanol extract  and assayed for its antibacterial activity against Agrobacterium tumefaciens, Erwinia amylovora, and Pectobacterium atrosepticum by measuring the inhibition zones and minimum inhibitory concentrations (MICs). According to chemical analysis of M. oleifera SRRP, benzene:alcohol extractives, holocellulose, lignin, and ash contents were 7.56, 64.94, 25.66 and 1.53%, respectively, while for the produced unbleached pulp, the screen pulp yield and the Kappa number were 39% and 25, respectively. The produced papersheet showed tensile index, tear index, burst index, and double fold number values of 58.8 N m/g, 3.38 mN m2/g, 3.86 kPa m2/g, and 10.66, respectively. SEM examination showed that the average fiber diameter was 16.39 µm, and the mass average of for elemental composition of C and O by EDX were, 44.21%, and 55.79%, respectively. The main phytoconstituents in the extract (mg/100 g extract) by HPLC were vanillic acid (5053.49), benzoic acid (262.98), naringenin (133.02), chlorogenic acid (66.16), and myricetin (56.27). After 14 days of incubation, M. oleifera SRRP extract-wood treated showed good IFMG against R. solani (36.88%) and F. culmorum (51.66%) compared to fluconazole, where it observed 42.96% and 53.70%, respectively. Moderate to significant antibacterial activity was found, where the minimum inhibitory concentration (MIC) values were 500, 650, and 250 µg/mL against the growth of A. tumefaciens, E. amylovora, and P. atrosepticum respectively, which were lower than the positive control used (Tobramycin 10 µg/disc). In conclusion, M. oleifera SRRP showed promising properties as a raw material for pulp and paper production as well as for the extraction of bioactive compounds.In the present study, and for the waste valorization,  Moringa oleifera Lam. (family Moringaceae) is a fast-growing and drought-resistant tree, native to the Indian subcontinent with multipurpose uses1. Fruits of Moringa are three-sided pods with pendulous and linear shape, also, the pod generally has 250–450 mm long contains approximately 20 globular seeds2. From the literature survey, all the works are concentrated in how to use leaves, flowers, pods and roots of Moringa in different purposes5. Leaves and seeds of M. oleifera are promised as a first stage in the treatment for waste waters8 or for coagulant of primary treatment of paper mill effluent9. Acid activated from M. oleifera leaf was also prepared, which act as a good alternative adsorbent for dyes and heavy metal recoveries from aqueous solutions10. Petals of M. oleifera were used as a mediated green synthesis of gold nanoparticles11. Leaves and other parts from the tree were used as a source for antimicrobial and antioxidant agents as well as for pharmaceutical purposes14. In livestock application, leaves and seeds of M. oleifera are used for animal nutrition, where they have many nutritional compounds such as oils, carbohydrates, vitamins, fatty acids, amino acids, lipid, minerals and other chemical compounds16..2 and Trigo et al.17. Quercetin, myricetin glycosides, caffeoylquinic acid, coumaroylquinic acid, hydroxybenzoic acid, kaempferol, glucotropaeolin, glucosinalbin, glucoraphanin, glucomoringin, glucoiberin, glucosinolates, apigenin, luteolin, lutein, luteoxanthin, zeaxanthin, b-carotene and isothiocyonates were identified as the main compounds in the extracts from moringa19. Phenolic compounds from M. oleifera seed, including gallic acid, ellagic acid and kaempferol were observed good antioxidant activity21.Several bioactive compounds were isolated and identified from different parts of Moringa  and were summarized in the review articles of Chhikara et alM. oleifera, there are little works from the literature, i.e., kraft pulping yield of M. oleifera and M. concenensis (M. concanensis) stems showed satisfactory strength properties for wrapping and writing papers compared to those of conventional raw materials22. Also, some investigations showed that the fiber characterizations such as fiber length and diameter of M. oleifera stem indicated that stem-wood from the middle and base was best suited for pulp and paper production23, while among the collected stems from 1, 3 and 5 year olds Moringa oleifera, the fiber characteristics from 5 year old M. oleifera stem-wood showed the best suited for the production of pulp and paper24.For the production of pulp and paper from M. oleifera seeds-removed ripened pods in the production of papersheet and as source for bioactive compounds for antibacterial and antifungal activities.To the best of our knowledge, this is the first work showing the value-added of Moringa oleifera Lam. seeds-removed ripened pods (SRRP) were collected from Alexandria, Egypt, 2020. The plant was identified at the Department of Forestry and Wood Technology, Faculty of Agriculture, Alexandria University and a sample was deposited (voucher number Zidan0077). The SRRPs were ground into powder and screened (size 40–60 mesh), and then 100 g of this powdered size were extracted with ethanol (200 mL) by soaking method for 3 days25, where every day it was agitated at least three times for 5 min, and it should be noted that every day the amount of ethanol was replaced with the another amount (200 mL), therefore we used 600 mL ethanol for three days extraction. The extracted material was filtrated using Whatman filter paper no. 1 to get rid of residues and the dissolved extract was concentrated by evaporating the solvent using the rotary evaporator.This study is complied with relevant institutional, national, and international guidelines and legislation. This study does not contain any studies with human participants or animals performed by any of the authors, where Fusarium culmorum (Acc# MH352452), and Rhizoctonia solani (Acc# MH352450), were used for the bioassay28. Melia azedarach wood specimens (2 × 1 × 0.5 cm), that autoclaved (121 °C for 20 min) and left to cool, were treated with M. oleifera SRRP extract at the concentrations of 10,000, 20,000, and 30,000 µg/mL. Each wood sample was received 100 µL from each concentration of M. oleifera SRRP extract. Petri dishes contained PDA media were inoculated with 5 mm-disc diameter of each fungus and the treated wood samples were put directly over the media at the opposite side of the fungus disc30. The treated wood samples were compared with control treatment (autoclaved-untreated). The percentage of fungal inhibition was calculated with the formula of the inhibition percentage of fungal mycelial growth (IFMG %) = [(T0 − T1)/T0] × 100, where T0 and T1 are the average diameters (mm) of fungal colonies under the control and experimental treatments, respectively, after insuring that the growth of fungi in control treatment, the measurement was done according to the previous works33. The IFMG values were compared with the positive (25 µg of fluconazole) and negative (10% DMSO) controls34.Two fungi M. oleifera SRRP extract. Separation conditions can be found in previously published works38. The following standard phytochemical compounds with HPLC grade  were used; catechol, p-hydroxy benzoic acid, caffeine, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, benzoic acid, rutin, ellagic acid, o-coumaric acid, salicylic acid, cinnamic acid, myricetin, quercitin, rosmarinic acid, naringenin and kaempferol.HPLC 1260 Infinity Agilent System  equipped with a Quaternary pump and a Zorbax Eclipse Plus C18 column (100 mm × 4.6 mm i.d.) operated at 30 °C was used to identify the phytochemical compounds in Moringa oleifera SRRP , holocellulose, insoluble lignin content and Ash content were measured according to T204, T249, T222 om88, and T211, respectively.RRP Fig. a was colRRP Fig. b. For ch, 200-g oven-dried pieces of M. oleifera SRRP were swelled for one day, filtrated, washed several time with hot water. Kraft pulping was conducted in stainless steel vessel with capacity 2 L under rotation in oil bath. The conditions used for pulping of M. oleifera SRRP were: active alkalinity (11%), temperature (160 °C), reaction time (35 min) and the liquor ratio (liquid to M. oleifera SRRP ratio of 10:1). The solid residue was defibrated, washes with hot and cold water till neutral pH, and the resulted pulp  with grammage of about 60 g/m2 were obtained43. Papersheets were made and tested for the strength properties according to TAPPI test methods T218 and T220. The papersheets were tested for tensile resistance T404, tear strength T414, bursting strength T405 and double fold T423. Analysis of physical strength of pulp was performed according to TAPPI standard methods with sheets standard 60 g/m2. All the experimental works were performed in triplicate.Papersheet forming Fig. d was carM. oleifera SRRP pulp were studied by scanning electron microscopy (SEM), attached with energy dispersive spectrometry (EDX), JFC-1100E ion sputtering device  with acceleration voltage of 20.00 kV to show the elemental compotation and diameter of the fibers from three points and the average was taken. The measurements were taken from three parts of the paper sheets46.The produced papersheets Fig. d from M.M. oleifera SRRP pulp paper treated with three concentrations  from M. oleifera SRRP extract as well as the control treatment (DMSO 10%)25. Three plants pathogenic bacteria Agrobacterium tumefaciens (acc# MG706145), Erwinia amylovora (acc#LN876573) and Pectobacterium atrosepticum (acc#MG706146), were used for the antibacterial activity and were previously identified through molecular identification51. The agar disc diffusion method was employed for antibacterial activity determination of the extract by recording the inhibition zone52. All tests were performed in triplicate. Also, micro-dilution method with serial concentrations of 32–1000 µg/mL was measured and compared with the control (Tobramycin 10 µg/disc)53.Discs with approximate dimension of 1 × 1 cm were cut from the M. oleifera SRRP pulp paper were recorded as mean ± SD from three measurements. The measurements of antifungal and antibacterial activities were statistically analyzed with one way ANOVA using SAS system and comparisons among the means were recorded using LSD test at an alpha value of 0.0554.Tensile index, burst index, tear index, double fold number, brightness and optical measurements from the tested papersheet produced from This study is complied with relevant institutional, national, and international guidelines and legislation. “This study does not contain any studies with human participants or animals performed by any of the authors”.M. oleifera SRRP and the produced unbleached pulp are shown in Table M. oleifera SRRP is 64.94%, which indicates that it would be good sources of cellulose and hemicellulose. Furthermore, this content is well-compared with those reported by other studies, where the holocellulose content in M. oleifera stem was 65.5%55. While it was lower than those from other non-woody materials, i.e., Sorghum bicolor stalks (71.0%)56, Musa sapientum (73.43%), M. paradisiaca (72.60%) and Tithonia diversifolia (71.60%)57, bamboo (70.50%)58, Tunisian Alfa stems (68.2%)59, Date palm rachis (74.8%)61, Hesperaloe funifera (76.5%)62, Cotton stalks (72.9%)63, Canola straw (77.5%)64, Luffa cylindrica (83.0%)65, Hibiscus cannabinus (81.1%)66, Arundo donax (70.2%)63, and flax plant (70%)46. While it was higher than those from Zea mays stalks (62.33%) and Sorghum bicolor stalks (63.40%)67, lotus leaf stalks (53.8%)68, and Posidonia oceanica (61.8%)60.Chemical characteristics of M. oleifera SRRP was lower than the amount presented in Paulownia elongota wood (75.74%)69, Pinus pinaster wood (69.6%)63, Albizia lebbeck wood (78.60%)70, Eucalyptus globulus wood (80.5%)63, Acer rubrum wood (67.4%)71, Leucaena diversifolia wood (77.9%)63, and depithed Bagasse (72.38%)72. While it was higher than from those of Prosopis alba wood (63.6%)63, E. camaldulensis wood (56%) and Meryta sinclairii wood-branch (61%)25, and woods from Bougainvillea spectabilis (54.56%), Ficus altissima (54.73%), and F. elastica (53.37%)67.Comparing to the woody materials, holocellulose content in M. oleifera SRRRP was lower than from the reported in M. oleifera stem (20.5%)55. While it was equal to those found in lotus leaf stalks (25.4%)68, and were higher than those from rice husks (21.98%)73, rice hulls (20.44–23.33%)74, sugar beet (17.67%)75, stalks of Zea mays (19.9–20.1%)76, Sweet sorghum (21%)77, Corn stover (19%)78, Tall fescue (14.0%), and Miscanthus giganteus (17.8%)79, bamboo (24.5%)58, H. funifera (7.3%)62, Cotton stalks (21.4%)63, Canola straw (20.0%)64, Luffa cylindrica (15.2%)65, Kenaf (12.7%)66, Wheat straw (19.64%)80, A. donax (22.3%)63, flax plant (6.8%)46, depithed Bagasse (20.03%)72, Bagasse (23.33%)81, Cynara cardunculus stalks (16–19%)83 and Miscanthus × giganteus stalks (13%)84. While it was lower than amount from Nut shells (30–40%)85.Lignin content (25.66%) in M. oleifera SRRP was in the range of hardwood species (25–35%)86, i.e., in Albizia lebbeck wood (25.14%)70 and lower than those from Date palm rachis (27.2%)61, and Posidonia oceanica (29.8%)60. Compared to woody plant materials, it was lower than those from Pinus pinaster (26.2%)63, Acer rubruma (26.0%)71, and E. camaldulensis (27%)25, and higher than those from E. globulus (20.0%)63, L. diversifolia (19.1%)63, P. alba (19.3%)63, and M. sinclairii (23%)25.The content of lignin from M. oleifera SRRP (1.53%) was lower than the amount in stem (3.5%)55, while the Alcohol-benzene solubility (7.56%) was higher from the measured in the stem (3.16%)55.The ash content in M. oleifera SRRP pulp , and Kappa number (16.2–21.7)55.The unbleached lp Table  showed tM. oleifera SRRP pulp, where the tensile index (58.8 N m/g), tear index (3.38 mN m2/g), burst index (3.86 kPa m2/g), double fold number (10.66), brightness (32%) and opacity (67%).Table 87, flax material (42.66 N m/g)46, and oil palm empty fruit bunches pulp (20.4 N m/g)88. While it was lower than from the papersheet produced from depithed Bagasse pulp (60 N m/g)72. The tear index value (3.38 mN m2/g) was lower than from papersheets manufactured from pulps of rice straw (6.49–7.49 mN m2/g)87, depithed Bagasse (5.0 mN m2/g)72, flax plant (4.33 mN m2/g)46 and palm oil empty fruit bunches (7.20 mN m2/g)89, while it was partially equal to the measured from wheat straw (3.86 mN m2/g)90 and higher than of sunflower stems (2.04 mN m2/g)91.The tensile index value (58.8 N m/g) was higher than those reported from papersheet produced from rice straw pulps (38.0–55.2 N m/g)2/g) was in the range of the value reported from papersheets manufactured from pulps of rice straw (2.43–5.34 kPa m2/g)87, but lower than from depithed Bagasse (4.8 kPa m2/g)72. Double fold number (10.66) was lower than the value reported from the papersheets derived from pulps of rice straw (35–173)87, and depithed Bagasse (26–42)72.The burst index value  was higher than the reported from unbleached stem pulp (25.4–29.5%)55.Tensile, burst, and tear indices from papersheets produced from refined unbleached Kraft pulp from M. oleifera SRRP pulp, SEM–EDX technique was used. The images of SEM–EDX were taken from three places of the produced papersheet. The SEM images showed that the average fiber diameters was 18.52 µm  of C and O is 44.04%, 55.96% Fig.  Spc_001,Figure M. oleifera SRRP extract and inoculated with Rhizoctonia solani and Fusarium culmorum after 14 days from the inoculation are shown in Fig. R. solani and F. culmorum, respectively  as shown in Fig. A. tumefaciens, E. amylovora and P. atrosepticum, respectively, were lower than of the positive control (Tobramycin 10 µg/disc) 32–64 µg/mL.Table 98. However, the activity of plant extracts have been classified as significant (MIC < 100 μg/mL), moderate (100 < MIC ≤ 625 μg/mL) or weak (MIC > 625 μg/mL)100. In addition, Tamokou et al.101 proposed new threshold values of MIC for extracts as follow; highly active (MIC < 100 μg/mL), significantly active (100 ≤ MIC ≤ 512 μg/mL), moderately active (512 < MIC ≤ 2048 μg/mL), low activity (MIC > 2048 μg/mL), and not active (MIC > 10 mg/mL). According to these classifications, the activities M. oleifera SRRP extract were moderate to significant against A. tumefaciens and P. atrosepticum and weak to moderate against E. amylovora.It is important to note that a MIC value between 100 and 200 μg/mL was considered as positive for plant extracts102. Several phytochemical compounds were identified in different parts of M. oleifera including quercetin, ellagic acid, gallic acid and kaempferol103.Total polyphenols (13.7 g/100 g extract dry weight) and total flavonoids (69.0 g/100 g extract dry weight) were reported from the podsSalvadora persica root-bark acetone extract showed inhibition zones (IZs) against A. tumefaciens (13.6–18.6 mm), P. atrosepticum (15.3–23 mm)51. Chloroform leaf extracts from Lantana camara Duranta plumieri variegata and Citharexylum spinosum showed IZs with the range of 8.3–24.3 mm, 8–13.6 mm, 8–11.6 mm, against A. tumefaciens, and 6.6–9.6 mm, 0–9.3 mm, and 9.6–13.6 mm against P. atrosepticum, respectively50. Callistemon viminalis flowers acetone extract observed IZ value 15.0 mm against the growth of A. tumefaciens49.Revealed to the concentration used, Moringa oleifera SRRP extract-treated wood showed potential antifungal activity against F. culmorum  and R. solani . Also, the present results showed that the FMIP against F. culmorum was lower than the standard biofungicide Fluconazole (25 μg), which observed IFMG 53.70% and higher than Fluconazole (42.96% against R. solani) when applied to wood samples34. Previously, different parts of M. oleifera plant extracts have been observed to inhibit some phytopathogenic fungi including Alternata burnsi, Aspergillus niger, A. paracitic, A. flavus, Candida Albicans, F. oxysporum and Trichoderma harzanium104. Comparing to other natural extracts applied to wood samples as biofungicide preservatives, i.e., Haplophyllum tuberculatum whole plant extract with its main compounds resveratrol, kaempferol, myricetin, rutin, quercetin, and rosmarinic acid showed potential antifungal activity against F. culmorum and R. solani when applied to Melia azedarach wood105. The extracts from Coccoloba uvifera with its main compounds of gallic, benzoic, ellagic, and o-coumaric acids applied to Pinus roxburghii wood observed good activity against R. solani, Botrytis cinerea, and F. culmorum37. Flower extract from Acacia saligna-treated M. azedarach wood, with the presence of quercetin, naringenin, benzoic acid, o-coumaric acid, caffeine and kaempferol compounds observed antifungal activity against F. culmorum, R. solani, and Penicillium chrysogenum26. An antimicrobial potential activities against R. solani, F. culmorum and A. tumefaciens, were observed as wood-treated with Musa paradisiaca peel extract, where the HPLC analysis of the extract identified gallic acid, ellagic acid, naringenin, rutin, and myricetin as main compounds27. Furthermore, salicylic acid, rutin, vanillic acid and myricetin were found in Withania somnifera fruit extract that showed good wood-biofungicide activity against F. culmorum and R. solani wood-bactericide against A. tumefaciens, E. amylovora, and Pseudomonas cichorii106. M. oleifera SRRP, has been previously possessed potential antibacterial activities107, also myricetin and rutin were observed potent antifungal agents against Candida albicans and C. parapsilosis108. A. flavus and A. parasiticus were completely inhibited in terms of their growth and the production aflatoxin by vanillic and caffeic acids at 0.2 mg/mL109. Also, phenolic compounds of Stenoloma chusanum extract including vanillic acid showed potential antifungal activity110.Myricetin which found in the amount of 56.27 mg/100 g extract ofAs from the present study and commercially, moringa, the fast growing with multipurpose uses, and after obtaining the ripened seed, the seeds-removed pods have been shown some important properties. It acts as a raw material for the production of pulp and paper due to limited wood resources, where the mechanical and physical properties of the produced papersheet were comparable with those reported from the literature from woody and non-woody materials. Also, from the HPLC analysis of phytoconstituents profile, some important phenolic compounds vanillic, benzoic, syringic, and ferulic acids and flavonoid compounds myricetin, naringenin and kaempferol were identified. This study showed the maximizing the utilization of moringa residues in the pulp industry and the production of bioactive chemicals.


While the properties of the metalcenter are crucial for the ability of the complexes to electrochemicallyactivate CO2, systematic variations of the metal withinan identical, redox-innocent ligand backbone remain insufficientlyinvestigated. Here, we report on the synthesis, structural and spectroscopiccharacterization, and electrochemical investigation of a series of3d transition-metal complexes  coordinated by a new redox-innocent PNP pincerligand system. Only the Fe, Co, and Ni complexes reveal distinct metal-centeredelectrochemical reductions from M(II) down to M(0) and show indicationsfor interaction with CO2 in their reduced states. The Ni(0)d10 species associates with CO2 to form a putativeAresta-type Ni-η2-CO2 complex, where electrontransfer to CO2 through back-bonding is insufficient toenable electrocatalytic activity. By contrast, the Co(0) d9 intermediate binding CO2 can undergo additional electronuptake into a formal cobalt(I) metallacarboxylate complex able topromote turnover. Our data, together with the few literature precedents,single out that an unsaturated coordination sphere (coordination number= 4 or 5) and a d7-to-d9 configuration in thereduced low oxidation state (+I or 0) are characteristics that fosterelectrochemical CO2 activation for complexes based on redox-innocentligands.Coordination compoundsof earth-abundant 3d transition metals areamong the most effective catalysts for the electrochemical reductionof carbon dioxide (CO 2) activation.A series of 3d transition-metal complexes coordinated by a new redox-innocent PNP pincer ligandsystem were synthesized and structurally as well as electrochemicallyanalyzed to illuminate the role of the metal center in molecular electrochemicalcarbon dioxide (CO The thermodynamic challengeof this approach can be virtuously addressed in supplying the requiredenergy for the conversion of CO2 by renewable electricity.To overcome the kinetic barriers, molecular complexes of earth-abundant3d transition metals are heavily investigated, ranging among the mosteffective and efficient electrochemical CO2 reduction (eCO2R) catalysts.16 In a recent literature survey,we analyzed the main reaction pathways of homogeneously catalyzedCO2 electroreduction from an organometallic perspective.We classified them into mechanisms traversing the direct coordinationand activation of CO2 to the metal center  and those requiring theprevious formation of a metal hydride  as the reactive intermediate  and product  binding affinity,23 define the prevailingmechanistic route, which is directly linked to the catalytic performance.However, only a limited number of studies report on the systematicvariation of the metal center within an identical ligand frameworkin relation to CO2 electroreduction  or relaying protons .30 The resulting complex interplay of ligand- and metal-centered processesrenders deconvolution of the individual contributions rather challenging.31 By contrast, systematic variations of the metalcenters with redox-innocent ligands, such as pincer platforms,39 remain insufficiently investigated in the frame of CO2 electroreduction before the addition of chlorodiphenylphosphine and stirring at 65°C for 18 h. NMR spectroscopic analysis , high-resolution mass spectrometry (HRMS),and elemental analysis confirmed the structure and purity of ligand L received in 67% yield after workup . This planarity might resultfrom a steric hindering of the rotation around the C–N bondlinking the pyridine core and the bulky aniline moiety or from conjugationof the lone pairs of the aniline N atoms with the heterocycle, inferringin-plane triangular geometry at these N atoms. We also note that the pinching character of L is marked with a relativelyshort P–P distance (dP–P = 4.226 Å), likely by virtue of the steric constraint imposedat the aniline N atoms, making the C–N–P angles narrow(119.5° and 115.7°).Even before the coordination of a metal atom, the molecular structureof the ligand 4A exhibiL with 3d transition-metal precursors in yields rangingfrom 52 to 95%, as indicated in the MX complexes are presented in Pincer transition-metal complexes were obtained by the metalationof FeCl), Co (CoCl), Ni (NiCl),and Zn (ZnCl) but remained elusivefor Mn and Cu (vide infra). In these cases, complexes MnBr and CuCl were derived from the Mn(CO)5Br and CuClprecursors, respectively, in their +I oxidation state. Along withthese complexes, we also made variations to less electronegative ormore weakly coordinating anions and prepared FeMeCN, CuI, and ZnOTf.For a systematic comparison, we first targeted pincer complexesbearing metal centers in their +II oxidation state and bis-chloridecoordination. These structures were successfully obtained for Fe , as representedin Single crystals couldbe obtained for Characteristic features of the structures 4C, calcu2.1.22 at room temperature(rt) in THF, as reported for related pyridine-based PNP pincers ,37 did not succeed with our ligand nor did othervariations in the conditions . The only reported [Mn(PN3P)Cl2] structurewithin a pyridine-based pincer framework displays an almost idealsquare-based-pyramidal (SBP) geometry (τ5 = 0.04)with two P atoms in the trans position of the basal plane and relativelydistant from Mn (dMn–P = 2.574and 2.590 Å and dP–P = 4.886Å), in a high-spin, five-unpaired-electron configuration (μeff ≈ 6).37 We surmise that L does not allow such significant elongation between the twoP atoms, likely required in a putative [MnLCl2] complex. The electron-withdrawing character of the phenyl substituentat the P atom may also disfavor the coordination of a Mn(II) fragment.In the case of Mn, metalationwith MnClI(CO)5Br precursor effectively yields the Mn(I) complex MnBr, suggesting stabilization of the loweroxidation state by L. This 18-electron Mn complex crystallizesin a distorted octahedral (Oh) coordination environment, as is commonly encountered forrelated pincer manganese(I) carbonyl bromide complexes.42 In this particular case, XRD analysis revealeda mixture of the cis and trans configurations of the CO ligands ina 95:5 ratio in the crystal.By contrast, coordination of the Mn6 low-spin MnBr complex exhibits distinguishable,but overlappingsets of 1H NMR signals for the chemically inequivalentaromatic protons of the RN and RP phenyl moietiespositioned syn or anti of Br in the predominant cis-CO configuration .The diamagnetic d2.1.3FeCl crystallizes in a stronglydistorted SBP structure, as reflected by a τ5 valueof 0.38. The PNP coordination sites of L exhibit relativelyextended bond distances of 2.327 Å for Fe–N as well as2.429 and 2.426 Å for both Fe–P bonds. Interestingly,the steric demand of the RN = Ph group in L seems to prevent the formation of bis-PN3P-coordinatedspecies, which readily occurs for RN = H.331H NMR analysis of FeCl in CD2Cl2 exhibitsthe contact-shifted peaks diagnostic of a paramagnetic Fe(II) center,signals at 57.50 and −11.79 ppm are assigned by integrationto pyridinic protons in the meta and para positions, respectively,and indicate a high level of symmetry in the structure .Although 57Fe Mössbauer spectrum of FeCl recorded with a powder sampleat 80 K showed a quadrupole doublet with high isomer shift, δ= 0.80 mm·s–1, and large quadrupole splitting,ΔEQ = 3.33 mm·s–1 . The values are typicalof high-spin Fe(II) with S = 2. Accordingly, thecompound was electron paramagnetic resonance (EPR)-silent in a THFsolution at X-band frequencies . Moreover,the effective magnetic moment of solid FeCl was μeff = 4.9 μB at 270K , in agreement with the spin-onlyvalue expected for S = 2. The axial zero-field splittingparameter obtained from the temperature variation of the magneticsusceptibility χT versus temperature T is in the usual range for Fe(II), S =2 with D = 4 cm–1.The zero-field 6 high-spin electronic configuration of Fe(II)in FeCl, the highest occupiedmolecular orbital (HOMO) is a dx2–y2 one, which is expectedto be stabilized by distortion of the SBP structure to high θapic angle values (up to 117°).43 Similar distorted SBP geometries were observed on a series of relatedPN3P-coordinated iron bis-chloride complexes, for whichextended Fe–N and Fe–P distances were related to a high-spinstate at Fe.34In the3d2.1.4FeCl inacetonitrile  results in a color changefrom yellow to red , indicative of a substantial change in the coordinationsphere. Corroborating this point, new diamagnetic signals build upbetween 5.6 and 8.0 ppm in 1H NMR spectra taken in CD3CN.Dissolving IIL}2+ fragment in MeCN by reacting in this solvent the ligand L with the Fe(OTf)2 precursor bearing weakly coordinatinganions. The resulting FeMeCN complexexhibits a distorted octahedral geometry, where L occupiesthree meridional positions of the inner coordination sphere, completedby three MeCN ligands. The P1–Fe–N bond anglesare close to the expected 90° for the two axial solvent ligands(91.40 and 89.75°) but are significantly distorted for the equatorialMeCN (98.56°), as is also reported for Fe(II) PN3Ptris-MeCN complexes .321H NMR analysis of FeMeCN  revealed equatorial and axial MeCN signals  in CD2Cl2 and the disappearanceof these signals by exchange with the deuterated solvent in CD3CN . The Mössbauerspectrum of solid FeMeCN showeda significantly lower isomer shift than FeCl with δ = 0.34 mm·s–1, as well as weak quadrupole splitting of 0.87 mm·s–1 . These parameters readilyexclude a high-spin configuration but reveal a diamagnetic d6 low-spin configuration of FeMeCN with S = 0, as was already inferred from the NMRresponse.To further elucidate the identity of the formedspecies, we assessed the structure of an {FeFeCl in CD3CN with that of an authentic FeMeCN sample in the same solventpoints toidentical coordination environments. The conversion of FeCl in MeCN was further supported by crystalsgrown from an MeCN solution of FeCl that reveal an inner-sphere molecular structure  identical with that of FeMeCN.Comparing the NMR spectra of FeCl into FeMeCN in MeCN is incomplete in solution;the two species are inequilibrium with other intermediates . We, therefore, use FeMeCN for further electrochemical analysis in MeCN to analyze a singlewell-defined species.Further NMR studies yet showedthat the conversion of 2.1.5CoCl coordinatesin an almostideal SBP structure with τ5 very close to zero. Forcomparison, with RN = H, Rösler et al. reporteda structure also resembling the ideal SBP (τ5 = 0.01).36CoCl displaysa bond length of 239 pm between Co and the apical (apic) chlorideatom , which suggests that this ligand is only weakly bound.However, crystals prepared from the strongly coordinating MeCN solvent showed neither formation ofthe outer-sphere chlorido complex nor substitution of the ligand bya solvent molecule but only a fractional elongation of the Co–Clapic bond to 242 pm.CoCl exhibits heavily broadened 1H NMR signals in the5–12 ppm chemical shift region  that are in accordance with a paramagnetic d7 electron configuration. Although integration of the peaks sums tothe expected 33 protons, the paramagnetic character of the substanceprohibited further NMR spectroscopic analysis on other nuclei.CoCl gave an effectivemagnetic moment of 1.9 μB at 270 K , consistent with the spin-only value for S = 1/2 (1.73 μB)and in line with related complexes in the literature (an effectivemagnetic moment of 2.3 μB was reported for RN = H).36 The corresponding 3d7 low-spin configuration of CoCl was further corroborated by the X-band EPR spectrum of thecompound in an MeCN solution at 10 K, showing distinct anisotropic g splitting and 59Co hyperfine splitting .Solid CoCl is favored over distortions tohigher values of τ5 because the corresponding structureswould stabilize the singly occupied dz2 orbital but concomitantly destabilize the fully occupied dyz and dxz orbitals.43We posited that the nearlyideal SBP geometry observed for 2.1.6NiCl displaysan SBP structure,with a distortion intermediate of FeCl and CoCl. Electronicstabilization of the dz2 HOMOprocured by a θapic value of up to 110°43 is particularly favorable for the 18-electrond8 configuration of NiCl. Here, the apical chlorido ligand is weakly bound to the metalas well, manifesting in a bond length of 255 pm. Although the formationof a cationic almost ideally square-planar (SP) d8 [Ni(PN3P)Br]Br complex (with RN = H) was observed in methanol(MeOH),32NiCl crystals grown from MeCN  maintain the apical chlorido ligand coordinated with a marginallyelongated bond distance.31P singlet peak at 85.0 ppm  and well distinguished sets of 1H signals  for the different aromatic positions,most indicative for the pyridinic protons in the meta position thatexhibit a characteristic doublet at 5.73 ppm . We suspect in situ reduction of Cu(II) to Cu(I) upon coordination to L with simultaneous oxidation of the ligand ,as was already reported in the literature.47 We thus aimed for the Cu(I) complex CuCl, which could be successfullysynthesized starting directly from CuCl. The identical NMR spectraand crystal structures (data not shown) of the products synthesizedfrom CuCl2 and CuCl supported this approach. Similarly, CuI was synthesized from CuI to introducethe less electronegative iodo ligand.For Cu, the reaction of 10 complexes both exhibit a distorted tetrahedral (Td) structure, with τ4 values of 0.78 (chloride) and 0.77 (iodide) being intermediatebetween that of the ideal tetrahedron (τ4 = 1) andthat of a butterfly/seesaw structure (τ4 ≈0.43). The coordination geometries found are in accordance with knownbromide analogues .35The tetracoordinated dFigures S19–S24) reflects the similarcoordination structure, with the 31P peaks and 1H peaks  only marginally deviatingCl complexes, leaving the complex in an almostideal tetrahedral coordination geometry (τ4 = 0.95).The 18-electron valence of this d10 configuration at theZn center likely favors tetracoordinated structures over pentacoordinatedstructures. ZnCl preferentiallyadopts a tetrahedral structure over a SP one, possibly because HOMOsin the former  are significantly more stabilizedthan the HOMO in the latter . Additionally,the structural constraints of the pincer ligand might disfavor formationof the SP configuration. A bromide analogue with RN = Mealso exhibits the tetrahedral structure.38In ZnOTf retainspentacoordination and forms a 20-valence electron (VE) complex ofdistorted trigonal-bipyramidal (TBP) structure, as reflected in theτ5 value of 0.82. 20-electron complexes are onlyaccessible in very weak field complexes, where the antibonding molecularorbital hosting two electrons is sufficiently low in energy, withthe TBP geometry stabilized compared to SBP.43ZnOTf is the only example ofa pyridine-based PNP pincer complex derived from the Zn(OTf)2 precursor to the best of our knowledge. Therefore, the closest relatablesystem is the terpyridine (tpy)-coordinated Zn complex reported byBocian et al., which crystallizes in a seesaw/butterfly structure(τ5 = 0.47).48 Here, thebond between Zn and the N atom of the pyridine core is significantlyshorter than the Zn–N bond in ZnOTf (204 vs 233 pm), indicating that the N is loosely coordinatedin the latter system.By contrast, ZnCl and ZnOTf also can be seen in the recorded NMR spectra, with a differenceof 2.1 ppm forthe 31P nucleus  and 0.09 ppm  for the meta protons of the pyridine unit.Distinctions in the molecular structuresof 2.2L and MX will be discussed individually in thissection. The cyclic voltammograms under an Ar atmosphere of the MCl complexes bearing chlorido ligandsand FeMeCN are depicted in The electrochemical behaviors of 2.2.1L was found to be electrochemically inert in the window of potentialsfrom −2.5 to −0.1 V versus Fc+/0 (abbreviatedVFc in the following) in MeCN and N,N-dimethylformamide , thus falling in the category of redox-innocent systems.2.2.2MnBr shows an irreversible reductionwave at Ep,c = −2.64 VFc  in CV. For related Mn complexes[Mn(tpy)(CO)2Br]49 bearing the tris-chelating planar tpy ligand, a metal-centered reductionto a Mn(0) species with concomitant halide loss and subsequent fastdimerization was suggested. Nevertheless, the lack of an anodic wavefor the reoxidation of a putative dimer of reduced MnBr makes such a metal-centered reductionevent appear unlikely. In addition, MnBr is already in an 18-VE, alleged d6 low-spin configuration,and reduction would populate a high-lying eg orbital. Wetherefore propose that reduction is centered on the ligand at thestrongly cathodic potential observed here  higher than 20 V·s–1, suggesting that follow-up chemical events are fast. The occurrenceof structural reorganization upon reduction is also supported by theobservation of a reoxidation wave at a considerably more positivepotential  that follows reversal of the potential scan between the first andsecond reduction waves . Thisoxidation wave vanishes at low scan rates when the scan is reversedafter the second reduction wave . We thus suggest that FeMeCN reduces to a [FeIL(MeCN)n]+ complex (n = 3), which undergoesfast MeCN loss into short-lived [FeIL(MeCN)n]+ with n = 1or 2 (I2L2(MeCN)n2]2+ compound, in the form of an Fe–Fe-bondedor an MeCN-bridged one. We tentatively attribute the oxidation waveat −1.24 VFc to the oxidation of such dimeric species.The second reduction wave is then assigned to reduction of the putative[FeI2L2(MeCN)n2]2+ dimer into an [Fe02L2(MeCN)n'2] species. When the scan reversal is negative to this second reduction,the disappearance of the reoxidation wave at −1.24 VFc for low sweep rates suggests the evolution of the postulated [Fe02L2(MeCN)n'2] dimer into monomeric Fe(0) species or otherdegradationproducts . The reversibility of the second reductionwave is gradually established when the scan rate is raised (from 5V·s–1), corresponding to reoxidation of thetransient Fe(0) complex, from which the follow-up chemical step isonly moderately fast.1.96 VFc 5, which on waves S44. This2.2.4CoCl (E0 = −1.21 VFc (ΔEp = 94 mV) and an irreversible wave at Ep,c = −1.98 VFc can be observed, to which we assignthe CoII/I and CoI/0 couples, respectively.Although the molecular structure obtained for CoCl from an MeCN solution confirmed the inner-spherecoordination of both chlorido ligands in the solid state, we hypothesizethat a dynamic ligand exchange between Cl– and MeCNtakes place in solution.50 Likely, a cationic[CoIIL(MeCN)nCl]+ (n = 1 or 2) complex forms upon the dissolutionof CoCl into MeCN. We postulatethat the complex reversibly reduces into a neutral [CoIL(MeCN)nCl] species  suggests that the follow-up chemical step is fast. We attributethis fast chemical step to the dissociation of a chloride ligand,forming a putative [Co0L(MeCN)n'] complex.In the cyclic voltammogram of CoCl 5,a part species 6C. Furth2.2.5NiCl shows a voltamperometricpattern at 100 mV·s–1 similar to that of CoCl with a reversible reduction waveat E0 = −1.16 VFc, followedby an irreversible process at a more negative potential of Ep,c = −1.65 VFc nCl]+ (n = 1 or 2) complex. Thereduction of [NiIIL(MeCN)nCl]+ is proposed to generate a neutral [NiIL(MeCN)nCl] species  species is here also coupled to a chemical step (EC), likelya Cl– elimination producing a low-valent [Ni0L(MeCN)n'] complex.In that case, however, the reversibility of the NiI/0 couplecan be recovered by elevating the scan rate , suggesting a slow chemical step. A standard potentialof E0(NiI/0) = −1.59VFc is recovered from the reversible wave at a fast scanrate. The Ep,c versus log(ν) graphdecays linearly in the irreversibility region with a −22.1 mV·dec–1 slope , in reasonable agreement with thetheoretical value for an EC mechanism expected at −29.6 mV·dec–1.51 From this data, theapparent forward rate constant kf,app ofthe coupled chemical step was estimated at kf,app(Ar) = 3.6 × 10–1 s–1.1.65 VFc 5, to whi species 6D. As stEp,c = −2.41 and −2.75 VFc . We assign the wave at −2.41VFc to a ligand-centered reduction, but the second reductionwave has remained unassigned so far.Two more negative irreversible reduction waves appear at 2.2.6CuCl exhibits anirreversible reduction wave at Ep,c =−2.35 VFc . Referring to the challenging synthesisof the Cu(II) species, it is interesting that the +II oxidation stateapparently can be accessed by electrochemical means. The Cu(II) complexis possibly only stable in the time scale of the CV experiment anddecomposes during the reaction time of the synthesis.2.35 VFc 5. At fir2.35 VFc 6E. MoreoCuI shows almost identical redox featurescompared to CuCl with an irreversiblereduction wave at Ep,c = −2.27VFc , which we assumeis a ligand-centered reduction as well .2.27VFc S41, whic2.2.7ZnCl at Ep,c = −2.56 VFc. d10 Zn ions are usually redox-innocent in accessible reduction windows,and the corresponding complexes are thus commonly used to identifythe redox behavior of the coordinated ligand system.54 In particular, ZnCl is alreadyin a stable 18-electron configuration with metal 3d orbitals fullyoccupied (d10), making a metal-centered reduction unlikely.We therefore posited that the reduction observed at low potentialis centered on the ligand , whose trace is crossed by that of the backwardanodic scan and followed by a sharp oxidative desorption peak at higherpotential . The observation of a line crossing and a desorption wave is diagnosticof electrodeposition upon reduction. Therefore, we assume that thereduction wave of ZnOTf representsa two-electron reduction to Zn(0) with concomitant decomposition in(nanoparticulate) deposits on the electrode surface . With a focus on the second reduction wave, anoticeable change in the reaction mechanism is observable from thechange in the peak shape. While dimer formation is likely under Aratmosphere, this pathway might be inhibited by the coordination ofCO2 at this stage.Under a COCoCl, the CoII/I redox wave remains unaffectedby CO2, discarding thereaction with this substrate at the Co(II) and Co(I) states. By contrast,the CoI/0 irreversible wave shifts by approximately 50mV in the anodic direction upon CO2 saturation. This observationevidences CO2 coordination upon the reduction to Co(0)in an EC mechanism. Even at elevated scan rates, the CoI/0 wave remains irreversible , evidencing a fast CO2 association rate. The lack ofknowledge of EAr0(CoI/0) (vide supra) yet prevented quantification of the rate constant of CO2 association under our electrochemical conditions. Interestingly,however, a slight increase in the cathodic peak current  is also observed and suggests electrocatalyticactivity. Although reductive disproportionation of CO2 intoCO and CO32– is conceivable under thedry conditions used here, we suspect that traces of protons  are involved in the CO2 electroreduction.For NiCl, the NiII/I couple is also left unaffected by CO2, discarding coordinationat the corresponding +II and +I oxidation states. On the contrary, NiCl exhibits a very strong anodicshift (∼130 mV) at the NiI/0 wave under CO2, the largest observed at the MI/0 couples of the analyzedcomplexes. At variance with the observation under an Ar atmosphere,the reversibility of the NiI/0 reduction wave cannot berecovered by a faster scan rate . These observations marka clear indication of a fast CO2 association at the Ni(0)stage (EC). Corroborating this process, Ep,c = f[log(ν)] linearly decays by −38.7mV·dec–1 in the KP zone . Prior knowledge of EAr0(NiI/0) allows one to evaluate the CO2 associationapparent rate constant (section 2.2), foundat kf,app(CO2) = 4.2 ×103 s–1, which gives a bimolecular rateconstant of kA,f(CO2) = 1.5× 104 M–1·s–1 (taking a saturation concentration at 1 atm of CO2 of[CO2] = 0.28 M in an MeCN solution23).For –1, an additional oxidation wave  becomes visible. We associate this anodic wave with the oxidationof a [NiIIL(MeCN)n(CO2H)]+ or [NiIIL(MeCN)n(CO)]2+ (n =1 or 2) adduct only accessible at elevated scan rates due to the limitedlifetime of the species. We can, however, not fully exclude that thiswave corresponds to the oxidation of a putative [NiIIL(MeCN)nH]+ hydridespecies formed by the reaction of a Ni(0) intermediate with residualprotons.Furthermore, from scan rates above 10 V·s33.1MCl , the M–Pand M–N bonds shorten while the 3d row is incremented, as expectedfrom the decreasing metal ionic radii in the 3d block.55 The trend is overruled as other factors suchas the auxiliary ligands and coordination geometries are varied.The bond distancesbetween the coordination sites and the donoratoms (P and N) of the ligand scaffold are depicted in 31P Δδvalues, defined as the differencebetween the chemical shift of the free versus coordinated ligand (seethe Supporting Information for details),show a systematic variation along with the metal series. The valuedecreases when the electron count is incremented from the left (MnBr) to the right (ZnCl/OTf) of the 3d row  likely because of the combined saturationof the 3d metal orbitals and the valence shell (Fc), irrespective of the metalcenter, confirming the redox-innocent nature of this ligand. Nevertheless,below these potentials, reduction of the ligand is possible.In the seriesof complexes under scrutiny, metal-centered reductions are generallyprevented with a 3dce shell 9. The li10 configuration, thed6 low-spin MnBr complexalso exhibits a ligand-centered reduction event only. We posited thatthe strongly field-splitting carbonyl ligands destabilize the unoccupiedeg orbitals of the metal center to the extent where ligandreduction is energetically more favorable than metal reduction, althoughat very cathodic potentials (<−2.5 VFc). Onemay thus conclude that CO-coordinated Mn complexes generally requireredox-active ligands  or a cationic state57 to be active inCO2 electroreduction. This point is in sharp contrast tothermochemical CO2 reduction passing through the Mn–Hpathway, where manganese(I) carbonyl complexes based on redox-innocentligands are highly efficient catalysts.61Although deviating from a d10 state unlocks metal-centered reduction processes,as observed in the case of FeMeCN, CoCl, and NiCl , in our series, a comparison with the other complexes allows assignment of the first two reductions, indicating coordination atthe Fe(I) state. With FeMeCN adoptinga distorted octahedral geometry in a d6 low-spin configuration,the one-electron reduction from Fe(II) to Fe(I) populates an eg antibonding orbital. In the presence of CO2, thefast chemical step deduced from the irreversibility of the FeII/I wave is consistent with the high reactivity of Fe(I) species.The activation of CO2 at Fe(I) centers reported previously,namely, in eCO2R, has been mainly observed with complexesbearing redox-active ligands,64 which arethus best described as ligand-reduced Fe(II) complexes. For the redox-inactivepincer ligand used here, we postulate that the metal-centered radicalbinds CO2 to form [FeIL(MeCN)(CO2)]+ as the primary intermediate. We note that oneof the rare reported metal-centered Fe(I) radicals is known to resultin the bimolecular cleavage of CO2 into a (μ-CO)(μ-O)-bridgedFe(II) dimer65 and a similar bimolecularCO2 activation with FeMeCN also seems possible.Complex CoCl, CO2 activation occurs onlyat the M(0) state instead of M(I). The slightly distorted SBP geometrydetermined for CoCl in the solidstate is expected to be retained in an MeCN solution, possibly withchloride and solvent in dynamic exchange at the apical position. TheHOMO is consequently a single occupied dz2 one at the d7 configuration of paramagnetic CoCl. The first reduction is expectedto fill this dz2 orbital, producinga diamagnetic [CoIL(MeCN)Cl] complex (MeCN)]+ .66 The large potential gap to the second reductionin a Co(0) species  is consistent with the need for the injection of a second electroninto a high-lying antibonding dx2–y2 orbital. This secondreduction is followed by an irreversible chemical step (EC), mostprobably the expulsion of Cl– concomitant with MeCNassociation to generate a neutral [Co0L(MeCN)]complex. Calculations on similar [Co0(PNP)(MeCN)] complexessuggest a η2 π-bonded MeCN in a structure bestdescribed as a cobalt(II) cycloimine.66 We postulate that MeCN binding rather than Cl– expulsion is rate-determining. The positive shift of the irreversibleCoI/0 reduction  under CO2 indicates that the associated chemicalequilibrium is more shifted or accelerated forward with that substrate.The electrophilic nature of the C atom of CO2 likely makesthe association faster compared to that of MeCN. Although formallymetalloradicals, Co(0) species do not commonly exhibit single-electron-transferreactivity but undergo two-electron oxidative chemistry.67 We thus favor formulation of the CO2 activation product as a d7 [CoIIL(CO2)] complex. The potential at which this intermediateis accessed is low enough for further reduction into a 16-electron[CoIL(CO2)]− complex,refilling the low-lying dz2 orbital  two-electronreduction from [CoILCl] to [CoIL(CO2)]−.Moving from Fe to Co in  complex 10A. Rele orbital 10A. ThisNiCl also displaysa SBP coordination geometry and similar to Co interacts with CO2 only at the 0 oxidation state. NiCl is reduced down to a Ni(0) species by two consecutiveelectron injections expected to fill the dx2–y2 orbital (3P)Br2] complex bearing a close PN3P ligand .68 Comparedto CoCl, the potential gap betweenthe +I and 0 oxidation states is tighter [E0(NiI/0) – E0(NiII/I) = −430 mV]  PNP halide complexes.69 Upon reductionto the Ni(0) state, we assume Cl– dissociation andMeCN association similar to that proposed for Co(0). However, theapparent rate of these follow-up chemical steps  is decelerated compared to the Co(0) complex, as witnessed by reversibilityof the NiI/0 wave at an elevated scan rate. This pointalso agrees with the formation of an intermediate closed-shell 18-electronNi(0) species, more stable than the 17-electron Co(0) congener. Thebinding of CO2 at this stage is, however, largely favored,with NiCl displaying the largestcathodic shift ΔEp,c2Ar→CO(MI/0) of 130 mV. This shift translates into a follow-up chemical step4 orders of magnitude faster upon CO2 addition , corresponding to the binding of the electrophilicCO2 substrate. CO2 activation at a Ni(0) centersupported by a neutral PPP ligand was shown to build a pentacoordinatedNi(0) species displaying η2-CO2 binding,70 as crystallographically shown in the famousAresta complex.71 Such species are betterdescribed as Ni(0) species with minor electron transfer to the boundCO2. We suggest that NiCl results in a similar [Ni0L(CO2)] adduct. The reoxidation wave at Ep,a = −0.39 VFc observed at an elevated scanrate (ν = 50 V·s–1) could then relateto the oxidation of a subsequent intermediate, for instance, a [NiIIL(CO2H)]+ hydroxycarbonylcomplex formed by the protonation of [Ni0L(CO2)].Complex  orbital 10B, a fa−430 mV] 8C, consi3.42 coordination on aseries of nickel(II) and cobalt(II) cyclam-like complexes.75 The CO2 association equilibrium [KA(CO2)] and forward rate  constants at the +I oxidation state werefound to follow a general trend, increasing as the MII/I standard potential becomes more negative.74 The present series of complexes FeMeCN, CoCl, and NiCl exhibit reduction potentials sufficientlynegative to afford CO2 coordination. While interactionwith CO2 occurs at the +I oxidation state for FeMeCN, both CoCl and NiCl require reductionto a formal M(0) state to proceed to CO2 activation. Thispoint is in sharp contrast to complexes of the same metals with redox-innocentligands, where CO2 coordination already occurs at the formalM(I) state. In particular, [Ni(cyclam)]+, which has a NiII/I reduction potential [E0(NiII/I) = −1.14 VNHE75 corrected to approximately −1.77 VFc76] close to the NiI/0 one of NiCl [E0(NiI/0) = −1.59 VFc], exhibits a NiI-CO2 association rate constant  exceeding by several orders of magnitude thatfound for Ni0-CO2 with NiCl . We note here that equilibrium and rate constantsfor CO2 association at 3d centers in their 0 oxidationstate are scarcely reported (we only found reported an order of magnitudeof 102–103 s–1 forthe CO2 association rate constant at the electroreduced0 state of a [CoII(PN3P)(MeCN)2]2+ complex).66 Nevertheless, thisdifference likely reflects the higher kinetic barrier for CO2 association at the closed-shell [Ni0L(MeCN)n'] complex compared to the open-shell17-VE[NiI(cyclam)]+, underlining the importance ofthe electronic configuration for binding and activating CO2.The groupsof Lewis and Fujita have emphasized the relationship between the reductionpotential of the metal center and CO0L(MeCN)n'] binds CO2 at faster ratesthanthe 18-VE complex [Ni0L(MeCN)n']. A larger degree of CO2 activationis also suggested by the increase in the CoI/0 cathodicpeak current upon contact with the substrate. This interpretationis in line with findings for the series of cyclam-like complexes inwhich Co(I) transfers up to two electrons to the bound CO2, whereas Ni(I) does not substantially activate the substrate.75 The electrochemical CO2 activationby CoCl and NiCl within the redox-innocent ligand structureappears to follow the same trend. The two-electron reduction of NiCl at mildly negative potentialgenerates a [Ni0L(η2-CO2)] complex that cannot be reduced further at the potentialof the NiI/0 wave, whereas [CoIIL(η2-CO2)] is further reducible at thepotential of the CoI/0 event and can lead to electrocatalyticturnover.In agreement with this consideration, the present resultsindicate that the 17-VE complex ,1,3-bis(diphenylphosphino)propane , and potassium tert-butoxide  were placed in a Schlenk tube in the glovebox, andtoluene was added (15 mL). The suspension was stirred at 100 °Cfor 18 h and cooled to rt before dichloromethane  wasadded. The organic phase was washed with brine (20 mL), the resultingaqueous phase was extracted with DCM (3 × 10 mL), and the combinedorganic layers were dried over anhydrous magnesium sulfate. Afterremoval of the organic solvents in vacuo, the crudeproduct was purified by column chromatography on silica using a mixtureof pentane and ethyl acetate (85:15). The desired product was receivedas an orange solid . The obtained analyticaldata are consistent with those previously reported in the literature.40The synthesiswas performedaccording to a modified procedure of Wagaw and Buchwald.n-Butyllithium  was added dropwise to an orange solution of N2,N6-diphenylpyridine-2,6-diamine in THF (35 mL) at −78 °C.The solution was allowed to warm up to rt and stirred for 1 h beforecooling down again to 0 °C and dropwise addition of chlorodiphenylphosphine. The solution was allowed to warmup to rt and stirred at rt for 1 h, then at 65 °C for 18 h. Aftercooling to rt, the solvents were removed in vacuo, and the brown residue was washed with MeOH (4 × 10 mL), diethylether , and pentane (3 × 5 mL).Removal of the residual solvents in vacuo yieldedthe desired product L as a white solid . Crystals suitable for XRD were obtained from a concentratedTHF/pentane (3:1) solution at −35 °C. 1H NMR: δH 7.28–7.16 , 7.15–7.07 , 7.02–6.95, 6.82–6.76 , 5.81 . 13C{1H} NMR : δC 160.3–159.7 , 143.1–142.8 , 139.5–138.8 , 138.6 (PyCp), 133.9–133.3, 130.9 (NPhCo), 129.1 (NPhCm), 128.8 (PPhCp), 128.0 , 126.4 (NPhCp), 101.5 (PyCm). 31P{1H} NMR : δP 52.8. HRMS (ESI+). Calcd for C41H33N3P2 + H+: m/z 630.22225. Found: m/z 630.22213. Elem anal. Calcd for C41H33N3P2: C, 78.21; H, 5.28; N, 6.67. Found: C,78.36; H, 5.15; N, 6.64.5.2.2General procedure: L  and the respectivemetal precursor  were placed in a Schlenktube in a glovebox, and the respective solvent (2 mL) was added. Theresulting solution was stirred at the indicated temperature for 16h before removal of the solvent in vacuo. Purificationsteps and deviations from the general procedure are explicated belowfor each complex.L(CO)2Br] (BrMn): Reaction conditions: Mn(CO)5Br,toluene, 110 °C. The desired product MnBr was obtained as a yellow solid after the residuewas washed with DCM (2 mL) and pentane (3 × 2 mL) prior to removalof the solvent in vacuo. Yield: 30.8 mg, 0.0375 mmol,75%. In concentrated solutions of DCM and under exposure to light,a change of color to brown, possibly upon CO loss and dimerization,was observed. Crystals suitable for XRD were obtained from the vapordiffusion of pentane into a concentrated DCM solution under exclusionof light. 1H NMR : δH 7.77 , 7.47–7.36 , 7.36–7.27 ,7.26–7.13 , 6.81 ,5.79 . 13C{1H} NMR : δC 178.0 (CO),164.1 , 141.3 (NPhCq), 139.9 , 139.1 (PyCp), 137.5 , 132.5, 131.8,131.7 , 131.5, 130.6, 130.3, 130.2, 130.1,128.8, 128.2 , 127.4 , 102.9 (PyCm). 31P{1H} NMR : δP 138.9. HRMS (ESI+). Calcd for C43H33BrMnN3O2P2+: m/z 819.06064. Found: m/z 819.06076. Elem anal. Calcd for C43H33BrMnN3O2P2·0.1C7H8: C, 63.26; H, 4.11; N, 5.06. Found: C,63.66; H, 4.44; N, 4.99.[MnLCl2] (ClFe):Reaction conditions: FeCl2, DCM, rt. The crude productwas purified by precipitation from a DCM solution with pentane. FeCl was obtained as a yellow solidafter the precipitate was washed with pentane (3 × 2 mL) andthe solvent was removed in vacuo. Yield: 30.8 mg,0.0476 mmol, 95%. Crystals suitable for XRD were obtained from a concentratedDCM/pentane (3:1) solution at −35 °C. 1H NMR: δH 57.14 , 14.38 , 8.61 , 3.52 , 3.13 , −4.92, – 5.45 , −11.61 . 13C{1H} NMR : δC 282.7, 258.5,199.5, 155.0, 152.1, 116.9, 108.6. HRMS (ESI+). Calcd forC41H33ClFeN3P2+: m/z 720.11821. Found: m/z 720.11849. Elem anal. Calcd for C41H33Cl2FeN3P2:C, 65.10; H, 4.40; N, 5.56. Found: C, 65.31; H, 4.79; N, 5.28.[Fe[FeL(MeCN)3](Cl3FeOFeCl3) were obtained from a concentrated MeCN/Et2O (3:1) solution of FeCl at −30 °C under air.Crystals of L(MeCN)3](OTf)2 (MeCNFe): Reactionconditions: Fe(OTf)2, MeCN,rt. The desired product FeMeCN was obtained as an orange solid after washing with Et2O (2 mL) and pentane (3 × 2 mL) prior to the removal of thesolvent in vacuo. Yield: 48.9 mg, 0.0442 mmol, 88%.Crystals suitable for XRD were obtained from a concentrated MeCN/Et2O (3:1) solution at −35 °C. 1H NMR: δH 7.75–7.59 , 7.60–7.50 , 7.48–7.37 , 7.34 , 7.10 , 5.99 , 2.43 , 1.73 . 13C{1H} NMR : δC 166.0 , 141.8 (PyCp), 139.2 , 138.7 (CH3CNeq), 138.1 (CH3CNax), 134.1 , 132.9 (PPhCp), 131.1 (NPhCm), 130.5 (NPhCo), 129.9 (NPhCp), 129.7 , 129.5 (PPhCq), 129.3, 104.5 , 5.4 (CH3CNeq), 4.7 (CH3CNax). 31P{1H} NMR : δP 129.2. HRMS (ESI+). Calcd for C43H36FeN4P22+: m/z 363.08768. Found: m/z 363.08752. Elem anal. Calcd for C49H42F6FeN6O6P2S2·0.25 C4H10O: C, 53.37;H, 3.99; N, 7.47. Found: C, 53.04; H, 4.33; N, 7.12.[FeLCl2] (ClCo):Reaction conditions: CoCl2, THF, rt. The crude productwas purified by precipitation with pentane from a THF solution. CoCl was obtained as a dark-red solidafter the precipitate was washed with pentane (3 × 2 mL) andthe solvent was removed in vacuo. Yield: 34.0 mg,0.0448 mmol, 90%. Crystals suitable for XRD were obtained from thevapor diffusion of pentane into a concentrated THF solution or froma concentrated MeCN/Et2O (3:1) solution at −35 °C. (ClNi):Reaction conditions: NiCl2·DME, DCM, rt. The desiredproduct NiCl was obtained asa red solid after the crude product was precipitated from a solutionof DCM with pentane, followed by washing of the residue with pentane(3 × 2 mL) and removal of the solvent in vacuo. Yield: 34.2 mg, 0.0450 mmol, 90%. Crystals suitable for XRD wereobtained from a concentrated DCM/pentane (3:1) solution or a concentratedMeCN/Et2O (3:1) solution at −35 °C.+: m/z 722.11862. Found: m/z 722.11871. Elem anal. Calcd for C41H33Cl2N3NiP2·0.2 CH2Cl2: C, 63.75; H, 4.34; N, 5.41. Found: C, 63.55; H, 4.57;N, 5.43.LCl] (ClCu):Reaction conditions:CuCl, THF, rt. The desired product CuCl was obtained as a light-yellow solid after precipitation froma solution in DCM with pentane and washing of the residue with pentane(3 × 2 mL) before removal of the residual solvent invacuo. Yield: 23.9 mg, 0.0327 mmol, 66%. Crystals suitablefor XRD were obtained from a concentrated DCM/pentane (3:1) solutionat −35 °C. (ICu):Reaction conditions:CuI, THF, 65 °C. The desired product CuI was obtained as a white solid by precipitating thecrude product from a DCM solution with pentane, washing the residuewith pentane (3 × 2 mL), and removing the solvent invacuo. Yield: 21.3 mg, 0.0260 mmol, 52%. Crystals suitablefor XRD were obtained from a concentrated THF solution at −35°C. (ClZn):Reaction conditions: ZnCl2, THF, rt. The desired product ZnCl was obtained as a white solidby precipitating the crude product from a solution of DCM with pentane,followed by washing of the residue with pentane (3 × 2 mL) andremoval of the solvent in vacuo. Yield: 33.3 mg,0.0434 mmol, 87%. Crystals suitable for XRD were obtained from a concentratedTHF/pentane (3:1) solution at −35 °C.+: m/z 728.11242. Found: m/z 728.11180.Elem anal. Calcd for C41H33Cl2N3P2Zn·0.25 CH2Cl2: C,62.94; H, 4.29; N, 5.34. Found: C, 62.67; H, 4.30; N, 5.33.L(OTf)2] (OTfZn): Reaction conditions: Zn(OTf)2, MeCN, 80 °C. Thedesired product ZnOTf was obtainedas a white solid by precipitating the crude product from a solutionof DCM with pentane, washing the residue with pentane (3 × 2mL), and removing the solvent in vacuo. Yield: 47.4mg, 0.0477 mmol, 95%. Crystals suitable for XRD were obtained froma concentrated THF solution at −35 °C.+: m/z 842.09559. Found: m/z 842.09613. Elem anal. Calcd for C43H33F6N3O6P2S2Zn: C, 52.00; H, 3.35; N, 4.23. Found: C, 52.26;H, 3.68; N, 4.32.