Cannabis is the fourth psychoactive substance to be legalized which are of far-reaching significance to the world. We analyzed data from the 2019 Global Burden of Disease Study (GBD) to estimate the incidence and prevalence of cannabis use disorder (CUD) and calculated the disease burden of CUD in 204 countries and territories and 21 regions over the past three decades. We reported disease burden due to CUD in terms of disability-adjusted life years (DALYs), age-standardized rate (ASR), estimated annual percentage change (EAPC), and analyzed associations between the burden of CUD and sociodemographic index (SDI) quintiles. Globally, the number of incidence cases of CUD was estimated to be increasing by 32.3% from 1990 to 2019 and males are nearly double higher than that of female. DALYs increase 38.6% from 1990. Young people aged 20–24 years old with cannabis use disorder have the highest DALYs in 2019, followed by those younger than 20 years old. India, Canada, USA, Qatar, Kenya, and high SDI quintile areas showed a high burden of disease. Nearly 200 million individuals are cannabis users worldwide, and CUD is a notable condition of GBD. The global cultivation of cannabis, rooted in different cultures, diversified access to cannabis, legalization in controversy, the promotion of medical cannabis, and many other factors promote the global cannabis industry is constantly updated and upgraded. It deserves more discussion in the future in terms of pathophysiological mechanisms, socioeconomics, law, and policy improvement.The online version contains supplementary material available at 10.1007/s11469-022-00999-4. According to a report . THC can produce a desire for repeated use, which in some users develop into cannabis use disorder (CUD)  and the number of years of healthy life lost due to disability (YLDs) . The crude rates, including incidence, prevalence, and DALYs, are extracted by the occurrence of a certain disease in a whole population of one country which could affected by its age distribution. Age-standardized rates are used to compare rates of health outcome  between different populations. To calculate ASR, the crude rate (the outcome rate observed in the population) is adjusted for the age structure of the population in each country and region.EAPC is a summary and widely used measure of the ASIR, ASPR, and ASDR trends over a specific time period. To take ASIR as an example, a regression line was fitted to the natural logarithm of the ASIR values, which can be represented as y = α + βx + ϵ, where y = ln (ASIR) and x = calendar year. The EAPC was calculated as 100 × (exp (β) − 1), and its 95% confidence interval (CI) was extracted from a linear regression model . We obtained incidence number, prevalence number, DALYs, ASIR, ASPR, and ASDR of CUD from 1990 to 2019 according to sex, 21 regions, and 204 countries and territories. Based on the SDI which combines information about the economy, education, and fertility rate of countries around the world, as a representation of social and economic development present in 2019, the 204 countries or territories were divided into five quintiles: low, low-middle, middle, high-middle, and high. The general methods used in GBD 2019 are described in detail on the official website , which also provides the SDI values from 1990 to 2019 at the global, regional, and national levels. To analyze global trends, we also assessed the trends of CUD according to the following age stratification used in GBD 2019: < 20, 20–24, 25–29, 30–34, 35–39, 40–44, 45–49, 50–54, 55–59, 60–64, 65–69, 70–74, 75–79, and 80 plus years old which will generally be reflecting adult CUD, for adolescent combined into < 20 group.The GBD of CUD estimation process including case definition, input data, age and sex splitting, data adjustment, and modeling strategy is clearly defined in the appendix 1 of systematic analysis for the Global Burden of Disease Study 2019   in 1990 to 48.8/100,000 persons  in 2019 and 303.3/100,000 persons  in 1990 to 303.4/100,000 persons  in 2019, respectively. The ASDR was almost unchanged  for males and 0.24 million (95% CI = 0.14 to 0.37 million) for females in 2019, respectively. What can be clearly seen is the continual growth of ASIR and ASPR in male and that steady decline in female. The ASDR for males increased slightly and decreased in female, EAPC revealed 0.04% (95% CI =  − 0.05–0.13%) and − 0.21% (95% CI =  − 0.28–0.14%), respectively. The ASDR was much higher for males than for females in any year: in 2019; it was 11.4/100,000 persons  for males and 6.1/100,000 persons  for female  and China increased slightly (6.9%) among the five most populous countries. An obvious reduction in the number of incidence cases was in Bosnia and Herzegovina (− 54.2%) and Italy (− 47.0%) (Supplementary Table CI = 157.1 to 208.6) and , followed by the USA  and , respectively. The lowest ASIR and ASDR were Turkey  and , followed by the Togo  and , respectively. ASPR shares the similar trend with ASIR and ASDR.In 2019, ASIR and ASDR were the highest in Canada  and 1.21% (95% CI = 0.98 to 1.44%), respectively. In contrast, Australia and Italy have had the most significant declines in ASIR (Supplementary Table CI = 1.26 to 1.63%) and 1.41% (95% CI = 1.12 to 1.70%), respectively. However, Italy and Australia experienced the greatest decline in ASPR (Supplementary Table EAPCs =  − 1.02% (95% CI =  − 1.25 to − 0.79%) , with an EAPC of 1.33% (95% CI = 0.77 to 1.46 million), prevalence cases , and DALYs  in 2019 , while it was lowest in Western Sub-Saharan Africa at 21.9/100,000 persons . ASPR of these two regions showed the same trend with ASIR. The ASDR in all regions was relatively stable, with only slightly fluctuating. For those five regions that incidence, prevalence cases and DALYs decreased which mentioned above, and the ASDR in these regions showed steady decreased as well. Australasia, Central Europe, Eastern Europe, Western Europe, High-income Asia Pacific, High-income North America, and Southern Sub-Saharan Africa demonstrated negative percentage change in all age DALYs. In addition, the ASDR was highest in High-income North America, at about 28.7/100,000 persons  and lowest in Western Sub-Saharan Africa, at about 3.7/100,000 persons  in 2019.The ASIR and ASPR were stable over 30 years in most regions, with only slightly waxing and waning. High-income North America showed the highest ASIR in 2019, at about 149.3/100,000 persons (95% CI = 0.38 to 0.55%) and 0.64% (95% CI = 0.56 to 0.71%), while the lowest were Australasia, at − 0.80% (95% CI =  − 0.96 to − 0.65%) and − 0.81% (95% CI =  − 1.01 to − 0.62%), respectively. EAPC of DALYs showed the similar trend with ASIR and ASPR  to 6.40 million (95% CI = 4.73 to 8.41 million) . The rest of the regions had ASIRs ranging from 35.5 to 48.5/100,000 persons , Australia and New Zealand (10.6%), and West and Central Africa (9.3%). The decline in the incidence and prevalence of cases in the appealed regions may be due to the increase in the number of cannabis users worldwide, while these regions have remained stable, and statistics have shown a relative decline  survey, have increased over the last decade Supplementary file2 (XLSX 47 KB)Supplementary file3 (XLSX 43 KB)Supplementary file4 (XLSX 16 KB)Supplementary file5 (XLSX 16 KB)Supplementary file6 (XLSX 16 KB)Below is the link to the electronic supplementary material.


MT treatment in both mango cultivars significantly delayed weight loss, firmness, respiration rate, and decay incidence. However, MT did not influence the TSS, TA, and TSS:TA ratio regardless of the cultivar. Moreover, MT inhibited the decrease in total phenol and flavonoid content and AsA content while delaying the increase in the MDA content of mango during storage in both cultivars. In addition, MT dramatically inhibited the enzyme activity of PPO. In contrast, an increase in the activities of antioxidant enzymes (SOD and APX) and PAL and their genes’ relative expression was noticed in MT-treated fruits versus control in both cultivars. However, MT treatment was cultivar dependent in most parameters under study. These results demonstrated that MT treatment could be an essential postharvest treatment in minimizing decay, maintaining fruit quality, and extending mango fruits’ postharvest shelf life by enhancing the physiological and metabolic processes during cold storage.The postharvest deterioration of mango fruits is a critical issue limiting mango storage and preservation due to its climacteric nature. This study evaluated the storage behavior of two mango cultivars and their response to exogenous melatonin (MT, 1000 μmol L Mangifera indica) is one of the most vital tropical fruits globally due to its unique features: its pleasant flavor, attractive color, distinct taste and aroma, and rich nutritional value  [Mango (phenols) ,2. In adphenols) ,3.Most fruits, including mango, that contain very high water content are highly vulnerable to the attack of microbes, which ultimately cause decay in the harvested produce and reduce the shelf life and quality. Therefore, it is crucial to protect them from postharvest decay caused by microbes, bruises, and mechanical damage while improving shelf life and maintaining postharvest quality. Melatonin , a derivative of tryptophan, is a vital indoleamine hormone widely found in various plant species, including apple, strawberry, grape, cherry, banana, kiwi, etc. . MT play−1 maintained fruit firmness, ascorbic acid content (AsA), the content of phenolic compounds, and the antioxidant capacity of mango during storage. Similar research by Liu et al. [PpSODs, PpCATS, PpAPXs, and PpGRs) expression in peaches’ antioxidant defense [MaACO and MaACS genes and delays sharp changes in quality indices in bananas [SOD, CAT, APX, POD) and PAL resulting in increased tolerance to drought stress in Chinese hickory plants [Recently, exogenous MT treatment has been tested as an effective postharvest treatment in different fruits. Some researchers found that MT was effective in promoting postharvest banana ripening , reducinu et al.  stated tu et al. . Howeveru et al.  discoveru et al. , which d defense . In addi defense . MT redu bananas . Furthery plants .‘Guiqi’ and ‘Tainong 1′ used in this research are two of Guizhou province’s most cultivated mango cultivars. ‘Guiqi’, also known as ‘Gui re 82′, is a mango cultivar with a thin peel and is rich in aroma content, TSS, and TA . ‘Tainonv/v) by immersing them for 10 min. Then, they were rinsed using distilled water and air-dried.Mango fruits of the early maturing ‘Tainong 1′ and late-maturing ‘Guiqi’ were harvested from Guizhou Mountain Mango Base farm in Wangmo county, Guizhou province , on 21 July and 8 August, respectively. In total, 240 fruits of ‘Tainong 1′ and ‘Guiqi’ each were harvested at the physiological green mature stage and packed in cardboard boxes. The fruit of each layer was separated with soft fabric to avoid compression injury. After sorting to choose fruits of uniform weight with no visible blemishes, they were transported to the laboratory within 3 h using cardboard boxes via a van with air conditioning at 25 ± 1 °C and 50–60 ± 0.5% relative humidity (RH). Upon arrival at the laboratory, the harvested fruits were treated using sodium hypochlorite  with three replications was used in this research. Each treatment was assigned 56 fruits and replicated three times. According to our pre-experiment, 1000 μmol LThe fruit weight loss was measured using an electronic scale and measured in grams. The weight loss was measured every three days using 20 fruits from each treatment, compared with the initial weight, and expressed in percentage.n = 3). A peel from the mango fruit was removed from both ends, and the firmness was measured in the equatorial region using a 3.5 mm probe. Firmness was expressed as a force in Newton (N).A handheld penetrometer  was used to determine the firmness of the mango fruits  relative to the total number of fruits and expressed in a percentage (percentage). Twenty fruits from each treatment were used to measure decay incidence, and a visual inspection was carried out every three days.2·kg−1·h−1 FW was determined as modifications described by [2, which was measured by a CO2 gas analyzer .The respiration rate expressed as mg COribed by . Six (6)TSS was measured using a refractometer  at room temperature and expressed as Brix percentage. For the measurement of TA, 20 μL of mango juice was pipetted with 980 μL of distilled water, and the concentration of TA was determined using a refractometer. The results were expressed in percentage (%). The ripening index was calculated as the ratio of TSS:TA according to .3 was then added and mixed well with the vortex. The mixed solution was placed in the dark at 30 °C for 60 min, and the absorbance was recorded at 534 nm against the blank. Ascorbic acid was used as the standard, and the AsA content was expressed in a 100 g sample (fresh weight), i.e., mg·100 g−1 FW.In total, 0.5 g of pulp sample was weighed and homogenized with 2 mL of 50 g/L trichloroacetic acid (TCA) in a mortar and grounded in ice condition. The homogenate was centrifuged at 12,000 rpm for 20 min, and the supernatant was collected and used for the assay of the AsA content. Briefly, 1 mL of the sample extract was mixed with 1 mL of 50 g/L TCA solution, and 1 mL of anhydrous alcohol was added and mixed well by vortex. Afterward, 0.5 mL of 4% phosphoric acid was added and vortexed again. After 5 min, 1 mL of 5 g/L BP was added and mixed well, and 0.5 mL of 0.3 g/L FeClv/v) methanol. The homogenate was sonicated for 30 min, followed by centrifuging at 11,000× g for 20 min at 4 °C. Then, the supernatant was transferred to a new tube and stored at −20 °C, used to determine the total phenol and flavonoid content.One gram (1 g) of frozen mango samples was ground using a mortar and pestle and extracted in 5 mL of 95% . After being allowed to stand for 5 min, the mixture was combined with a 10% AlCl3 solution (0.3 mL), while 1 M sodium hydroxide (NaOH) (1.5 mL) and distilled water (1.9 mL) were added after 5 min. The absorbance of the reaction mixture was recorded at 510 nm. Quercetin (QE) was used as a standard, and the results were expressed as mg QE·100 g−1 FW.The total flavonoid content was determined using modifications by the colorimetric method . Methanow/v) TCA solution and centrifuged at 10,000× g for 20 min at 4 °C. Then, 2 mL supernatant and 3 mL 0.67% (w/v) thiobarbituric acid (TBA) were mixed and heated for 20 min in a boiling water bath at 50 °C. After cooling, the absorbance of the supernatant was measured at 450, 532, and 600 nm. Finally, the MDA content was calculated using the formula and expressed as nmol·g−1 FW basis:The MDA content was assayed according to the thiobarbituric acid method  with sli−1, pH 7.8) containing 1% (w/v) polyvinyl polypyridine (PVP). Then, the homogenate was centrifuged at 12,000× g for 30 min at 4 °C, following which the supernatant was divided into 1.5 mL centrifuge tubes and stored at −70 °C as a crude extract for PPO, SOD, and APX determination.First, 0.5 g mango samples were rapidly homogenized on the ice with 8 mL ice-cold sodium phosphate buffer  following the manufacturer’s instructions and absorbance measurement at 290 nm and expressed in U·min−1 sodium phosphate buffer, pH 7.8, 0.3 mL of 130 μmol L−1 methionine solution, 0.3 mL of 750 μmol L−1 NBT solution, 0.3 mL of 100 μmol L−1 EDTA-Na2 solution, 0.3 mL of 20 μmol L−1 riboflavin solution, and 0.1 mL of enzyme extract. The reaction mixture was exposed under a 4000 lx fluorescent lamp for 10 min before measuring the SOD activity at 560 nm. The reaction system’s inhibition of the photochemical reduction of NBT was expressed as one SOD activity unit (U) per min per gram of fresh weight at 50%.The reaction mixture includes 1.7 mL of 50 μmol L−1 H2O2 solution was added to initiate the enzymatic reaction and immediately mixed. The absorbance at 290 nm of the initial reaction was recorded from 15 s after then at 30 s interval for 3 min. The APX enzyme activity was expressed in U·min−1·g−1 FW.A 2.6 mL reaction buffer was added to 0.1 mL of enzyme extract in a test tube. Then, 3 mL of 2 μmol LPPO, PAL, MnSOD, and APX were obtained from National Center for Biotechnology Information (NCBI) and designed by their nucleotide sequence using PRIMER 6 software, and then synthesized by a biological company (Tsingke Biotech (Guiyang) Co., Ltd., Guiyang, China). The qRT-PCR analysis of PPO, PAL MnSOD, and APX genes in mango was performed using gene-specific primers  CO., LTD DP140916, Beijing, China) and further assessed for quantity and quality with a NanoDrop One. Then, 2 μg of extracted RNA were further treated with FastKing-RT SuperMix and further converted into cDNA with the FastKing gDNA Dispelling RT SuperMix according to the manufacturer’s instruction (TIANGEN BIOTECH (BEIJING) CO., LTD; KR170801). Specific primers of  primers  in a totp < 0.05 was used as the statistical difference between means using Tukey’s test. The storage period, cultivar, and treatment were used as the source of variations in this research.Microsoft Excel , OriginPro , and SPSS  were used as the statistical software to analyze the results obtained from the experiment. ANOVA analysis was used to compare the mean difference. p < 0.05).During the storage period, weight loss measured in percentage increased steadily in both cultivars regardless of the treatment A,B. Howep < 0.05). MT treatment delayed fruit firmness in ‘Guiqi’ almost thrice that of the control at 27 d (8.47 ± 1.83 and 2.72 ± 1.47), respectively, while in ‘Tainong 1′, the delay difference between MT and control at 24 d was almost twice (13.52 ± 1.03 and 7.91 ± 1.09), respectively.The firmness of both cultivars showed a constant decreasing trend, which signifies that the fruits became softer with an increased storage period C,D. Howep < 0.05). The highest decay percentages were 65% and 85% for MT treatment in ‘Guiqi’ and ‘Tainong 1′, and 75% and 95% for the control, respectively, for 24 d and 27 d storage periods, respectively.Fruits started to show signs of decay incidence after 12 d in the ‘Guiqi’ cultivar and 3 d in ‘Tainong 1′, and the trend continued to increase until the end of the storage period A,B. MT tp < 0.05). While in ‘Tainong 1′, the difference was significant from 12 d to 24 d, in which MT-treated fruits showed lower respiration at most stages of the storage period. The respiration peak in MT-treated fruits was observed on 18 d (94.56 ± 0.05 mg CO2·kg−1 FW·h−1) in ‘Guiqi’, while in ‘Tainong 1′, it was observed on 12 d (59.96 ± 0.06 mg CO2·kg−1 FW·h−1) as compared to the control with a peak at  in ‘Guiqi’ and ‘Tainong 1′, respectively.Both treatments showed a similar respiration pattern during storage in both cultivars C,D. Howep < 0.05) between the treatments in each cultivar at the end of the storage period (p < 0.5). A continuous increase in the TSS:TA ratio in both cultivars was observed, indicating increasing maturity and ripening during storage. At the end of the storage period, MT treatment did not influence the TSS:TA ratio reduction in both cultivars . At the 27 d, the AsA in ‘Guiqi’ was 0.62 ± 0.03 mg/100 g FW for MT treatment as compared to 0.51 ± 0.02 mg/100 g FW for the control, while at 24 d in ‘Tainong 1’, 0.27 ± 0.01 mg/100 g FW for MT was observed compared to 0.19 ± 0.14 mg/100 g FW for the control. Moreover, the interaction between the treatment and storage period was more significantly affected by MT treatment in ‘Guiqi’ than in ‘Tainong 1’ (p < 0.05) during most stages of the storage period.A continuous decrease in the AsA content was observed in both cultivars throughout the storage period regardless of the treatment A,B. Howep < 0.05) in both cultivars. The lowest content of total flavonoid in the MT treatment was (22.07 ± 1.08 and 18.82 ± 0.26 mg QE/100 g FW) compared to the control (17.13 ± 1.03 and 14.52 ± 0.75 mg QE/100 g FW) in ‘Guiqi’ and ‘Tainong 1’, respectively. A similar trend was noticed in the total phenol content, in which the MT treatment showed higher mean values (32.38 ± 1.04 and 21.80 ± 1.06 mg GAE/100 g FW) than in the control (23.78 ± 2.76 and 18.13 ± 1.06 mg GAE/100 g FW) in ‘Guiqi’ and ‘Tainong 1’, respectively. The interaction between the treatment and the storage period was similar in both cultivars but slightly more pronounced in ‘Tainong 1’ than in ‘Guiqi’.−1) than in the control (1.89 ± 0.04 nmol g−1) on 27 d. In ‘Tainong 1’, MDA slightly increased until 15 d, where a massive increase occurred until the final day of the storage period. However, MDA was lower in the MT-treated fruit (1.01 ± 0.02 nmol g−1) than in the control fruit (1.06 ± 0.02 nmol g−1) on 24 d. The difference between MT and the control in MDA content inhibition was more pronounced in ‘Guiqi’ than in ‘Tainong 1’ at the final stage of storage.MDA, the main product of membrane lipid peroxidation, was measured in both cultivars during the 27 d for ‘Guiqi’ and 24 d for the ‘Tainong 1’ storage periods A, B. In −1 g−1 FW for ‘Guiqi’ and ‘Tainong 1’, respectively) than in the MT-treated fruits  at the end of the storage (p < 0.05). However, the difference between MT and control treatments in response to the PPO activity was more pronounced in ‘Tainong 1’ than in ‘Guiqi’.p < 0.05) than the control treatment, regardless of the cultivar  did not significantly influence the treatment effect and storage period in ‘Guiqi’.−1 g−1 FW) than the control (13.82 ± 0.14 U min−1 g−1 FW) (p < 0.05). At the end of the storage period, the significantly higher APX between the MT treatment and the control was more pronounced in ‘Guiqi’ than in ‘Tainong 1’.As depicted in PAL and PPO genes is depicted in PAL gene (PPO gene (p < 0.05) in the relative expression of both genes. The cultivars displayed slightly different gene expression profiles; ‘Tainong 1’ was more responsive to the PPO gene than ‘Guiqi’. Nevertheless, MT displays a typical pattern of browning-related gene expression in both cultivars.The expression of the PAL gene A,B and dPPO gene C,D durinMnSOD genes (APX genes (p < 0.05) versus the control. Moreover, a slight difference in the gene expression profiles displayed by the two cultivars was observed but the difference was insignificant.OD genes A,B and APX genes C,D in boMango is a seasonal fruit; one of its primary constraints for continuous supply in the market is its climacteric nature. As a result, an attempt to minimize postharvest fruit deterioration, extend shelf life and maintain quality are critical in the supply chain of mango fruits. Concerning storage characteristics, our results showed that MT treatment delayed the increasing weight loss and slowed down the decreasing firmness of fruits compared to the control treatment in both cultivars A–D. Our These above results are supported by the fact that MT treatment significantly improves preservation via a mechanism involving the inhibition of respiration compared to the control in both cultivars C,D. HoweTSS and TA are critical parameters related to fruit ripening. Sugars represent a fundamental component of fruit’s edible quality, predominantly conferring sweetness and significantly influencing consumer satisfaction. According to the present results, MT does not significantly affect the TSS and TA content A–D in boOxidative damage can affect the quality of harvested fruit, which is a process of physiological deterioration . This stMango fruit is rich in several other types of antioxidant phytochemicals, such as phenolic ,35. TherTo determine the physiological and molecular mechanism of the MT effect in two cultivars concerning fruit postharvest quality, an analysis of the enzyme activities related to browning and their relative gene expressions was determined during storage. MT treatment significantly delays the PPO enzyme activities compared to control fruits in the current study, regardless of the cultivar A,B. HoweThe PAL enzyme, responsible for synthesizing polyphenols in plants, is located at a branch point between primary and secondary metabolism . It entePAL genes and inhibited the PPO gene during storage  in both cultivars were higher in MT-treated fruits than in control during storage (MnSOD and APX may be the significant genes regulating fruit’s antioxidant system. The high expression of antioxidant-related genes was essential for maintaining the storage quality of fruit by delaying fruit decay and enhancing the antioxidant system of mango fruits. It was reported that the upregulated expression of SOD, CAT, and POD might contribute to prolonging the shelf life of kiwifruit [CAT, APX, Mn-SOD, and Cu/Zn-SOD in two cultivars of sweet cherry led to improved fruit quality during cold storage [Specific genes associated with antioxidant enzyme activities were unclear in mango and their roles in modulating fruit decay and maintaining quality. To understand the role of genes in the antioxidant system by MT treatment, the expression patterns of two genes were assessed in both mango cultivars during cold storage. The transcription levels of these genes ( storage A–D, but iwifruit , which a storage . The resTo simplify the understanding of the mechanism of MT’s ability to delay mango fruit decay and enhanced the antioxidant system, the results of the experiment are summarize in the schematic diagram  includinIn conclusion, it was observed that the storage behavior of the two cultivars differs slightly in response to MT treatment. ‘Guiqi’ lasted for a 27 d storage period while ‘Tainong 1’ lasted for 24 d. The study also discovered that MT maintained the quality of mango during storage by delaying weight loss, firmness, respiration rate, and decay incidence more than the control treatment in both mango cultivars. This result is evident with reduced PPO activity and enhanced PAL enzyme activity. However, MT did not affect TSS, TA, and the TSS:TA ratio in this study in both cultivars. Furthermore, MT was also found to delay the MDA content, while increasing SOD and APX enzyme activities, which correlates with maintaining a higher concentration of total phenols, flavonoid content, and AsA content during cold storage. The inhibition of the PPO enzyme and increase in the activities of the PAL, SOD, and APX enzymes by MT treatment correlates with the gene expressions. MT treatment was also cultivar dependent on specific parameters being studied. Therefore, this study could give a broader insight into the role of MT in the postharvest technology of mango fruits during storage.


Ziziphus jujuba Mill is the dried ripe fruit of the Rhamnaceae family; it is widely distributed in Shandong, Henan, Liaoning, and other places in China. In folk medicine, it was used to restore vital energy, as a blood tonic, and for the treatment of spleen deficiency. To date, a complete investigation of the compounds of Z. jujuba has rarely been performed. Therefore, a reliable strategy based on UHPLC‐Q‐Exactive Orbitrap MS, combined with trace data acquisition mode  and multiple data processing methods, is necessary for the characterization of compounds in the Z. jujuba. Ultimately, 295 compounds, including 69 flavonoids, 60 alkaloids, 82 phenylpropanoids, 52 organic acids, and 32 other components, were identified in the Z. jujuba; of these, 270 have been reported in Z. jujuba for the first time. This study provides deep insights into the chemistry of Z. jujuba and could be useful for further studies aimed at identifying the factors contributing to the health benefits attributed to this fruit. Z. Jujuba. Eventually, 295 compounds were identified in Z. jujuba; of these, 270 have been reported in Z. jujuba for the first timeIn this study, a reliable strategy based on UHPLC‐Q‐Exactive Orbitrap MS combined parallel reaction monitoring scanning mode was established to characterize the chemistry in  Quinic acid, oleanic acid (OA), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (CGMP), trilobatin, and ferulic acid were acquired by Chengdu Purechem‐Standard Co., Ltd. . The purity of the standard compounds was no less than 98% based on LC‐UV. Z. jujuba was provided by Sun Ten Pharmaceutical Co., Ltd. .Catechin, quercetin, luteolin 7‐O‐glucoside, quercitrin, hyperoside, isoquercitrin, sinomenine, kaempferol‐3‐O‐rutinoside, kaempferol‐3‐O‐neohesperidoside, and quercetin 3‐O‐rutinoside were purchased from the Chengdu Refine Biology Co., Ltd. . Caffeic acid, magnoflorine, phlorizin, procyanidin B2, and The HPLC‐grade acetonitrile and methanol were purchased from Mackin company. The MS grade formic acid was received from the Thermo Fisher Scientific Co., Ltd. (USA). The deionized water was purchased from Watsons Water . Other solvents were of an analytical grade.2.2Ziziphus jujuba (1 g) was accurately weighed and sonication with 20 ml of methanol/deionized water (7:3) was performed for 1 hour; the extracting solution was centrifuged  to obtain the supernatant and dried under the stream of nitrogen at room temperature to obtain the residues, which were reconstituted in 0.1 ml of methanol. Finally, the supernatant was added to the LC–MS for further analysis.The 21 reference standards were accurately weighed and dissolved in methanol to obtain reference standard solutions (0.1 mg/ml).2.32 data of Z. jujuba.An Dionex Ultimate 3000 UHPLC  and the Q‐Exactive Focus Orbitrap MS, equipped with an electrospray ionization (ESI) source, were used for acquiring the MS and MSAn Thermo Scientific Hypersil GOLD™ aQ  was applied for chromatographic separation with a column temperature of 40°C with a flow rate of 0.3 ml/min. The mobile phase consisted of water containing 0.1% formic acid as eluent A and acetonitrile containing 0.1% formic acid as eluent B. The flow rate was set at a linear gradient as follows: 95% A at 0–2 min; 95–85% A at 2–5 min; 85–65% A at 5–20 min; 65–45% A at 20–30 min; 45–20% A at 30–40 min; 20–5% A at 40–45 min; 5–95% A at 45–45 min; and 95% A at 45–50 min. The sample injection volume was 2 μl.2 spectra were obtained by PRM triggered by inclusion ions list .MS analysis was running in both positive and negative ionization modes using an electrospray ionization (ESI). The key parameters were as follows: spray voltage, 3.5 kV (+); spray voltage, 3.2 kV (−); the sheath gas flow rate, 35 arb; aux gas flow rate, 10 arb; capillary temperature, 320°C; heater temperature, 350°C; S‐lens RF level, 60. The scan modes were full MS with a resolution of 70,000 and MS2.43), oxhydryl (OH), methoxyl (OCH3), glucose , arabinose , rhamnose , rutinose , lactose , gentianose , glucuronic acid , and xyluronic acid . Therefore, the molecule could be predicted based on this strategy . Secondly, the sample was injected into UHPLC‐Q‐Exactive Orbitrap MS to gain high‐resolution MS data for the trace components in Z. jujuba; the MS2 data can be obtained by the PRM mode triggered by the inclusion ions list. Thirdly, the data of Z. jujuba were processed by Compound Discover version 3.0 modified metabolism workflow and expected compounds predicted. Finally, the components were identified based on standard fragmentation ions, retention time, and bibliography.To screen and identify components systematically in 3.22 data mining of trace components. The components were identified using Xcalibur version 4.2 and comparing the obtained precursor ions and corresponding fragment ions with the standard and documents.The raw data were captured on the UHPLC‐Q‐Exactive Orbitrap MS with the PRM acquisition strategy, which combines the characteristically fast scanning capability and MSThis study investigated three collision energies: 40%; 60%, and stepped; and normalized collision energies 20%, 40%, and 60%. The results showed that, when the collision energy was 20%, 40%, and 60%, more suitable desugared or dehydrated fragment ions were generated, which was beneficial for detection. In addition, MS and MS/MS features, including flavonoids, phenylpropanoids, and organic acids, were quite abundant in negative ion mode, and alkaloids were found in positive ion mode. Therefore, we decided to choose two ion scan modes for this qualitative analysis. In addition, adding 0.1 formic acid to the mobile phase not only improves the chromatographic peak but also generates more adduct ion, which is helpful for the identification of compounds.3.3Z. jujuba based on UHPLC‐Q‐Exactive Orbitrap MS combined with PRM, the mass spectrum information and retention time of compounds  were identified in the ds Table . Figures3.3.1− at m/z 577.1350 , 289.0715 , 609.1458 , 463.0880 , 463.0880 , 447.0928 , 593.1510 , 593.1512 , 447.0929 , 435.1292 , 435.1297 , 301.0350 , and 593.1297 , respectively; these were characterized as procyanidin B2, catechin, quercetin‐3‐O‐rutinoside, hyperoside, isoquercitrin, luteolin‐7‐O‐glucoside, kaempferol‐3‐O‐neohesperidoside, nicotiflorin, quercitrin, phlorizin, trilobatin, quercetin, and proanthocyanidins, respectively, by comparing the retention times and MS and MS2 information with those of the standards.Compounds 77, 80, 212, 215, 219, 220, 223, 231, 237, 245, 258, 268, and 284 were eluted at 5.32, 5.51, 9.69, 9.27, 9.38, 9.57, 9.92, 10.50, 10.88 11.38, 11.38, 14.26, and 27.02 min, respectively, with the deprotonated molecular ion [M‐H]Compounds 76 and 134, which possessed the same quasi‐molecular ions and characteristic fragment ion as compound 80, were characterized as catechin isomer. Likewise, compounds 103, 135, 208, 211, 224, and 250 were the isomers of procyanidin B2, phlorizin, quercetin‐3‐O‐rutinoside, isoquercitrin, phlorizin, and luteolin‐7‐O‐glucoside, respectively.− at m/z 435.0933 (C20H19O11) and their fragment ions [M‐H]− at m/z 289.0716, 109.0282, and 125.0231, respectively; this indicates that they possessed the catechin moiety. Considering the neutral loss of xyluronic acid reside , they were identified as catechin‐O‐xyluronate. Likewise, compounds 59 and 100 were catechin‐O‐glucoside , 343.0827 ([M‐H‐90]−); they were identified as naringenin‐C‐glucosides  and tentatively identified as naringenin‐O‐glucoside , and further yielded the characteristic fragments ion at m/z 271.0248 and 151.0027, which indicate that they possessed quercetin moiety. Then, they were tentatively characterized as quercetin‐O‐glucoside‐rutinoside ; they were deduced as methylquercetin‐O‐rutinosides —and were identified as myricetin‐O‐rutinoside by referring to the literature , which further produce the characteristic fragment ions at m/z 255.0292, 227.0341. Therefore, they were deduced as kaempferol‐O‐glucoside  were eluted at 6.10, 7.15, and 7.77 min, respectively; compound 101 was accurately characterized as caffeic acid by comparison with the reference standard. Compounds 151 and 175 were caffeic acid isomers. Compounds 46, 52, 61, 70, 84, and 86 yielded the same quasi‐molecular ion [M‐H]− at m/z 341.0878 and were eluted at 3.97, 4.40, 4.78, 5.05, 5.59, and 5.67 min, respectively. All these compounds showed the fragment ions at m/z 179.0338 [M‐H‐glu]−, 161.0232 [M‐H‐glu‐H2O]−, 135.0438 [M‐H‐glu‐CO2]− and might be considered caffeic acid‐O‐glucoside  through the neutral loss of lactose group , gentianose group , arabinose+rhamnose group , lactose+glucuronic acid group .Compounds 101, 151, and 175 with the same deprotonated ions [M‐H]r  and generated the characteristic fragment ion of ferulic acid at m/z 134.0360, 149.0595, 178.0261. Therefore, they were considered ferulic acids‐O‐glucoside , and were characterized as caffeoyl ferulic acid‐O‐glucosides. Similarly, compounds 221 and 251 were identified as caffeoyl ferulic acid‐O‐lactoside and dicaffeoyl ferulic acid‐O‐rhamnoside, respectively.Compound 192 was found at 8.47 min, possessing the quasi‐molecular ion [M‐H]−m/z 325.0929. The main fragment ions m/z 163.0388 were generated based on the neutral loss of glucose reside , and further produced the ion at m/z 119.0488, which was identified as coumaroyl hexose as reported in the literature  and C4H9N group (71 Da), respectively; through data comparison with the literature, they might be considered (s)‐4‐keto‐magnoflorine isomers ‐tembetarine isomers according to the MS and MS/MS spectra , which further gave rise to the product ions at m/z 265.0865 based on the neutral loss of H2O residue (18 Da). These compounds were tentatively proposed as desmethyl magnoflorine , H2O group (18 Da); they were identified as n‐methyllaudanidinium isomers ; they were deduced as nuciferine and chiloenamine, respectively . Based on previously reported literature, it was deduced as coclaurine ; they were identified as coclaurine‐O‐glucosides. Compound 150 was norcoclaurine‐O‐glucoside.Compound 136, detected at 6.82 min, possessed the quasi‐molecular ion [M + H]+ at m/z 282.1489 and the fragment ions m/z 265.1229, 250.0994, and 219.0809; it was identified as O‐nornuciferine, consistent with the literature , was detected at 0.89 min and characterized as quinic acid by comparing the retention time, MS, and MS2 information with the standards. Compounds 9 and 12, possessing the same quasi‐molecular ions and characteristic fragment ion as compound 4, were characterized as quinic acid isomers.Compound 4, possessing the deprotonated ion [M‐H]−m/z 151.0401 and were deduced as vanillin isomers . Likewise, compound 96 was 4‐hydroxybenzoic acid‐O‐rutinoside. Compounds 17, 20, 28, and 30 were observed at 1.95, 2.10, 2.47, and 2.62 min, respectively, possessing the same quasi‐molecular ions [M‐H]− at m/z 153.0193; they were identified as dihydroxybenzoic acid ; these were tentatively identified as dihydroxybenzoic acid‐O‐glucosides.Compounds 44, 45, 205, and 217, with a molecular mass of − at m/z 191.0197 (C6H8O7) and the fragment ion at m/z 111.0073, suggesting that they were citric acid isomers , 328.0453 , and 455.3526 , respectively, and were unambiguously characterized as CGMP, CAMP, and OA, respectively.By comparing the retention time and fragment ions with reference compounds, Compounds 7, 8, and 295 generated a deprotonated molecular ion [M‐H]−m/z 490.0981, generated the main fragment ion at m/z 328.0432 by the loss of a glucose group , and was identified as CAMP‐O‐glucoside. Compounds 1, 3, and 5, found at 0.81, 0.89, and 1.08 min, respectively, possessed the same quasi‐molecular ion at [M‐H]−m/z 341.1089 and were tentatively identified as sucrose isomers based on a previous study (Zhang et al., Compound 10, eluted at 1.37 min, exhibited a quasi‐molecular ion at [M‐H]3.4Z. jujuba. Among them, phenylpropanoid compounds such as ferulic and caffeic acid have attracted wide attention in improving diabetes and treating cardiovascular and cerebrovascular diseases (Chao et al., proanthocyanidins, and quercitrin, have anti‐inflammation, antioxidant, and antibacterial activity (Ivanišová et al., Z. jujuba as it improves immunity and delays aging.Various constituents, including phenylpropanoids, flavonoids, alkaloids, organic acids, and other components, were discovered in Z. jujuba. The result is helpful for the development of Z. jujuba in food, medical, and pharmaceutical industries.As nitrogen‐containing substances of natural origin and of limited distribution, alkaloids possess a complex structure and show significant pharmacological activity. For example, sinomenine and magnoflorine possess anti‐inflammatory, analgesic, antitumor, and other biological active properties, and can be used to treat acute and rheumatoid arthritis (Qian et al., 4Z. jujuba. In this study, 295 compounds including 69 flavonoids, 60 alkaloids, 82 phenylpropanoids, 52 organic acids, and 32 other compounds were tentatively identified via the accurate mass determination of the quasi‐molecular ion and MS2 fragmentation pattern, and of these, 270 have been reported in Z. jujuba for the first time. To our knowledge, this is the first report describing the comprehensive characterization of the chemical constituents of Z. jujuba.In the present study, UHPLC‐Q‐Exactive Orbitrap MS combined with PRM has been confirmed to be a powerful analytical technique for the separation and detection of chemical compounds in All contributing authors have no conflicts of interest to declare.


The adsorption mechanisms of Pb(II) by CMBC include ion exchange, physical adsorption, electrostatic attraction, chemical precipitation, surface complexation, and cation–π bond interaction. Based on the residual phosphorus content and adsorption effect, complexation rather than the chemical precipitation had a greater contribution toward adsorption. Besides, as the concentration of Pb(II) increased, the main adsorption mechanisms likely transformed from chemical precipitation to ion exchange and complexation. CMBC not only had a good effect on Pb(II) removal in the solution, but also immobilized the Pb(II) in soil to restrain plant uptake as well as promote plant growth. The main novelty of this work is providing more insights to the cow manure bio adsorbent on Pb immobilization and phosphorus release. This study is expected to serve as a basis and reference for analyzing the release effects of inherent nutrients and the interfacial behaviors with heavy metals when using CMBC and other nutrient–rich carbon–based fertilizers for pollution control.Cow manure derived biochar (CMBC) can serve as a promising functional material, and CMBC can be regarded as an ecofriendly approach compared to conventional ones. CM bioadsorbent can be employed for heavy metal immobilization (such as for lead) as well as an amendment to increase soil fertility . Few studies have examined the surface interactions between pollutants and bioadsorbents when inherent nutrient release is present. In this work, CMBC was prepared and applied for Pb(II) removal, and the vital roles of released phosphorus from CMBC were comprehensively disclosed. Furthermore, CMBC could immobilize part of the Pb(II) in soil and promote plant growth. CM400 was an effective adsorbent whose calculated  In recent decades, the risks posed by heavy metals have garnered widespread global attention owing to the persistence, toxicity, and bioaccumulation of these metals. Among them, lead (Pb) is persistent in the environment and can result in both decreased growth and reproduction in plants and animals, in addition to neurological effects in vertebrates, while the potential harm of these effects has attracted growing concerns . In Chin43− and CO32− contained and functional groups improve the adsorption via complexation and precipitation [2PO4 or NaOH can significantly improve adsorption [Over recent years, the production of cow manure (CM) has increased with the developments of the large-scale and intensive cattle breeding industry in China . CM is apitation . Thus, cpitation ,18. Compsorption ,20.Even though the presence of phosphorus in cow manure derived biochar has been found , few stu3)2·3H2O, HNO3 (65–68%), and NaOH were used, and all chemicals were of analytical grade. Further, CM was collected from the Laohadu Demonstration Ranch in Mengzi City, Yunnan Province, China.Pb(NO−1) were mixed. Secondly, the mixture was shaken with ultrasound for 30 min and stirred for another 2 h. Thirdly, the mixture was hydrothermally heated at 200 °C for 24 h and then rinsed with acetone. Finally, the mixture was dried at 85 °C. CMBC was obtained via pyrolysis under the protection of N2 at 400 °C, 600 °C, and 800 °C for 1 h, denoted as CM400, CM600, and CM800, respectively.The preparation process for the CMBC is illustrated in 2 adsorption isotherms at 77 K using a Micropore Analyzer . The electron binding energy and elemental valence were analyzed using X-ray photoelectron spectroscopy (XPS) . The functional groups were qualitatively examined using a Fourier transform infrared spectrometer (FTIR) . The amount of the elements in the solutions was determined by inductively coupled plasma mass spectrometry (ICP) . The contents of the elements in the soil and crop were determined via atomic absorption spectrophotometry .The elemental composition of biochar was analyzed with elemental analyzer . The morphology was analyzed with scanning electron microscopy (SEM) and SEM–EDX at 5 kV . The specific surface area (SSA) of biochar was detected by N−1, 25 °C), 5 mL solution was filtered (0.22 μm) at predetermined times , and the phosphorus or Pb contents were determined via ICP. The adsorbent dosage was 1 g·L−1, then 20 mL Pb(II) solution was added , and HNO3 and NaOH solutions were used to adjust the pH of solution (pH = 7.0). After 24 h of oscillation (200 r·min−1), the sample solution was filtered (0.22 μm), and the Pb(II) content was determined via ICP. When the adsorbent dosage was 1 g·L−1 and the initial concentration of Pb(II) was 100 mg·L−1, the effects of pH  and temperature  on the adsorption was explored.Twenty mg of biochar was weighed, then 20 mL deionized water or Pb(II) solution was added. After oscillation (200 r·minThe adsorption capacity of Pb(II) was calculated using Equation (S1) and the experimental results were fitted using the adsorption dynamic model (Equations (S2)–(S5)) and the isothermal adsorption model (Equations (S6) and (S7)). The equations and symbols in the equation are described in the 1: 1 g CM400 application, T2: 1 g CM600 application, T3: 1 g CM800 application) and two control groups  were set up in the pot experiment. Firstly, 2 kg screened soil (5 mm) was placed in a plastic pot. Secondly, to simulate the actual conditions of Pb(II) pollution in Yunnan Province, China [2 were sprayed with Pb(NO3)2 solution , and CK1 was sprayed with 100 mL water. We then ripened the loaded soil by pouring a permeable and dried it naturally for 40 days. Thirdly, Chinese cabbage seeds were directly sown into pots (10 seeds per pot), and 1 g CM400, CM600, or CM800 were applied to three experimental groups, respectively, and then covered with 1–2 cm of soil. Each pot was irrigated daily with 150 mL water, and the budding and growth of Chinese cabbage were observed and recorded. Once the Chinese cabbage was mature enough, the Pb(II) and P contents in roots, stems, and leaves were determined.Chinese cabbage is a type of common model plant, and it has small, fast growth, growth ability, etc. . Three ee, China , the expThe basic elements of CMBC were shown in 2·g−1 for CM400, CM600, and CM800, respectively, which showed that the SSA of CM800 was higher than that of CM400 and CM600. In the range of 400–800 ℃, the SSA of CMBC increased with the preparation temperature, and in the range of 400–600 ℃, SSA increased greatly, while in the range of 600–800 °C, SSA increased steadily but slightly. The SSA is associated with physical properties and significantly affects the physical adsorption capacity. Hence, CM800 had better physical adsorption capacity than the two other types of biochar [The SSA is presented in  biochar . MoreoveThe SEM and SEM-EDS images are shown in 2 (26.6°) (PDF#70–2517), the substances included a combination of embedded metal cations (Ca2+ and Mg2+) and anions (PO43– and CO32–), such as Ca3(PO4)2 (PDF#09–0169), Mg3(PO4)2 (PDF#48–1167), and CaCO3 (PDF#70–0095) [3(PO4)2, Mg3(PO4)2, and Mg3PO4OH (PDF#47–0957), as the high-temperature pyrolysis was conducive to the formation of cationic and anionic conjugates, namely, CM400 was more likely to release inherent substances into solution. The peak of CaCO3 in CM800 decreased, which may be because high pyrolysis temperature (>600 °C) likely caused the decomposition of CaCO3 [The XRD spectrum is depicted in 70–0095) . The hig−1), the C=C stretching peaks (1566 cm−1 and 1455 cm−1), the C–O stretching vibration peak (1384 cm−1), and the C–O–C peak (876 cm−1) were observed [32− and PO43− were located at 1415 cm−1 and 1096 cm−1 [−1 and 780 cm−1 [32−, PO43−, and SiO2 were retained in CMBC. As shown in the FTIR spectra . Although the strong acid condition promoted the release of PO43−, which can form precipitation with Pb(II) [eQ of CM400 was only 42.09 mg·g−1. Therefore, H+ likely competed with Pb(II) for the adsorption sites, and the protonated surface functional groups also exhibited electrostatic repulsion [− was involved in adsorption, such as Pb10(PO4)6(OH)2. The eQ of CM400 was almost unchanged from pH = 5 to pH = 11 (97.01 vs. 98.47 mg·g–1). This not only showed that CM400 functions well in a wide range of pH, but also showed that although the adsorption sites increased steadily, eQ did not vary because Pb(II) was adsorbed. In conclusion, the CMBC had the best adsorption effect for Pb(II) in an alkaline environment.The effect of pH on adsorption is shown in h Pb(II) , Qe of Cepulsion . Under aepulsion . Besides−1, the removal rates were not affected by temperature (98.38% vs. 99.80% at 25 and 45 °C). Once the initial concentration was 500 mg·L−1 or 1000 mg·L−1, the adsorption was enhanced with temperature. For example, when the initial concentration was 1000 mg·L−1, eQ of CM600 was 422.59 mg·g−1 and that of CM800 was 308.63 mg·g−1 at 25 °C, but eQ of CM600 and CM800 were 425.1 and 320.1 mg·g−1 at 45 °C. Therefore, the adsorption by CMBC was endothermic, and CM600 and CM800 were more significantly affected by temperature.The temperature had an impact on adsorption . The eff2 values of three kinds of CMBC were all less than 0.5. Thus, the surface adsorption energy was not uniformly distributed during adsorption, and the internal diffusion is not the sole rate-determining factor as C ≠ 0. For CM400 and CM600, the pseudo-second-order kinetic model fitted the adsorptions better (0.9997 and 0.9998 vs. 0.9999 and 0.9999), by contrast, for CM800, the pseudo-first-order kinetic model described the adsorption slightly more accurately (0.9998 vs. 0.9988). Therefore, physisorption and chemisorption likely coexisted during adsorption by CM400 and CM600, while physisorption dominated Pb(II) adsorption by CM800 [eQ of CM800 than that of CM400 (98.21 vs. 81.28 mg·g−1). Compared with other research data, three kinds of CMBC were all effective Pb(II) adsorbents; among them, CM400 showed the best effect, as its eQ reached 691.34 mg·g−1  better. Therefore, the adsorption of Pb(II) by CMBC was monolayer adsorption [The isothermal adsorption model was presented in Qe, the solution phosphorus content reached 0.620, 0.495, and 0.329 mg·g−1. The results were summarized in 2 values of the pseudo-first-order model for phosphorus release by CM400, CM600, and CM800 were 0.9786, 0.9667, and 0.9296, respectively. Simultaneously, those of pseudo-second-order model were 0.9954, 0.9870, and 0.9786, respectively. Therefore, the phosphorus release by CMBC involved both physical and chemical reactions, which was consistent with the phosphorus release from reed straw biochar modified with phosphate [Pyrolysis temperature was a decisive factor for morphology and crystal structure of phosphorus in biochar . In watehosphate .−1 at 45 °C vs. 0.045 mg·L−1 at 25 °C), which inferred that phosphorus release was endothermic.During Pb(II) adsorption, the process of phosphorus release from CMBC was also affected by the pH and temperature . A highe−1 for CM400, CM600, and CM800, respectively, while when pH = 11, they were 0.423, 0.274, and 0.197 mg·L−1 for CM400, CM600, and CM800, respectively. Under the initial alkaline reaction conditions, phosphorus mostly existed as HPO42− and PO43−, whereas phosphorus mostly existed in the form of HPO42− and H2PO4− under neutral conditions [43− from CMBC, while in an alkaline environment, OH− also precipitated with Pb(II) [The higher the pH, the more of the phosphorus content remained . When pHnditions . Strong h Pb(II) . Thus, tConsidering the adsorption capacity mentioned above, the adsorption capacity significantly exceeded that at pH = 3 and the residual phosphorus content at pH = 11 considerably exceeded that at pH = 3. Thus, under alkaline conditions, the chemical precipitation of phosphate likely contributed less toward adsorption, whereas complexation of functional groups and Pb(II) had a considerably greater contribution. −1, eQ of CM400 was more than that of CM800 (98.38 vs. 81.19 mg·g−1), however, the residual phosphorus content of CM400 was less than that of CM800 (0.05 vs. 0.09 mg·L−1). Thus, when Pb(II) was less than 100 mg·L−1, the main adsorption mechanism likely was chemical precipitation, while the main mechanisms transformed into ion exchange and complexation. When Pb(II) solution was 100 mg·L−1, the residual phosphorus content increased rapidly within 6 h and then decreased. Finally, the residual phosphorus content remained stable 12 h after the onset of adsorption. Because the solution became alkaline when CMBC was placed in, the inherent phosphorus was released slowly at first. Further, Pb(II) was adsorbed by electronegative CMBC via electrostatic attraction, and underwent a complexation reaction and ion exchange with the functional groups. During the adsorption, OH− was consumed, and the reaction solution tended to be neutral. Thus, the phosphorus release accelerated, and the remaining content remained stable.Excluding the pH and temperature, the Pb(II) concentration also affected the phosphorus release . For exa3(PO4)2 (PDF#73–0834), which confirm that inherent phosphorus released and reacted [3 (PDF#74–1101) and PbCO3 (PDF#85–1088), indicating that CMBC adsorbed Pb(II) via precipitation and ion exchange [3 was 3.3 × 10−14, while those of CaCO3 and MgCO3 were 2.8 × 10−9 and 2.6 × 10−5. Therefore, a few Pb ions were exchanged with the precipitates. Moreover, the peak at 27.4° represented C3H6O3Pb, and the peak at 27.8° represented CH2O3Pb (PDF#14–0831), indicating an ion exchange [3(CO3)2(OH)2 (PDF#72–1144) and Pb10(PO4)6(OH)2 (PDF#87–2478), verifying that the hydroxyl group participated in adsorption [The XRD of Pb(II)-loaded CMBC is shown in  reacted . The chaexchange . The preexchange . There wsorption .−1 disappeared, possibly because the oxygen–containing functional group ligand complexed with Pb(II) [−1 shifted, indicating that H+ provided by the carboxyl and hydroxyl groups exchanged with Pb(II) [The FTIR of Pb(II)-loaded CMBC is shown in h Pb(II) . The peah Pb(II) . The C–Oh Pb(II) .The XPS of Pb(II)-loaded CMBC were depicted in + from the basic functional groups. The inherent phosphorus reacted with Pb(II) to form precipitates, and the oxygen-containing functional group ligands on the surface of tCMBC were complexed with the Pb(II). CMBC adsorbed Pb(II) in the solution rapidly and efficiently. The adsorption mechanisms include physical adsorption, ion exchange, cation–π bond interaction, chemical precipitation, electrostatic attraction, and surface complexation . Pb(II) The adsorption mechanisms differed under diverse reaction conditions. Under alkaline conditions, the complexation had a greater contribution than precipitation, and the main adsorption mechanisms transformed from chemical precipitation to ion exchange and complexation.The main adsorption mechanisms for CM400 were chemical precipitation, surface complexation, and ion exchange. By contrast, CM800 had a rich pore structure and a large pore size; hence, its physical adsorption ability was strong.1, T2, T3, and CK1, three, three, two, and three buds were noted, however, none of the CK2 groups sprouted at that time. Application of CMBC can reduce the germination inhibition of Chinese cabbage growing in lead-contaminated soil. It was speculated that CMBC can reduce the biotoxicity of Pb(II) to Chinese cabbage growth by solidifying soil heavy metals during the germination stage of Chinese cabbage. After the Chinese cabbages ripened, the roots, stems, and leaves were cut for digestion and detection. For CK2, the Pb(II) content in leaves was 0.04 mg·L−1, while it was 0.03, 0.02, and 0.00 mg·L−1 for T1, T2, and T3, a reduction of 26.82%, 40.50%, and 99.72%, respectively. The Pb(II) content in stems was 0.04 mg·L−1 for CK2, while it was 0.03, 0.02, and 0.01 mg·L−1 for T1, T2, and T3. Furthermore, the Pb(II) content in roots for CK2 was 0.86 mg·L−1, while it was 0.06, 0.07, and 0.04 mg·L−1 for T1, T2, and T3, a reduction of 92.57%, 91.48%, and 94.92%, respectively. After the Chinese cabbage planted in lead-contaminated soil ripened, by comparing the data of CK2, T1, T2, and T3, it was found that the Pb(II) content in three parts of the Chinese cabbage in the four groups was different. When biochar was applied, the Pb(II) content in the cabbage decreased. The cabbage treated with CM800 (T3) had the least Pb(II) content. In other words, if biochar was applied to lead-contaminated soil and regardless of the type of biochar applied, both had an effect on the Pb(II) content in Chinese cabbage. The application of CMBC to lead-contaminated soil can achieve decontamination, and CM800 was most effective. Therefore, it is speculated that CMBC can reduce the bioenrichment of Pb(II) in Chinese cabbage by solidifying soil heavy metals during the growth stage of Chinese cabbage. In short, the biochar likely immobilized Pb(II) in the soil to reduce the amount of plant uptake [The effects of CMBC on soil decontamination and fertilizer improvement were also investigated, and the contents are shown in −1 for T1, T2, T3, CK1, and CK2, respectively. The phosphorus content in stems was 1.85, 1.88, 1.37, 2.27, and 0.41 mg·L−1. The phosphorus content in roots was 1.83, 1.65, 1.14, 2.39, and 0.62 mg·L−1, respectively. Compared with CK2, application of CMBC increased phosphorus in plants. In summary, the application of CMBC adsorbed Pb(II) in soil, reduced the Pb(II) content in crops, and promoted plant growth effectively.The phosphorus content in leaves was 3.85, 1.96, 1.88, 6.65, and 1.41 mg·LeQ reached 691.34 mg·g−1, and it rapidly adsorbed 98.36 mg·g−1 of Pb(II) within 30 s. The adsorption mechanisms of Pb(II) by CMBC include ion exchange, physical adsorption, electrostatic attraction, chemical precipitation, surface complexation, and cation–π bond interaction. CMBC released phosphorus and the phosphorus released from CM400 reached 0.620 mg·g−1. The phosphorus release was endothermic, and the higher the pH, the more the phosphorus content remained. Based on the residual phosphorus content and adsorption effect, complexation rather than chemical precipitation had a greater contribution toward adsorption. Besides, as the concentration of Pb(II) increased, the main adsorption mechanisms likely transformed from chemical precipitation to ion exchange and complexation. CMBC not only had a good effect on Pb(II) removal in the solution, but also immobilized the Pb(II) in the soil to restrain the plant uptake and promoted plant growth.CM400 proved to be an effective adsorbent; its


Vibrio alginolyticus LHF01 was engineered to efficiently produce poly-3-hydroxybutyrate (PHB) from starch in this study. Firstly, the ability of Vibrio alginolyticus LHF01 to directly accumulate PHB using soluble starch as the carbon source was explored, and the highest PHB titer of 2.06 g/L was obtained in 18 h shake flask cultivation. Then, with the analysis of genomic information of V. alginolyticus LHF01, the PHB synthesis operon and amylase genes were identified. Subsequently, the effects of overexpressing PHB synthesis operon and amylase on PHB production were studied. Especially, with the co-expression of PHB synthesis operon and amylase, the starch consumption rate was improved and the PHB titer was more than doubled. The addition of 20 g/L insoluble corn starch could be exhausted in 6-7 h cultivation, and the PHB titer was 4.32 g/L. To the best of our knowledge, V. alginolyticus was firstly engineered to produce PHB with the direct utilization of starch, and this stain can be considered as a novel host to produce PHB using starch as the raw material. Escherichia coli has been extensively studied as the host bacterium, but the range and efficiency of the biosynthesis applications begin to be limited due to its intrinsic capabilities. Interestingly, Vibrio strains are gaining more and more attentions due to their rapid growth rates. For example, the doubling time of Vibrio alginolyticus and Vibrio parahaemolyticus is reported to be 12-14 min  is an environment-friendly alternative to replace petroleum-based plastics due to its biodegradability, and the most common of which is poly-3-hydroxybutyrate (PHB) . Generalcultures . Recentloduction . Studiesoduction . The mosoduction . There aoduction .Geobacillus sp.  medium, while V. alginolyticus was cultured in TYS50 medium. TYS50 medium contained (g/L) NaCl 50, KCl 0.7, CaCl2·2H2O 1.4, MgSO4·7H2O 6.8, MgCl2·6H2O 5.4, NaHCO3 0.2, yeast extract 1, and peptone 5. When required, 1.5% (w/v) agar was added to obtain the corresponding solid medium. In the process of culturing V. alginolyticus LHF01 for PHB production, soluble starch was firstly used as carbon source, and the shake flask experiments were performed at 37°C and 200 rpm. The culture conditions were optimized in terms of pH, soluble starch concentration, and culture time. The pH was set at 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, and 10, while the starch concentration gradient was 20, 30, and 40 g/L. The samples were taken every 2 hours to determine the cell growth, PHB accumulation, and starch consumption.During all experiments, V. alginolyticus LHF01 was extracted using a Blood & Cell Culture DNA Midi Kit  according to the manufacturer’s protocol. DNA concentration and purity were determined via Qubit fluorometer and Nanodrop 2000 spectrophotometer . DNA integrity was assessed by 1% agarose gel electrophoresis. Whole genome sequencing was performed on the MGISEQ-2000 platform and PacBio Sequel II system at BGI .The genomic DNA of V. alginolyticus LHF01 by conjugation. In brief, the plasmid was firstly transformed into E. coli S17-1 to prepare the donor strain. E. coli S17-1 and V. alginolyticus strains were cultured in LB and TYS50 medium, respectively, with the relevant antibiotics to an OD600 of 1.5-2. Afterwards, 1.5 mL of donor and recipient cells were respectively placed in a centrifuge tube and centrifuged at 6,000 rpm for 5 min. The supernatant was discarded, and the cell pellets were washed once with 1 mL of the corresponding medium, and then suspended in 50 μL of TYS50 medium at a ratio of 1:1. The suspension was cultured for 4-8 h in a 37°C incubator. Finally, an inoculation loop was dipped into the bacterial solution, and then streaked on the TYS50 medium plate with appropriate antibiotics. The plate was cultured in a 37°C incubator for 36-48 h. A single clone was picked out, and put into the TYS50 medium with antibiotics for 24-48 h, and the bacterial solution was verified by 16S rDNA sequencing and stored for follow-up research.The target plasmid was transferred into After treating with boiling water, the soluble starch can be dissolved and its solution is transparent at room temperature. Since starch is turned to blue when exposed with iodine, its consumption could be indicated by adding the iodine solution. To measure PHB accumulation, the strain culture was centrifuged at 10,000 g for 10 min, and washed twice with deionized water. Then the cell pellet was lyophilized to determine the cell dry weight (CDW) and PHB content. CDW was calculated based on the weight of the empty centrifuge tube, the weight of the centrifuge tube containing the cell pellet, and the volume of freeze-dried culture. The lyophilized cell was placed into an esterification tube, reacted with 2 mL of chloroform and 2 mL of esterification solution at 100°C for 4 h, and then shaken with 1 mL of deionized water, and set for stratification. The chloroform phase was taken for gas chromatography (GC) analysis as reported previously .V. alginolyticus strains when overexpressing the PHB synthesis operon, the wild type LHF01 and engineered recombinants were cultured in TYS50 medium with 20 g/L soluble starch for 18 h and observed by transmission electron microscopy (TEM). The shake flask culture was centrifuged to obtain the cell pellet, washed twice with pre-cooled phosphate buffered solution, slowly added with pre-cooled fixative solution, and then fixed for 12 h to obtain the cell samples. Then, the sample was sliced and observed according to the methods reported previously . From the 4,882 predicted genes identified, a total of 4,639 protein-coding sequences (CDSs) were determined, and 128 tRNA genes, 37 complete rRNA genes , and four ncRNAs were annotated.Whole genome sequencing has the highest resolution in bacterial genetic research, and is widely used in microbial traceability, transmission, and population structure identification. Therefore, the genomic characterization of bler 8.3 . The com plasmid . The fulV. alginolyticus LHF01 was constructed, and results indicated that they are all α-amylase and belong to the glycoside hydrolase family 13  was verified by 16S rDNA sequencing.The genetic modification of host cells is one of the most powerful tools in engineering bacteria for microbial fermentation. To begin with, molecular biology research requires introducing foreign plasmids into the target hosts. Generally, developing DNA transformation methods for non-model organism poses unknown challenges. Fortunately, the broad host pBBR1 origin was reported to be transformable into jugation . No chloolyticus , thus plagenesis . The elet strain . Overexpt strain . For exaendocina . To explphaBAPC operon and the wild type control. As shown in phaBAPC can well promote the conversion of soluble starch to PHB, and it also demonstrates the success of genetic manipulation in V. alginolyticus. In addition, the cells of V. alginolyticus LHF01 and LHF01 (phaBAPC) grown on soluble starch were observed by TEM. It was obvious that electron translucent inclusions can be observed in both bacterial cells, yet LHF01 (phaBAPC) has significantly more intracellular particles than V. alginolyticus LHF01 , LHF01 (03416), and LHF01 (03713) were obtained by conjugation. Shake flask experiments were performed to study the consumption rate of soluble starch. As shown in Next, the three amylase genes of V. alginolyticus LHF01, LHF01 (03713) and LHF01 (03713-phaBAPC) all reached the maximum at 18 h, but it was obvious that LHF01 (03713-phaBAPC) had the PHB highest titer of 4.20 g/L at this time, and the PHB content reached 30.66%  was constructed to explore the synergistic effect of PHB synthesis operon and amylase on substrate consumption and PHB accumulation. The PHB titer of d 30.66% . Furtherus LHF01 . It can V. alginolyticus. Strain LHF01 and LHF01 (03713-phaBAPC) were cultivated in TYS50 medium supplemented with 20 g/L corn starch for 18 h, and starch consumption and PHB titer were recorded. Glucose was also employed as the control. Due to the insolubility of corn starch, the accurate CDW cannot be measured. The PHB titer of wide type strain LHF01 was 1.70 g/L, a litter higher than that of 1.33 g/L when glucose was used as the carbon source. In terms of corn starch consumption, starch was not completely consumed after 8 h cultivation by LHF01, showing that the utilization ability of corn starch was weaker than that of soluble starch. It is worth mentioning that, the PHB titer of LHF01 (03713-phaBAPC) reached 4.32 g/L with corn starch, slightly lower than that of 5.85 g/L using glucose as carbon source (It had to be admitted that the cost of soluble starch is relatively high due to the reliance on chemical or other means to denature starch. Therefore, soluble starch was replaced with insoluble corn starch to explore the direct utilization of starch by n source . In addin source .Aeromonas, Bacillus cereus, Massilia, Halogeometricum borinquense, and Halolamina (E. coli and H. bluephagenesis, which cannot assimilate starch naturally. The recombinant E. coli carrying amylase gene from Panibacillus sp. and PHB synthesis genes from R. eutropha accumulated 1.24 g/L of PHB using starch (H. bluephagenesis harboring an amylase with suitable signal peptide was able to grow on corn starch to 9.5 g/L cell dry weight containing 51.5% PHB (V. alginolyticus LHF01 indicated the fastest starch consumption rate. For example, the engineered V. alginolyticus could exhaust 20 g/L corn starch in 7 h and accumulate 4.32 g/L PHB after 18 h cultivation, while the shake flask experiments for PHA production by other reported strains using starch were usually performed for 48 h or even longer. These results demonstrated V. alginolyticus to be a superior chassis for production of PHB from low-cost corn starch. Further development of genetic manipulation techniques and rational engineering will help to improve PHB production from starch by V. alginolyticus.”Several strains have been reported to utilize starch for PHA production, including lolamina . Generalg starch . Enginee1.5% PHB . CompareV. alginolyticus LHF01, and based on this, the utilization of starch by the strain and the overexpression of PHB synthesis operon and amylase were investigated. V. alginolyticus LHF01 was able to consume 20 g/L soluble starch in 5 h shake flask cultivation, achieving PHB titer of 2.06 g/L. Afterwards, the genome of V. alginolyticus LHF01 was sequenced and analyzed. With the overexpression of its phaBAPC operon, PHB titer was improved to 4.55 g/L, and the consumption of soluble starch was increased with the overexpression of its native amylase. Notably, the engineered V. alginolyticus harboring extra amylase and PHB synthesis operon was successfully used to degrade 20 g/L corn starch in 7 h shake flask cultivation, with PHB titer of 4.32 g/L. The use of V. alginolyticus points to the possibility of rapid cell growth for production of chemicals and biopolymers using starch, a sustainable resource with cost much lower than glucose. Further engineering of V. alginolyticus may open new applications in metabolic engineering and synthetic biology research.In this study, a genetic manipulation platform based on conjugation was established for the first-time in


A multiple linear regression analysis revealed a positive association between the plasma retinol concentration and the TBLH BMC . In conclusion, an appropriate plasma retinol concentration and greater intakes of dietary VA and β-carotene may enhance the bone mineral status of children who are aged 6–9 years.The bone mass increases that occur during the period of childhood are of great significance for maximizing the peak bone mass in adults and preventing for osteoporosis. Studies have reported that VA can improve the bone health in adults. Moreover, limited studies have assessed such associations in children. In this cross-sectional study including 426 children, we assessed the children’s plasma retinol concentration by liquid chromatography–mass spectrometry and the dietary intake of VA and carotenoids using a structured Food Frequency Questionnaire. Their bone mineral content and bone mineral density (BMD) were measured using dual-energy X-ray absorptiometry. After adjusting for potential confounders, the restricted cubic spline revealed an inverted U-shaped association between plasma retinol concentration and BMD; the estimated effects on the TBLH BMD per μmol/L increase in the plasma retinol concentration were 1.79 × 10 Osteoporosis is a systemic bone disease that develops when the bone mineral density and quality decrease, the bone microstructure is destroyed, and bone fragility increases . In receStudies have reported that bone homeostasis is influenced by many dietary factors, including vitamin A (VA) ,6,7,8,9.Therefore, in this study, we aimed to assess whether the plasma retinol concentration, dietary VA intake, and carotenoids intake are positively associated with the bone mineral status among healthy children who were aged 6–9 years.This cross-sectional study was approved by the ethics committee of the School of Public Health at Sun Yat-sen University (No. 201549). Written consent was obtained before the enrollment for each participant through his or her parent or legal guardian. We recruited healthy children who were aged 6–9 years from kindergartens and primary schools in Guangzhou between 2015 and 2017 . The recWhole-body dual-energy X-ray absorptiometry (DXA) was performed using the Hologic Discovery W System  according to the manufacturer’s instructions to determine the total body (TB) and the total body less head (TBLH) bone mineral content (BMC), the bone mineral density (BMD) as well as the total body fat (TBF). The coefficients of variation between five consecutive measurements of the TB BMC (BMD), TBLH BMC (BMD) and TBF conducted on 33 children on the same day were 1.09% (1.58%), 1.37% (2.04%) and 1.7%, respectively.Venous blood samples were obtained from the children in the morning after a 10 h of fasting. The plasma was separated within 2–4 h and stored at −80 °C until the analysis was performed. The plasma retinol was isolated and quantified using liquid chromatography–mass spectrometry which was conducted using the Qlife Lab 9000plus system  with the MassHunter Workstation Data Acquisition platform . A pooled plasma sample was analyzed with batches of study samples to monitor them with analytical precision, with approximately day-to-day coefficient of variation values of 7.7%.The children’s dietary intake over the past year was assessed using a structured Food Frequency Questionnaire (FFQ) involving 79 food items that were classified into 20 food groups. The dietary intakes of energy, protein, calcium, phosphorus, VA, and β-carotene were calculated using the 2009 China Food Composition Table .−1·h−1) for each type of physical activity after multiplying it by its duration per day [Detailed sociodemographic data  and information regarding dietary habits  were obtained through the face-to-face interviews of parents and children, and these were conducted by trained interviewers. The child delivery mode was defined as a binary variable: cesarean or vaginal. Their feeding patterns during infancy were classified as breastfeeding or artificial feeding. The household income per month was classified into four categories: ≤8000 Yuan, 8000–15,000 Yuan, >15,000 Yuan, and unknown. The parental education level was classified as secondary or less, university, and postgraduate or above. The weights and heights were measured while the children were wearing light clothes and no shoes. Their physical activity was assessed using a 3-day physical activity questionnaire and calculated by combining the metabolic equivalent  if they are normally distributed or as medians with interquartile ranges if they are not normally distributed. The categorical variables are presented as percentages. The continuous variables were assessed using Student’s The dietary protein, calcium, phosphorus, VA and carotenoids intake levels were adjusted using the residual energy adjustment method. The regression of restricted cubic spline with three knots at the plasma retinol of 10th, 50th and 90th centiles was used to flexibly model the association between the plasma retinol concentration and the bone mineral status after adjusting for age, gender, height, TBF, delivery mode, feeding pattern, maternal and paternal educational level, household monthly income, use of calcium and multivitamin supplement, physical activity, total energy, and energy adjusted dietary intakes of protein, calcium and phosphorus. Multiple linear regression models using the enter method were applied to examine whether the dietary VA (carotenoids) intake were significantly associated with the children’s BMC and BMD. Model 1 was only adjusted for age and sex; model 2 was adjusted using the all of the same confounders as were used in the restricted cubic spline. All of the statistical analyses and plots were performed using R version 4.1.2. The significance level was set at 0.05.2 and 0.609 ± 0.063 g/cm2, respectively. The plasma retinol concentration, dietary VA, α-carotene, β-carotene, and β-cryptoxanthin daily intakes were 1.11 ± 0.28 μmol/L, 676 ± 354 μg RE, 860 ± 883 μg, 2408 ± 1615 μg and 97.6 ± 103 μg, respectively.In total, 426 children who were aged 8.0 ± 1.0 years were included in this cross-sectional study. As presented in −2 g/cm2, 95% CI: 4.21 × 10−3–3.16 × 10−2 g/cm2) until 1.24 μmol/L, and then, it started to decline  afterwards . Consistently, there was also an inverted U-shaped relationship which was observed for the TB BMD ; the estimated average effects per μmol/L increase in the plasma retinol concentration increase were 3.05 × 10−2 g/cm2 (95% CI: 1.15 × 10−2–4.95 × 10−2 g/cm2) below 1.17 μmol/L and −2.37 × 10−2 g/cm2 (95% CI: −4.58 × 10−2 – −1.52 × 10−3 g/cm2). These similar association did not have statistical significance for the TB BMC . However, a significantly positive linear relationship between the plasma retinol concentration and the TBLH BMC was detected; per μmol/L increase in the plasma retinol concentration resulted in a 1.89 g (95% CI: 1.64 × 10−1–3.62 g) increase in the TBLH BMC (p = 0.032).In −3 g/cm2 (95% CI: 1.56 × 10−3–1.02 × 10−2 g/cm2) increase in the TB BMD (p = 0.008). The TB BMD increased by 4.47 × 10−3 g/cm2 (95% CI: 5.47 × 10−5–8.88 × 10−3 g/cm2) with every 1 SD increase in the dietary β-carotene intake (p = 0.048). However, no significant association was observed between the dietary α-carotene, β-cryptoxanthin intake and the bone mineral status.As presented in This study evaluated the associations of plasma retinol concentration and dietary VA and carotenoids intake with the bone mineral status in children who were aged 6–9 years. Additionally, the results indicated that vitamin A nutritional status was a key determinant of bone mass in children.p < 0.001) [2 increase in the BMD of both the femur neck and the entire hip [Maggio et al. assessed 90 participants who were aged ≥ 55 years and reported that the plasma retinol concentration was lower among women with osteoporosis (2.020 μmol/L) than it was among the controls (2.290 μmol/L) (< 0.001) . Anothertire hip . In conttire hip . Michaëltire hip . Our restire hip ,24. In a2 increase in the total hip BMD of the premenopausal women (p = 0.026) [In terms of the dietary VA intake, which is consistent with our study, a case–control study including 1452 elderly Chinese patients with newly diagnosed hip fractures and control participants reported that a high dietary VA intake  was associated with a reduced risk of hip fracture  . However= 0.026) . HoweverThe following mechanisms may explain the associations that we observed in our study. An adequate VA intake (retinol or retinoic acid) is of great importance in maintaining bone homeostasis, which effectively suppresses the over-secretion of the parathyroid hormone ,29. It mTo the best of our knowledge, this study is the first to comprehensively describe the associations of plasma retinol concentration and dietary VA (carotenoids) intake with bone mineral status among children who are aged 6–9 years. However, there exist several limitations to our study. Firstly, the causality of the association between VA and bone mineral status could not be accurately demonstrated using the cross-sectional study design. Secondly, some nutrients from the supplements or fortified foods were not included in the calculation of the dietary nutrient intake in our analyses. However, the use of multivitamins and calcium supplements were applied as binary categorical variables for adjustment. Thirdly, the carotenoids contents in foods may differ, depending on the plant’s cultivation, harvesting, processing and storage conditions. Moreover, the dietary VA intake was calculated using the 2009 China Food Composition Table, and thus, the findings may not be generalizable to other countries. This may also have contributed to the heterogeneity among the different studies. Fourthly, our sample size was small, and all of the participants were from Guangzhou and they had a narrow age range, thus, caution should be exercised when one is extrapolating our results to children in other areas and of different ages. Fifthly, although many covariates were included in our analysis models, the residual confounding effects due to the unmeasured or incompletely measured factors cannot be excluded.In summary, an appropriate plasma retinol concentration and a greater intake of dietary VA and β-carotene may result in better bone health status among children who are aged 6–9 years, according to our study.