Sonchus arvensis L. leaf extracts both in vitro and in vivo.Malaria continues to be a global problem due to the limited efficacy of current drugs and the natural products are a potential source for discovering new antimalarial agents. Therefore, the aims of this study were to investigate phytochemical properties, cytotoxic effect, antioxidant, and antiplasmodial activities of S. arvensis L. leaf were prepared by successive maceration with n-hexane, ethyl acetate, and ethanol, and then subjected to quantitative phytochemical analysis using standard methods. The antimalarial activities of crude extracts were tested in vitro against Plasmodium falciparum 3D7 strain while the Peter's 4-day suppressive test model with P. berghei-infected mice was used to evaluate the in vivo antiplasmodial, hepatoprotective, nephroprotective, and immunomodulatory activities. The cytotoxic tests were also carried out using human hepatic cell lines in  (MTT) assay.The extracts from n-hexane, ethyl acetate, and ethanolic extracts of S. arvensis L. leaf exhibited good in vitro antiplasmodial activity with IC50 values 5.119 ± 3.27, 2.916 ± 2.34, and 8.026 ± 1.23 μg/mL, respectively. Each of the extracts also exhibited high antioxidant with low cytotoxic effects. Furthermore, the ethyl acetate extract showed in vivo antiplasmodial activity with ED50 = 46.31 ± 9.36 mg/kg body weight, as well as hepatoprotective, nephroprotective, and immunomodulatory activities in mice infected with P. berghei.The S. arvensis L. leaf ethyl acetate extract against P. falciparum and P. berghei as well as the antioxidant, nephroprotective, hepatoprotective, and immunomodulatory activities with low toxicity. These results indicate the potential of Sonchus arvensis L. to be developed into a new antimalarial drug candidate. However, the compounds and transmission-blocking strategies for malaria control of S. arvensis L. extracts are essential for further study.This study highlights the antiplasmodial activities of  Malaria is an endemic disease in tropical areas of Asia, Africa, and Central and South America, and is known to spread even in vaccinated populations . In 2020Cinchona succirubra L., as well as relatively newer medicines, such as artemisinin, which is produced from Artemisia annua L.  into each well of parasitized culture suspension. The parasitized cultures with 0.5% (v/v) of DMSO served as negative controls. The plates containing parasite cultures were incubated in an incubator  for 48 h. The antiplasmodial assay of each extract were carried out in three replicates (n = 3). Afterward, the suspensions were collected, thinly smeared on glass slides, fixed with methanol, and stained with 10% Giemsa. The number of parasites was counted under a microscope and compared with the negative control to determine the extent of parasite growth inhibition in 5000 of total erythrocytes. The equation for calculating parasitemia, inhibition, and growth percentage used the equation method as described in a previous study  2, 5-diphenyl tetrazolium bromide (MTT) assay as described by Fosenca et al. . MTT asssample was the absorbance sample at 560 nm-Absorbance sample at 750 nm and Acontrol was the absorbance DMEM medium. The percentage of cell viability was then plotted and regressed linearly to obtain the CC50 values. The selectivity index (SI) values were calculated based on the ratio between the CC50 value of cytotoxicity and antiplasmodial activity P. falciparum 3D7 from each extract (IC50).where AP. falciparum 3D7 was selected to subsequently analyze the antiplasmodial activity against P. berghei (the mice-infected Plasmodium) with Peter’s method [P. berghei ANKA was obtained from the Eijkman Institute of Molecular Biology . Blood infected with P. berghei ANKA was taken from mice with 20% parasitemia and diluted with phosphate-buffered saline. Swiss mice Mus musculus of BALB/c strain  were intraperitoneally injected with 0.2 mL blood (1 × 106 ANKA parasitized erythrocytes) and randomly divided (n = 7 per group) into four experimental groups and three control groups . The experimental groups were orally treated with 0.25 mL single dose of 1, 10, 100, or 200 mg/kg BW of leaf extract (in 0.5% sodium carboxymethyl cellulose (Na-CMC)) two times per day for four days for antiplasmodial assay and continued seven days for biochemical analysis. The negative and positive control groups were treated with 0.5% Na-CMC suspension and 10 mg/kg BW of chloroquine diphosphate, respectively. The normal control group was the uninfected and untreated mice group  was calculated with Probit analysis. The ED50 was calculated from each replication, and then averaged getting the mean and deviation standard.The extract with the highest antiplasmodial activity against s method . The strAfter seven-day treatments, blood samples (0.5–0.75 mL) were collected from the left ventricle of each mouse into 1.5-mL microtubes and left standing at room temperature for 2 h. Then, serum was isolated by centrifugation at 3000 rpm for 20 min. The levels of serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT) in obtained serum were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits  to assess the hepatoprotective effects of the selected extract on infected mice. For analyzing nephroprotective effects, blood urea nitrogen (BUN) and creatinine levels were measured using commercial ELISA kits . The level of tumor necrosis factor-alpha (TNF-α) and interleukin 10 (IL-10) in serum was also analyzed to investigate the immune response of treated/control mice using commercial ELISA kits . The replication of samples was four to seven times (Table 50 of antioxidant and CC50 of cytotoxicity were counted using regression linearly (Microsoft Excel). The Probit analysis was conducted to calculate the IC50 and ED50 values. Statistical significance was determined with the one-way analysis of variance (ANOVA) continued with Duncan Multiple Range Test (DMRT) for IL10 and TNFα, with a nonparametric independent t-test for SGOT and SGPT, and Kruskal–Wallis continued with Mann Whitney test for BUN and creatinine data. The level of significance was set at 0.05. All statistical analyses were conducted using IBM SPSS Statistics for Windows, version 20.0. .Data are expressed as the mean ± standard deviation (SD). The ICn-hexane extract . Under 366-nm UV light, there were five separate spots with Rf values of 0.14, 0.19, 0.24, 0.35, and 0.53. After staining using p-anisaldehyde sulfuric acid, three separate purple stains were seen, with Rf values of 0.31, 0.59, and 0.71  had the highest in vitro antimalarial activity, followed by the n-hexane and ethanol extracts.The ICSonchus arvensis L. extracts to hepatocytes was determined. From highest to lowest, the CC50 values of the extracts were 1420.88 ± 20.88, 437.39 ± 7.46, and 778.77 ± 10.53 µg/mL for n-hexane, ethanol, and ethyl acetate extract, respectively , nephroprotective (BUN and creatinine), and immunomodulatory (IL-10 and TNFα) effects . However, there was no different significant between normal control (40.76 ± 9.59 U/mL), positive control (32.92 ± 6.07 U/mL), and treatment groups (31.11 ± 13.16 U/mL at a concentration of 200 mg/kg BW)  and alanine aminotransferase (SGPT) into the blood to respond with liver injury. Therefore, raising the enzyme levels in the blood is the signaling to indicate the damage of liver. As compared to the negative control (201.87 ± 91.73 Ug/mL), serum SGOT levels of mice infected with p < 0.05)  and treatment with ethyl acetate extract at 1 mg/kg BW (32.37 ± 13.6 U/mL) and 10 mg/kg BW (22.74 ± 2.08 U/mL), serum SGPT levels were significantly decreased in the groups treated with 100 and 200 mg/kg BW  as well as the positive and normal control groups . Overall, the SGOT and SGPT levels significantly decreased in the treatment groups compared with the negative control group (Plasmodium berghei infected mice that was given Na-CMC) group (7.00 ± 0.26 mg/mL), the serum BUN levels of the infected mice were significantly decreased by treatment with the ethyl acetate extract at 1, 10 100, and 200 mg/kg BW , p < 0.05 . In addition, as compared to the negative control group (1.99 ± 0.29 mg/mL), serum creatinine levels were significantly decreased by treatment with the ethyl acetate extract at 1, 10, 100, and 200 mg/kg BW , p < 0.05 , while there was no significant difference as compared to the normal control group , serum IL-10 levels were significantly increased in the positive control group (131.09 ± 8.13 pg/mL) and slightly increased in the groups treated with the ethyl acetate extract at 1, 10, 100, and 200 mg/kg BW . There were also significant differences among the negative, normal control, and positive control groups . Collectively, these results suggest that ethyl acetate extract of S. arvensis L. enhances the immune response of mice against P. berghei infection , serum levels of TNFα were significantly increased by treatment with 10, 100, and 200 mg/kg BW , the use of traditional medicines continues to increase worldwide. Traditional medicines are rooted in Indonesian culture and history, although many traditional treatments have not been scientifically validated. Among the strategic objectives proposed by the WHO, the safety and efficacy of traditional medicines are primary goals before integrating traditional drugs in modern healthcare .S. arvensis L. is the seventh most popular medicinal plant for treating various diseases in Indonesia, especially in Java and Bali [S. arvensis L. callus is reported to possess antiplasmodial activities [S. arvensis L. leaf.and Bali . Althougtivities , 18, theS. arvensis L. leaf was extracted by successive maceration with n-hexane, ethyl acetate, and ethanol. From each solvent, different extract weights were obtained. Each of the S. arvensis L. extracts was screened for the presence of phytochemicals. The ethanol extract contained flavonoids, alkaloids, terpenoids, saponins, and polyphenols, while the ethyl acetate extract included flavonoids, alkaloids, terpenoids, and polyphenols, and the n-hexane extract contained flavonoids, alkaloids, and terpenoids.One kilogram of dried 3, the color of the solution changed to dark green, indicating the presence of tannins. Meanwhile, the presence of saponins was confirmed if the foam extract did not disappear after the addition of distilled water and shaking [The Wilstatter "cyanidin" test confirmed the presence of flavonoids, while testing of the extract showed the presence of alkaloids, as indicated by the formation of a white precipitate after the addition of Mayer reagent. The Liebermann–Burchard test results confirmed the presence of terpenoids, as indicated by the yellow color of the solution. After adding a few drops of 10% FeCl shaking .n-hexane extracts. These findings are consistent with similar studies conducted by Khan [n-hexane extract of S. arvensis L. [S. arvensis L. For example, flavonoid and phenolic compounds possess antioxidant activities [Polyphenols were present in the ethanol extract, whereas relatively large amounts of terpenoids were confirmed in the ethyl acetate and  by Khan  and Seal by Khan . Many trensis L. . Additiotivities , saponintivities , and tertivities .S. arvensis L extract showed antioxidant activities. The ethyl acetate and ethanol extracts exhibited potent antioxidant activities (IC50 < 50 µg/mL) [50 value 8.27 ± 4.93 µg/mL and 12.36 ± 10.40 µg/mL respectively. While n-hexane extract had moderate antioxidant activity (101 > IC50 < 250 µg/mL) [50 value (the antioxidant activity) of ethyl acetate extract was lower than ascorbic acid as standard (22.63 ± 1.40 µg/mL)  , with IC0 µg/mL) . The IC5tense L. , Callisiaf juice , and Cen L. leaf  that rep sources , 32.50 value less than 10 μg/mL is considered to indicate the high activity, while 10 < IC50 ≤ 25 μg/mL can be regarded as moderately active and values > 25 μg/mL are deemed inactive [50 values < 10 μg/mL; therefore, they were primarily considered as the new candidates for antimalarial-drug development. The IC50 values of the ethyl acetate extract, n-hexane, and ethanol extracts were 2.916, 5.119, and 8.026 μg/mL, respectively. The industry standardizes the IC50 value of in vitro antiplasmodial activity, a pure compound is said to be active as antiplasmodial activity if the IC50 value is below 10 μg/mL [The antioxidant activity of the ethyl acetate extract was as good as the in vitro antimalarial effect. In general, an ICinactive , 34. AllS. arvensis L. extracts were evaluated by calculating the ratio of cytotoxicity with human hepatic cell lines (CC50) to in vitro antiplasmodial activity expressed as IC50 (selectivity index SI = CC50/IC50). A higher SI, theoretically, indicates greater drug effectiveness and safety for the treatment of plasmodial infections. An ideal drug would be cytotoxic only at very high concentrations and have antiplasmodial activities at low concentrations, thus yielding a high SI value and eliminating the plasmodial target at concentrations well below the cytotoxic concentration [50 values of extracts toxicity were 1420.88 ± 20.88, 778.77 ± 10.53, and 437.39 ± 7.46, μg/mL, and then the SI value were 277.57 ± 5.77, 97.03 ± 13.13, and 150 ± 3.62 for n-hexane, ethanol, and ethyl acetate extract respectively. de Souza et al. [S. arvensis L.leaf extracts exhibited low toxicity. Nurianti et al. [S. arvensis L had no toxic effects, and Harun et al. [S. arvensis L. was not toxic to healthy male albino rats. The antioxidant activities suggest that these extracts are relatively non-toxic because oxidative stress represents an imbalance between the production of free radicals and the ability of a biological system to readily detoxify reactive intermediates or repair the resulting damage [The toxicity of ntration . The IC5a et al.  mentionen et al.  revealedS. arvensis L. was chosen for the assessment of in vivo antiplasmodial activity because it exhibited the highest antioxidant and in vitro antiplasmodial activities, with IC50= 8.27 and 2.92 μg/mL respectively. In vivo antiplasmodial activity can normally be classified as moderate, good, and very good if an extract displayed percentage inhibition equal to or greater than 50% at a dose of 500, 250, and 100 mg/kg BW per day, respectively [P. berghei-infected mice given orally 50–250 mg/kg/day of extract exhibiting inhibition percentage > 60% are considered to be active or very active, and those exhibiting inhibition percentage > 30% are considered to be moderately active [S. arvensis L. showed excellent in vivo antiplasmodial activity below 100 mg/kg/day with an ED50 of 46.31 ± 9.36 mg/kg. The ED50 of ethyl acetate extract of S. arvensis L. was higher than the ethanolic extract of H. annuus root has an ED50 value of 10.6 ± 0.2 mg/kg [Tagetes erecta L. and Synedrella nodiflora (L.) Gaertn. extract can significantly suppress parasitemia in malaria-infected mice by 50.82% and 57.67% respectively at 400 mg/kg BW dose [S. arvensis L. could be developed as an antiplasmodial agent.Moreover, the ethyl acetate extract of ectively . P. bergy active , 34. Bas.2 mg/kg  but loweS. arvensis L. Many studies have reported that S. arvensis L. extracts exhibited antioxidant [S. arvensis L. against P. berghei infection in mice.Furthermore, blood was collected from the experimental mice to determine the nephroprotective, hepatoprotective, and immunomodulatory activities after 7 days of treatment with ethyl acetate extract of ioxidant , hepatopioxidant , nephropioxidant , and immioxidant  activitiAn increase in SGPT and SGOT serum levels indicates liver damage , and a rS. arvensis L showed nephroprotective, hepatoprotective, and immunomodulatory activities in mice infected with P. berghei. The result is very interesting because the pathogenesis caused by P. berghei is multifactorial and has not been well characterized. There were several hypotheses suggesting that erythrocyte cytoadherence, proinflammatory response, nephrotoxicity, and oxidative stress are involved in the pathogenesis of P. berghei [P. berghei parasites will infect erythrocytes and activate cytokines of phagocytic cells and endothelial cells to produce TNF-α, IL-10, IFN-γ, and free radicals . Free radicals are molecules with one unpaired electron in their outer orbit which makes the molecule unstable [P. berghei, the use of antioxidants in the body will increase, causing the number of antioxidants in the body to decrease [P. berghei supported that the S. arvensis L. ethyl acetate extract is active as antiplasmodial. Particularly, the S. arvensis L. leaf ethyl acetate extract increases the mice immune response to P. berghei infections. It is very valuable for the further investigation of the in vivo antioxidant activity and cytotoxicity of the S. arvensis L. leaf as antiplasmodial drug candidate.Overall, the serum levels suggested that the ethyl acetate extract of  berghei , 45. Fre berghei . In addiunstable . Free raunstable . The invunstable . Oxidatiunstable . The numdecrease . The bioP. berghei. This study highlighted that S. arvensis L. crude extract had antimalarial activity. In summary, the results suggest that ethyl acetate extract of S. arvensis L. could be used to develop new antimalarial drugs in the future from a natural resource.The comprehensive tests and discussions in this study confirmed that ethyl acetate extract possessed antiplasmodial both in vitro and in vivo with nephroprotective, hepatoprotective, and immunomodulatory activities in mice infected with S. arvensis L. both in vitro and in vivo as well as the antioxidant, nephroprotective, hepatoprotective, and immunomodulatory activities with low toxicity. It was strongly suggesting the potential as an antimalarial drug. These findings lay a foundation for further investigations of new antimalarial compounds for future pharmaceutical applications. Further research, including bioassay-guided fractionation, was also recommended to identify new antimalarial drug candidates.The results of this study confirmed the antiplasmodial activity of ethyl acetate extract of


Bacillus pumilus HR10, inhibited the Sphaeropsis shoot blight disease of pine, and the fermentation broth of HR10 strain contained protein antifungal substances. The optimal formulation of the fermentation medium for the antagonistic substance of B. pumilus HR10 was finally obtained by single-factor test, Packett-Burman test, steepest ascent test and Box-Behnken Design (BBD) response surface test, and the best formulation of the fermentation medium for the antagonistic substance of B. pumilus HR10 was 12 g/L corn meal, 15 g/L beef extract and 13 g/L magnesium sulfate, with a predicted bacterial inhibition rate of 89%. The fermentation filtrate of B. pumilus HR10 cultured with the optimized medium formulation was verified to have an inhibition rate of (87.04 ± 3.2) % on the growth of Sphaeropsis sapinea by three replicate tests. The antagonistic crude protein of B. pumilus HR10 were further isolated and identified using HiTrap Capto Q strong Ion-Exchange Chromatography and LC–MS-MS, and it was speculated that glycoside hydrolase (Ghy), beta-glucanase (Beta), arabinogalactan endonuclease β-1,4-galactanase (Arab), and immunosuppressant A (ImA) are proteins with antagonistic activity against S. sapinea in the B. pumilus HR10.A previous study found that a biocontrol bacterium, The online version contains supplementary material available at 10.1186/s13568-022-01401-1. Fermentation medium with high antagonistic activity of HR10 strain was determined.Antagonistic crude protein of HR10 strain purified by Ion-Exchange Chromatography.Composition of the antagonist protein of HR10 strain was analyzed using LC-MS-MS.The online version contains supplementary material available at 10.1186/s13568-022-01401-1. Bacillus spp. has received much attention from researchers because of its high resistance, wide range of habitat and high safety, and plays an important role in the biological control of plant diseases  design, steepest ascent test combined with Box-Behnken Design (BBD) response surface methodology, and further identified the antagonistic substances of HR10 strain by acid precipitation method, ammonium sulfate precipitation method and organic solvent precipitation method using the screened efficient antagonist medium, and then identified the antagonistic substances of HR10 strain by chromatography and mass spectrometry, in order to screen the antagonistic protein of HR10 strain and lay the foundation for the work of controlling pine blight disease.In a previous study, the fermentation filtrate of Bacillus pumilus HR10 was originally isolated from the rhizosphere soil around mycorrhizal seedlings of Pinus thunbergii , No. 2 ,No. 3 , No. 4 , No. 5 , No. 6   Sa , and LB Different carbon sources The antagonistic activity of the fermentation filtrate of HR10 strain against S. sapinea was determined by replacing the yeast extract in LB medium with 5 g/L of sucrose, soybean meal, soluble starch, mannitol, glycerol, lactose, corn flour and corn paste, respectively, and using LB medium as the control.Different nitrogen sources The antagonistic activity of the fermentation filtrate of HR10 strain against S. sapinea was determined by replacing the tryptone in LB medium with 10 g/L of beef extract, ammonia water, soybean meal, peptone, ammonium nitrate, ammonium nitrite, ammonium sulfate and corn steep liquor, respectively, and using LB medium as the control.Different inorganic salts The antagonistic activity of the fermentation filtrate of HR10 strain against S. sapinea was determined by replacing NaCl in LB medium with 10 g/L of potassium dihydrogen phosphate (KH2PO3), magnesium sulfate (MgSO4), manganese sulfate (MnSO4), ferrous sulfate (FeSO4), calcium chloride (CaCl2), potassium chloride (KCl), zinc sulfate (ZnSO4), and calcium carbonate (CaCO3), respectively, and using LB medium as the control.The PB experimental design was performed using Minitab 16 software for the five components of the screened media formulations  was performed on the fraction with higher inhibition rate, as well as the protein concentration and protease activity were determined.Ammonium sulfate was added to the fermentation filtrate of HR10 strain to reach a final saturation of 90%, and the precipitate was collected by centrifugation at 10,000 r/min for 30 min at 4 °C for 24 h. The resulting precipitate was dissolved in 5 mL of 10 mmol PBS buffer (pH 7.5) and volume fixed to 20 mL, followed by overnight dialysis. The dialysate was purified using a protein purification system strong Ion-Exchange Chromatography (HiTrap Capto Q), and the column was equilibrated with PBS buffer (pH = 6.0) before loading, and then the fractions were collected by linear gradient elution with PBS buffer  at a flow rate of 1 mL/min  method. Then the protease activity of the fermentation filtrate of HR10 strain, the crude protein precipitated by 90% saturated ammonium sulfate and the protein purified by ion chromatography were determined by the Folin-phenol Reagent Method. The 1% casein solution was mixed with 125 μL of fermentation filtrate of HR10 strain, 90% saturated ammonium sulfate precipitated crude protein or purified protein by ion chromatography, held at 37 °C for 10 min, and 250 μL of trichloroacetic acid was added. The supernatant was extracted and added with 2.5 mL of sodium carbonate solution (0.4 mol/L) and 0.5 mL of Folin-phenol reagent, and the reaction was developed at 37℃ for 20 min, and the absorbance value at 680 nm was detected. The amount of enzyme that produces 1 μg of tyrosine per minute of hydrolysis of casein at 37 ℃ and pH = 7.4 is defined as one viability unit (U/mL). Specific enzyme activity is the viability unit per mg of enzyme protein (U/mg); the purification multiple is the ratio of specific enzyme activity after each purification step to the specific enzyme activity of the fermentation filtrate.g for 10 min, and the supernatant was taken for injection. Separation was performed by Thermo UltiMate 3000 UHPLC. The sample was first enriched in trap column and desalted, and then entered a self-packed C18 column  and separated at a flow rate of 300nL/min by the following effective gradient: 0–5 min, 5% mobile phase B ; 5–45 min, mobile phase B linearly increased from 5 to 25%; 45–50 min, mobile phase B increased from 25 to 35%; 50–52 min, mobile phase B rose from 35 to 80%; 52–54 min, 80% mobile phase B; 54–60 min, 5% mobile phase B. The nanoliter liquid phase separation end was directly connected to the mass spectrometer. The peptides separated by liquid phase chromatography were ionized by a nanoESI source and then passed to a tandem mass spectrometer Q-Exactive HF X  for DDA (Data Dependent Acquisition) mode detection. The main parameters were set: ion source voltage was set to 1.9 kV, MS1 scanning range was 350–1500 m/z; resolution was set to 60,000; MS2 starting m/z was fixed at 100; resolution was 15,000. The ion screening conditions for MS2 fragmentation: charge 2+ to 6+, and the top 30 parent ions with the peak intensity exceeding 10,000. The ion fragmentation mode was HCD, and the fragment ions were detected in Orbitrap. The dynamic exclusion time was set to 30 s. The AGC was set to: MS1 3E6, MS2 1E5.The identification of protein gel strips is to separate the sample proteins by gel electrophoresis, then obtain the protein gel strips at different positions on the film, extract the peptides after enzymatic digestion, and then uses mass spectrometry to obtain the mass spectrum of the proteins in these gel strips, and finally uses the protein identification software to identify the proteins in the samples. Digestion of 100 µg protein samples using trypsin, followed by freeze-drying of the digested and desalted peptide liquid. The dried peptide samples were reconstituted with mobile phase A , centrifuged at 20,000 The protein identification used experimental MS/MS data and aligned them with theoretical MS/MS data from database to obtain results. The whole process started from converting raw MS data into a peak list and then searching matches in the database with Mascot 2.3.02 software. The search results were subject to strict filtering and quality control, and possible protein identifications were produced. Finally, from the final protein identification list, GO functional annotation analysis and iBAQ quantification were performed.S. sapinea, therefore, the LB medium with yeast extract (5 g/L) as the main carbon source, tryptone (10 g/L) as the main nitrogen source and NaCl (10 g/L) as inorganic salt was selected as the basal medium for subsequent medium optimization. When replacing yeast extract in LB medium with different carbon sources, the inhibition rate showed different degrees of decrease  = 80.408 + 1.642A + 2.492B + 1.358C−0.14D + 1.225E. The P-value of the model was 0.002 and the complex correlation coefficient R2 = 0.9349. Among the five factors to be investigated, the effect of peptone on the inhibition rate of fermentation filtrate of HR10 strain was not significant, and the remaining four factors were, in descending order of importance, corn flour > yeast extract > beef extract > MgSO4, and all of them had positive effects , beef extract (C) and MgSO4 (E) as the influencing factors  = 1.53A−2.47B−22.20C−21.13AB−23.88AC−23.77BC−23.59A2−22.19B2−25.74C2.The BBD experimental design and the response values were performed using the inhibition rate of S. sapinea growth at 11.748 g/L for corn flour, 14.917 g/L for beef extract, and 12.860 g/L for magnesium sulfate, with a predicted value of 89%. In order to facilitate the preparation of culture medium, the best formulation of fermentation medium was developed as 12 g/L corn flour, 15 g/L beef extract, and 13 g/L MgSO4. The inhibition rates of the three replicate tests were 83.33%, 88.89%, and 88.89%, respectively, with the mean value of (87.04 ± 3.2) %, which was close to the predicted value of 89%, indicating that the model obtained from the response surface test could predict the actual situation. In addition, the inhibition of S. sapinea growth by the fermentation filtrate of HR10 strain was increased by 3% after fermentation medium optimization , beta-glucanase (Beta), immunosuppressant A (ImA), and arabinogalactan endo-1,4-galactanase (Arab).The LC–MS-MS protein spectra of peak III with antagonistic activity obtained by Ion-Exchange Chromatography were analyzed, and a total of 49,368 spectra were obtained, and the number of matched spectra was 1267 after the identification was completed by BLAST, and a total of 220 proteins with 756 peptides were identified  is much greater than that of carbon source with corn flour (64%), however, the importance of corn flour is greater than that of yeast extract by PB test, indicating that with the increase of corn flour addition, its antagonistic effect on the sterile filtrate of HR10 strain is more significant than that of yeast extract. Usually, in fermentation tests, the production rate and fermentation product yield of strains fermented with yeast extract as raw material are higher than those with corn flour as raw material, yet the cost of yeast flour is much higher than that of corn flour. Therefore, in some fermentation process optimization experiments, in order to ensure the effect of prevention and at the same time reduce the cost, the mixture of yeast extract and corn flour is also used as the carbon source . Finally, by analyzing the results of LC–MS-MS protein spectrum, it was hypothesized that the antagonistic protein with antagonistic S. sapinea in B. pumilus HR10 were one or more of Ghy, Beta, ImA and Arab. Ghy is a glycoside hydrolase with cellulase activity, a member of the family of endoglucanases capable of cleaving cellulose or related substrates, and most Bacillus spp. are capable of secreting cellulases to degrade the cell wall of pathogenic fungi (Mori et al. Pa. polymyxa WY110 and finally obtained β-1,3–1,4 glucanase, which antagonized Pyricularia oryze. The secreted protein produced by B. velezensis YB15, β-glucanase, antagonized Rhizoctonia violacea with up to a width of 10.6 mm, indicating that the antimicrobial substance of strain YB15 was β-glucanase (Xu et al. B. thuringiensis has been reported to specifically hydrolyze antimicrobial peptides produced by insects and disrupt the immune system of some insects (Kwon and Kim In this experiment, the crude protein of strain HR10 antagonistic to B. pumilus HR10 was 12 g/L corn flour, 15 g/L beef paste and 13 g/L magnesium sulfate by Plackett–Burman test, steepest ascent test and Box-Behnken design, which optimized the formulation of antagonist substance fermentation medium for HR10 strain to some extent and provided data to support the future application of B. pumilus HR10 biocontrol agent in production. And then the efficient antagonistic protein against S. sapinea was isolated and purified using optimized medium and ion exchange chromatography, and the antagonistic activity was increased from 45.5 U/mg initially to 283.3 U/mg, with a purification multiple of fivefold. The obtained protein was not a single substance, and four proteases with hydrolytic activity were initially postulated, laying the foundation for the development and application of engineered strains for the control of Sphaeropsis shoot blight disease of pine.In this study, the best formulation of protein antagonist fermentation medium for Additional file 1:Table S1. All peptide fragments antagonistic protein of B. pumilus HR10 obtained by LC-MS- MS.


This study develops bio-nano composite gelatin-based edible film (NEF) by combining nanogelatin, cellulose nanocrystal (CNC), and nanopropolis (NP) fillers to improve the resulting film characteristics. The NEF was characterized in terms of thickness, swelling, pH, water content, solubility, vapor and oxygen permeability, mechanical properties, heat resistance, morphology, transparency, and color. The results showed that the thickness and swelling increased significantly, whilst the pH did not significantly differ in each treatment. The water content and the water solubility also showed no significant changes with loadings of both fillers. At the same time, vapor and oxygen permeability decreased with addition of the fillers but were not significantly affected by the loading amounts. The heat resistance properties increased with the filler addition. Tensile strength and Young’s modulus increased for the films loaded with >3% CNC. The elongation at break showed a significant difference together with transparency and color change. The greater the CNC concentration and NP loading were, the darker the resulting transparency and the color of the NEF. Overall results show a considerable improvement in the properties of the resulting NEFs with the incorporation of CNC and NP fillers. Synthetic plastic production is estimated to increase by approximately 320 million tons annually . More thPackaging that actively alters the packed goods and extends their shelf life is identified as active biodegradable packaging ,11. EdibGelatin has hydrophilic-lipophilic properties and, when combined with polysaccharides, it can form films ,29,30 anBiodegradable active packaging is one of the solutions developed to improve the poor mechanical, thermal, and water barrier qualities of biopolymer-based films by incorporating nanofillers or nano reinforcements ,43. Bio-Plasticizers are low-volatility molecules introduced to biopolymer materials. They adjust functional qualities, such as elasticity, stiffness, extensibility, mechanical properties, and dispensability . Glycerodumbo catfish (Clarias gariepinus) skin, CNC filler from waste banana peduncles (Musa acuminata L.) and NP as the bioactive material under various compositions. The addition of NP imposed antibacterial properties. Hence, the prepared NEF was classified as active packaging. The characterization of the prepared NEFs included the thickness, water content and water solubility, swelling, pH, morphology, heat resistance, mechanical properties, water vapor and oxygen permeability, transparency, and color change.This study reports the synthesis and characterization of a NEF. It was fabricated using the gelatin matrix obtained from Clarias gariepinus) skin was obtained from local dumbo catfish farmers in Aceh Besar, Indonesia. The waste of Barangan banana peduncles was obtained from banana traders in Aceh Besar, Indonesia. The chemicals used were sulfuric acid , NaOH , H2O2 (35% food-grade), acetic acid , NaCl, carboxymethyl cellulose , glycerol , NP , and distilled water.Dumbo catfish (Barangan banana peduncles (Musa acuminata L.) were cut into pieces and pressed using a pressing machine to obtain the fiber. The alkalization process was performed using a microwave (Samsung ME731K) with a 1 M NaOH solution at 450-Watt power for 1 h. After polymerization, the bleaching was conducted for an hour using 35 wt% of H2O2 with a fiber-to-solution ratio of 1:15 (w/v) at 180 W microwave power and a temperature range of 60–65 °C. The CNC isolation procedure was carried out for 1.5 h by hydrolysis using 3 M sulfuric acid at a ratio of 1:15 (w/v) at 100 W microwave radiation under a temperature range of 45–52 °C. The sample was then rinsed with distilled water until a neutral pH was reached. Subsequently, the sample was centrifuged at 11,000 rpm for 30 min, and the water was changed twice, followed by the sonification for 4 h. It was then centrifuged for 30 min and dried at 60 °C until a maximum moisture content of 12% was reached before being crushed and sieved using a 500-mesh sieve according to ASTM No: 11. The prepared CNC had the following characteristics in terms of morphology, functional groups, crystallinity, heat resistance, particle size, and color. The FTIR spectra of CNC showed the most relevant molecular bands, namely O-H, C-H, and C-O, at the corresponding wavenumbers range of 3200–4000 cm−1, 2500–3200 cm−1, and 500–1500 cm−1, respectively. The thermogravimetric analysis showed that it decomposed at a temperature of 275.35–409.40 °C, with a weight loss range of 84–94%. The crystallinity index and crystal sizes ranged from 54% to 57% and 22.35% to 36.28 nm, respectively.dumbo catfish skin was soaked in water for 2 h, then cleaned and reduced in size. Next, it was soaked in 0.1 M NaCl solution for 6 h, followed by soaking in 0.2 M NaOH for 2 h, with a ratio of fish skin to a solution of 1:15 (w/v). The solution was changed every hour. The sample was then washed with distilled water until the neutral pH was achieved, followed by immersion in a 0.1 M acetic acid solution with a ratio of 1:15 (w/v) for 6 h. Subsequently, the fish skin sample was washed and extracted with distilled water in a ratio of 1:5 (w/v) for 4 h. The extraction was performed using a microwave (Samsung ME731K) at a power of 180 W in the temperature range of 60–65 °C. Next, the sample was dried at 50 °C to reach a maximum moisture content of 16%. The prepared NGC was characterized and posed the following properties. The proximate analysis resulted in protein, fat, ash, and water content of 71.69%, 9.33%, 1.42%, and 17.56%. The pH and viscosity of the NGC solution were 7.1–7.2 and 1.25–6 cP. The FTIR analysis identified dominant peaks at 2500–4000 cm−1 (amide A: OH and CH stretching), 1500–2000 cm−1 , and 500–1500 cm−1 (C-N of methylene). The NGC sample decomposed at a temperature range of 267.63–427.23 °C mainly due to the decomposition or degradation of organic substances, while the decrease in the NGC mass occurred until a temperature of 600 °C resulting in a residual fraction of 15.42–20.17%. The XRD peaks showed 2 θ ranging from 7.33° to 9.68°, which was <20°, indicating the amorphous structure, with a crystallinity range of 9–64%. The average particle size of the NGC was 236.2 nm.Fresh w/v) of gelatin (NGC), 1% of CMC (w/v) and the 1%, 3%, and 5% (w/w) of CNC in distilled water were prepared by stirred for 1.5 h under temperature of 60 °C. The NGC, CNC, and CMC solutions were then mixed, reheated on a hot plate at 60 °C for 30 min, then cooled. Subsequently, it was loaded with glycerol 0.25% (v/v) and NP 0.1% (w/v) by weight of the NGC under continuous stirring. The treatment design is shown in Solutions containing 1%  at four random points. The thickness data are presented as average ± standard deviation.An empty 50 mL bottle was coded and weighed as the initial weight ( and (2) .(1)WC . The mechanical properties of the NEF samples were tested in terms of its tensile strength (TS), elongation at break (EB), and Young’s modulus (YM).The morphology of NEF samples was assessed by scanning electron microscopy (SEM). The samples were observed using SEM  with 10-k magnification at 30 kV power. After coating, the specimen was mounted and when the height of the surface is the same as that of the holder, the SEM machine was turned on and scanning was carried out.−1 Pa−1 h−1) of the NEF was calculated using Equation (4) [2), P (about 4247 Pa) is the difference in partial vapor pressure between the two sides of the film at room temperature and 100% relative humidity.A 9 mL bottle was filled with 6 mL of distilled water and covered with a 30 mm × 30 mm NEF. The bottle was then weighed and placed in a desiccator at room temperature. This was carried out every 24 h for three days, while the WVP (in g mtion (4) .(4)WVP=5 Pa at 25 °C), while The oxygen permeability (OP) was measured using a procedure reported earlier  with sliHeat stability properties of NEF samples were analyzed using differential scanning calorimetry (Shimadzu DSC-60). A 3 mg sample was placed into an aluminium pan and then scanned at a temperature range from 30 °C to 600 °C at a scan rate of 40 °C per minute under nitrogen atmosphere at a constant flow rate of 20 mL per minute.T600 is the percent transmittance at 600 nm and L (m) is the average film thickness.Transmittance refers to the fraction of incident light that is transmitted. Transparency is the condition of being see-through, hence can be quantified using the transmittance. Light transmission of NEF samples was measured by a UV-Vis Spectrophotometer . The sample of 4 cm × 5 cm was subjected to measurement at a wavelength range of 200–800 nm. The transparency of the NEF sample was calculated according to earlier work  using EqCIELAB is the most comprehensive r spaces . The coltion (8) .(8)ΔE*=The test data obtained were in the mean ± standard deviation and were further analyzed using One-way ANOVA . Duncan’s test was also carried out at a significant level of 5%. All data including three repetitions were analyzed using SPSS ver. 24.p < 0.05 on the NEF thickness. α = 0.05. A significant difference was observed between NGC before and after being composited with CNC and NP, namely between the NCC00 sample and the rest. The NEF’s thickness was further increased when composited by NP, as can be seen by comparing NCC01 vs. NCC04, NCC02 vs. NCC05, and NCC03 vs. NCC06, in which the increments of the first two pairs were significant. The film thickness tends to increase with a rising dry weight of the material for filmmaking [lmmaking . This rep < 0.01 on the swelling of the NEF.The swelling of the NEF showed an increasing ratio when composited with CNC and NP as shown in The NCC00 sample was not statistically significantly different from NCC01 but differed from the other samples. Meanwhile, the NCC02 sample was not significantly different from NCC03, NCC04, and NCC05, but NCC06 was significantly different from all other treatments. Materials with hydrophilic properties will easily bind with water, causing large swelling . In contp-values > 0.05. As suggested from an earlier report, the water content of NEF is not affected by the concentration of CNC and the addition of NP, but it was significantly influenced by the temperature and drying time. A previous study found that the water content of gelatin-based bio-nanocomposite films ranged from 11.0% to 29.1% [to 29.1% .p-values > 0.05) on the water solubility. The finding shows that the addition of CNC filler reduced the water solubility of the resulting NEFs as seen by comparing water solubility data of NCC00 (with superscript b) to the rests (with superscript ab and b). Given that gelatin is hydrophilic, adding polysaccharide fillers, such as cellulose, reinforces the protein layer and increases its resistance to moisture [w/w [An important property of edible films is their behavior in contact with water molecules . Biodegrmoisture . The mosure [w/w .The range of water solubility of NEF reported in literature is 35.9–52.9%. According to Tessaro et al. , CNC as p-values > 0.05 from the ANOVA. Tessaro et al. [The pH values of the prepared NEFs are shown in o et al.  reportedp < 0.05). When loaded with CNC , the NEF had the WVPs of 2.9 ± 0.1 × 10−7 to 3.0 ± 0.2 g−1 m−1 Pa−1 h−1, which is significantly higher than the WVP of NCC00 . Introducing the NP to the film increased the WVP of the CNC containing film to a range of 3.0 ± 0.3 × 10−7 to 3.4 ± 0.4 g−1 m−1 Pa−1 h−1, but still lower than the WVP of NCC00. According to Azizi-Lalabadi et al. [Overall, the WVP of NEF decreased after adding CNC and NP fillers, as shown in i et al. , the fir−1 d−1 to 1.4 × 10−9–4.6 × 10−9 g−1 m−1 Pa−1 d−1, respectively. The highest values of the OPTR and OP were found in the NCC00 sample with 3.1 g m−1 d−1 and 4.6 × 10−9 g−1 m−1 Pa−1 d−1, while the lowest was in the NCC05 with 0.9 g−1 m−1 d−1 and 1.4 × 10−9 g−1 m−1 Pa−1 d−1, respectively. The low values of OPTR and OP are desirable for the NEF, suggesting the effectiveness of the CNC and NP loading in improving the resulting film properties. Loading of the CNC significantly reduced both OPTR and OP, but the effect of the CNC concentration within the tested range was in significant. Similarly, further loading of 0.1% of NP on CNC containing NEFs lowered both the OPTR and OP values, but only significantly at CNC concentrations of 3% and 5% (NCC005 and NCC006).The OP of the NEF is directly proportional to the OPTR, as shown in As the concentration of CNC or NP in the NEF increased, the OP first decreased, then increased again. According to Azizi-Lalabadi et al. , the fir3 m−2 d−1 atm−1 to 1.323 cm3 m−2 d−1 atm−1 as well as 7.8 × 10−4 to 6.7 × 10−4 g day−1 m−1 atm−1. In addition, Yadav et al. [−6 cm3 m−1 d−1 atm−1.When compared to literature, Ge et al.  reportedv et al.  found thp > 0.05, but a significant difference * p < 0.05 was found on EB and MY. The data show no significant impact of CNC and NP loadings on the TS (dumbo catfish skin which produced a much higher TS value, ranging from 114.67 to 164.91 MPa.The mechanical properties of the TS, EB, and YM of the NEF samples are shown in n the TS a. AccordThe EB value generally decreased significantly when the CNC filler was added. The EB-value of NCC00 was significantly higher than the EB-values NCC01, NCC02 and NCC03. When those NEF containing CNC samples was loaded with NP, their EB-values decreased, but only the NCC04 sample showed a significant increase b.Loading the CNC into the NEF generally increased the YM but only significant for the one with 3% loading. Loading CNC increased the YM-value, but the loading amount did not show any clear trend c. The ma3–77.19 × 103 J/kg, respectively. The Tg value increased after the addition of both CNC and NP fillers. However, the Tm value increased after adding CNC filler, but decreased when further loaded with the NP filler, while the enthalpy showed an irregular trend. The findings suggest that the addition of CNC and NP affected the thermal properties of NEF. The highest Tg, Tm, and enthalpy values were obtained in samples NCC03, NCC02, and NCC06 with values of 151.39 °C, 354.68 °C and 77.19 × 103 J/kg respectively. CNC has an anti-plasticizing impact on gelatin-based films due to the ultimate absorption of water and glycerol. However, it also acts as a reinforcement of the biopolymer matrix, reducing macromolecule mobility and contributing to an increase in Tg as reported earlier [The thermal resistance property of the prepared NEF samples is depicted from the DSC shown in  earlier . The add earlier . Recent The morphology of the NEF’s surface can be seen from the SEM images presented in The transparencies of the NEF samples are presented in p < 0.01. The highest transparency was found in NCC06 with 14.48 mm−1, while the lowest was in the NCC00 with 11.04 mm−1. The trend of transmittance and transparency values is in line with It was suspected that the NP contained phenolic compounds that contributed to the low NEF’s transparency values. As the concentration of particles in the NEF increased, its transparency decreased due to the rise in light scattering . HoweverL* parameter value was obtained in NCC00 at 16.079 indicating the brightest NEF color. There was an increase in ΔL* when CNC was added in different concentrations together with NP. In contrast, the Δa* and Δb* values decreased with the darkening of the film color. The ΔE* color difference is directly proportional to the parameter of ΔL*, while the trend of color parameter for ΔL*, Δa*, Δb*, and ΔE* corresponds to the transparency when loaded with the CNC and the NP. The color and transparency results are consistent when compared visually with pictures in p-values < 0.01. The segregation of color parameters into The brightness and the color change of the prepared NEF samples are summarized in pearance  is ΔE*. quantity . Further−7 to 3.6 × 10−7 g m−1 Pa−1 h−1 respectively. Loading both CNC and NP lowered the water solubility by 4.9% for all treatments. High CNC concentration lowered the WVP value but increased it in the presence of NP. The OPTR and the OP were correlated linearly and ranged between 0.9 and 3.1 g m−1 d−1 and 1.4 × 10−9 and 4.6 × 10−9 g m−1 Pa−1 d−1, respectively. The TS, EB, and YM values ranged from 114.67 to 164.91 MPa, 13.89 to 41.07%, and 380.00 to 1203.72 MPa, respectively, in which a significant increase in TS and YM values was observed for NEF containing 3% of CNC. Moreover, the Tg, Tm, and enthalpy ranged from 127.76 to 151.39 °C, 304.40 to 354.68 °C, and 16.76 × 103 to 77.19 × 103 J/kg, respectively. The Tg value increased after adding CNC and NP, while the Tm increased when loaded with CNC but decreased in the presence of NP. The NEF’s light transmission decreased in the presence of CNC while the transparency increased. The value of ΔL* increased when loaded with both the CNC and the NP. A future work on evaluation of the antimicrobial properties in protection against bacterial invasion and prolonging the shelf life of food is required to measure the additional benefit of NP filler on the NEF.Overall results demonstrated changes of the resulting NEF properties when loaded with the CNC and the NP. The thickness of the NEF increased significantly with increasing CNC concentration and NP addition, with the value ranging from 0.138 to 0.164 mm. Higher CNC concentration led to increased swelling of the resulting NEF ranged at 17.0–28.8%. The CNC and NP loading did not affect the pH, whilst the water content, the water solubility, and WVP were in the ranges of 19.0–22.3% and 34.4–39.3% and 2.9 × 10


To investigate the impact of the combination of type 2 diabetes (DM) and high myopia on inner retinal layer thickness of the macular area. The patients were divided into four groups: control (group 1), patients with DM without high myopia (group 2), patients with high myopia without DM (group 3), and patients with DM and high myopia (group 4). Ganglion cell complex (GCC) thickness was compared among the groups. Linear regression analysis was performed to identify factors associated with GCC thickness. A total of 194 eyes were enrolled: 59 in group 1, 52 in group 2, 49 in group 3, and 34 in group 4. The average parafovea GCC thicknesses were 113.9 ± 10.4, 112.4 ± 11.2, 112.2 ± 7.8, and 102.6 ± 15.1 μm (P < 0.001), and the average perifovea GCC thicknesses were 104.8 ± 13.2, 103.5 ± 10.8, 103.6 ± 8.8, and 93.9 ± 15.5 μm in groups 1, 2, 3 and 4, respectively (P = 0.001). In multivariate analyses, age , DM duration , and axial length  were significantly associated with parafoveal GCC thickness. The GCC was significantly thinner when high myopia and DM were combined, compared to either condition alone. Additionally, age, DM duration, and axial length were significant factors associated with GCC thickness. The combination of mechanical stretching and neurodegeneration would accelerate neural damage to the retina, resulting in greater inner retinal layer thinning. Early investigations of DR focused on retinal vascular abnormalities, but emerging studies have shown that retinal thickness and visual function are reduced in patients with DM, even in the absence of clinical DR, which would be associated with diabetic retinal neurodegeneration (DRN). Many studies have reported that DRN predominantly affects the inner retinal layer due to chronic hyperglycemia, oxidative stress, and the accumulation of advanced glycation end products, which can lead to increased glutamate and a loss of neuroprotective factors6.Diabetic retinopathy (DR) is the most common complication of diabetes mellitus (DM) and one of the leading causes of blindness among working-age adults in developed countries7. Additionally, high myopia is associated with retinal thinning, possibly due to retinal stretching8. Many studies have reported that myopia has thinner inner retina than normal individual11. Mwanza et al. reported a mean decrease in average ganglion cell-inner plexiform layer (GCIPL) thickness of 1.06% per millimeter increase in axial length12. Wang et al. also showed negative correlation between GCC thickness and axial length13.High myopia, defined as an axial length ≥ 26.0 mm, is characterized by axial elongation, and consequent stretching of the posterior eye wall can cause retinal detachment, macular holes, choroidal neovascularization, and retinoschisis15. Lim and colleagues found that simultaneous presence of diabetes and myopia was associated with greater peripapillary retinal nerve fiber layer (RNFL) damage than either pathology alone16. However, few studies have assessed inner retinal layer thickness in the macular area in individuals with both DM and high myopia.Both type 2 DM and high myopia, which are relatively common and increasing in prevalence, greatly accelerate inner retinal layer thinningThe purpose of this study was to investigate the combined impact of DM and high myopia on the inner retinal layer in the macular area, by comparing inner retinal layer thickness between individuals with both conditions and those with only one of them.17. The exclusion criteria were any ophthalmic disease that could affect the thickness of the retinal layer, such as glaucoma, retinal disease, or neuro-ophthalmic disease, any history of intraocular surgery except for cataract, an IOP ≥ 21 mmHg, and clinical evidence of DR such as retinal hemorrhages or microaneurysms. We also excluded high myopia patients with structural changes, such as large chorioretinal atrophy, retinoschisis, or posterior staphyloma, which could cause segmentation error when measuring ganglion cell complex (GCC) thickness.This retrospective, cross-sectional study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board/Ethics Committee of Konyang University Hospital, Daejeon, Korea. We reviewed the charts of patients who visited our retinal clinic from March 2018 to December 2021. The requirement for written informed consent was waived by the Institutional Review Board/Ethics Committee of Konyang University Hospital due to the retrospective nature of the study. We recorded the medical history, best-corrected visual acuity (BCVA), spherical equivalent (SE), intraocular pressure (IOP), and axial length  of each patient. The patients were divided into four groups: a control (group 1), patients with DM without high myopia (group 2), patients with high myopia without DM (group 3), and patients with DM and high myopia (group 4). We defined high myopia as an axial length ≥ 26.0 mm. All diabetic patients were initially diagnosed with type 2 DM at the Department of Internal Medicine of Konyang National University Hospital; the diagnosis of DM was made according to the criteria of the American Diabetes Association18. We analyzed the data after adjusting for the thickness of each retinal layer for ocular magnification effects based on axial length as in previous studies21. The actual fundus distance (t) is determined from the OCT measurement (s) by the equation t = p × q × s, where p and q are magnification factors related, respectively, to the imaging system of the OCT device and the eye. Factor q was determined from the Bennett formula: q = 0.01306 × , where 1.82 is a constant related to the distance between the corneal apex and the second principal plane. Factor p for Heidelberg OCT devices is 3.39, given a normal axial length of 24.385 mm.Optical coherence tomography (OCT) measurements were performed by a skilled examiner using the Spectralis OCT 2 device , which can perform 70,000 A-scans per second using a light source centered at 870 nm. A macular volume scan was obtained using spectral domain-OCT . Using the Early Treatment Diabetic Retinopathy Study(ETDRS) grid, the macular area was divided into three concentric rings measuring 1, 3, and 6 mm in diameter, centered on the fovea. The two outer rings, with diameters of 3 (defined as the area of parafovea) and 6 mm (defined as the area of perifovea), were divided into four sectors . Central macular thickness (CMT), defined as the average macular thickness in the central 1-mm area of the ETDRS map, was measured automatically using software bundled with the Spectralis OCT 2 device. Retinal layer segmentation was performed automatically in each horizontal scan. The thickness of the GCC was calculated as the sum of the thicknesses of the macular nerveo fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL) as previous studyDemographic characteristics and ocular parameters were compared between groups using one-way analysis of variance followed by a post-hoc test (Bonferroni test), as well as by the Chi-squared test. Univariate and multivariate linear regression analysrs were performed to identify factors associated with the GCC thickness. All statistical analyses were performed with SPSS software .A total of 194 eyes were included in this study: 59 in group 1, 52 in group 2, 49 in group 3, and 34 in group 4 , and the average perifovea NFL thicknesses were 38.2 ± 5.5, 38.3 ± 6.4, 39.7 ± 5.4, and 35.1 ± 8.9 μm (P = 0.012) in groups 1, 2, 3 and 4, respectively. The average parafovea GCL thicknesses were 50.6 ± 5.3, 49.8 ± 6.2, 49.0 ± 5.0, and 43.3 ± 11.0 μm (P < 0.001), and the average perifovea GCL thicknesses were 35.8 ± 3.1, 35.5 ± 3.6, 34.9 ± 3.4, and 31.0 ± 7.0 μm (P < 0.001) in groups 1, 2, 3 and 4, respectively. The average parafovea IPL thicknesses were 41.4 ± 3.7, 40.7 ± 4.0, 40.3 ± 3.2, and 36.6 ± 6.9 μm (P < 0.001), and the average perifovea IPL thicknesses were 31.0 ± 2.4, 29.7 ± 2.6, 29.0 ± 2.2, and 27.8 ± 3.5 μm (P = 0.002) in groups 1, 2, 3 and 4, respectively.The average parafovea GCC thicknesses were 113.9 ± 10.4, 112.4 ± 11.2, 112.2 ± 7.8, and 102.6 ± 15.1 μm in groups 1, 2, 3 and 4 , respectively (P < 0.001) , HTN , DM duration , BCVA , and axial length  were significant factors associated with GCC thickness Table .Table 3UIn multivariate analyses, age , DM duration , axial length  were significant.In group 4, there was a significant negative correlation between GCC thickness and age, with a relatively steep slope  Fig. . AdditioIn this study, we evaluated the GCC thickness of the macular area in control, high myopia, DM, and high myopia and DM groups. The GCC of parafoveal and perifoveal areas was significantly thinner when both diseases were combined. Additionally, age, DM duration, and AL were significant factors associated with GCC thickness in multivariate analyses.22 reported that the average GCIPL thickness of eyes with high myopia was significantly thinner than those with normal controls . Lim et al.23 found that the average GCIPL thickness was 84.23 ± 6.22 and 81.10 ± 4.47 μm in control and DM groups, respectively, which was significantly different (P = 0.001). In our study, groups 2 and 3 showed a tendency for thinner average and sectoral GCC thicknesses than group 1, however, no significant difference was evident in the post-hoc analyses. This discrepancy with previous studies may be due to the use of different OCT devices. The previously mentioned studies used the Cirrus HD OCT system (Carl Zeiss), which measures GCIPL thickness by identifying the outer boundaries of the RNFL and IPL within an annulus with inner vertical and horizontal diameters of 1 and 1.2 mm, and outer vertical and horizontal diameters of 4 and 4.8 mm, respectively. The difference in the area and layer analyzing the inner retina thickness may explain the discrepancies with our findings.Previous studies have reported inner retinal thinning in patients with DM and high myopia. Lee et al.16. Accelerated inner retinal thinning by the combination of mechanical stretching in high myopia and DRN would occur not only in the peripapillary area but also in the macular area. Globe elongation may stretch not only retinal tissue but also retinal microvasculature. The stretched microvasculature would be more vulnerable to various damages including pathologic pathways triggered by hyperglycemia compared to normal microvasculature. This can cause severe neurodegeneration by more breakdown of the blood-retinal barrier and neurovascular coupling impairment, which would result in severe inner retinal thinning. Meanwhile, greater peripapillary RNFL damage has been also reported when high myopia and hypertension were both present. Lee et al.24 hypothesized that, together, mechanical stretching caused by high myopia and ischemic damage induced by hypertension would lead to a greater reduction in peripapillary RNFL thickness than ischemic or mechanical damage alone. Overall, when high myopia is present together with systemic disease, which damages the inner retina, inner retinal thinning will be accelerated.Contrary to the above results, the GCC was significantly thinner in our cases with both DM and high myopia. An earlier study reported that the presence of both DM and myopia was associated with greater peripapillary RNFL damage than that observed with either pathology alone27. In our study, age was a significant factor associated with GCC thickness, consistent with previous studies. In our subgroup analyses, the GCC thickness of group 2 and 4 patients, who had DM, showed a significant association with age, especially in the latter group. Diffuse loss of neural tissue over time may be accelerated by DRN, especially in patients with high myopia causing mechanical stretching.Previous studies showed that retinal layer thinning occurs naturally over time due to the effects of aging. Thinning of 0.01–0.16 μm/y of the macular RNFL, 0.05–0.10 μm/y of the GCL, and 0.05 μm/y of the IPL have been described, and tends to be more prominent in older individuals2 found that the duration of DM was correlated significantly and inversely with GCL thickness; their results suggest that neurodegeneration is primarily caused by a prolonged disturbance of glucose metabolism, which may occur irrespective of the presence of vasculopathy. Lee et al.28 also reported that patients with DM duration ≥ 10 years had a thinner GCIPL and lower macular vessel density than those with DM duration < 10 years. Similarly. our study showed a significant association between DM duration and GCC thickness. Once DRN begins, neuronal apoptosis and glial dysfunction may persist and be accumulated over time, resulting in severe inner retina damage in patients with prolonged DM.Van Dijk et al.9 reported that the average GCC thickness was significantly associated with axial length. Seo et al.11 found a significant correlation between average GCIPL thickness and axial length . Our multivariate analyses also showed a significant association between axial length and GCC thickness, especilly in patients with DM. This accords with the fact that inner retinal layer damage is exacerbated in the presence of both DRN and mechanical stretching, as mentioned above.Axial length is a known significant factor associated with inner retinal layer thickness. Zhao et al.Our study had several limitations. First, it used a retrospective design; thus, the results may not be representative of the general population as selection bias cannot be ruled out. Second, disease duration was based on the date of clinical diagnosis, and thus may have been underestimated. Third, our study was concerned only with the duration of DM, i.e., not the degree of DM control. Despite these limitations, we identified the effects of high myopia and DM, alone and in combination, on inner retinal layer thickness in the macular area, which to our knowledge has not previously been reported.In conclusion, the GCC was significantly thinner in the presence of both high myopia and DM compared with either condition alone. Additionally, age, DM duration, and axial length were significant factors associated with GCC thickness. In subgroup analyses, the effect of age was greater in patients with both DM and high myopia. Togegher, mechanical stretching and DRN would accelerate neural damage to the retina, resulting in greater inner retinal layer thinning. Physicians should be aware of this when assessing inner retinal layer thickness in patients with DM.


Plukenetia are remarkably rich sources of polyunsaturated fatty acids, in particular α-linolenic acid. The lipid composition of the large-seeded, recently described ‘Mountain Sacha Inchi’ Plukenetia carolis-vegae is reported here for the first time, and compared with Plukenetia huayllabambana, two cultivars of Plukenetia volubilis, and a newly developed hybrid between P. volubilis and P. carolis-vegae. All species and cultivars had a very high content of polyunsaturated fatty acids, 82.6–86.7% of total fatty acids, and at least 46.6% α-linolenic acid of total fatty acids. The highest content was found in P. carolis-vegae which had 57.4%. The exceptionally high α-linolenic acid content suggests that P. carolis-vegae may be an important plant-derived dietary source of this essential fatty acid and that the species has considerable potential for further domestication and commercialisation of its seeds and seed oil. A TAG analysis was carried out for the two P. volubilis cultivars, in which LLnLn and LnLL were most prevalent, and for P. huayllabambana, in which LLnLn constituted the largest fraction, followed by LnLnLn, indicating that this large-seeded species also has interesting dietary properties.Several Amazonian species of  Plukenetia carolis-vegae Bussmann, Paniagua & C.Téllez is a recently discovered liana with large, edible, nut-like, oleaginous seeds1. Together with Plukenetia huayllabambana Bussmann, C.Téllez & A.Glenn and the new species Plukenetia sylvestris Card.-McTeag. & L.J.Gillespie it forms a high elevation species complex of ‘Mountain Sacha Inchi’, which are all native to the tropical regions of the Andes3. Plukenetia carolis-vegae and P. huayllabambana are two of five large-seeded species from the genus Plukenetia L. (Euphorbiaceae) which are known to have traditionally been cultivated for food and medicine, the remainder being Plukenetia volubilis L., ‘Sacha Inchi’ or ‘Inca Peanut’, and Plukenetia polyadenia Müll.Arg., ‘Compadre-de-azeite’, which are both found in the Amazon Basin, and Plukenetia conophora Müll.Arg., ‘Awusa’ or ‘African Walnut’, which is native to tropical central and west Africa4.Plukenetia has attracted increasing attention in recent years due to remarkably high amounts of polyunsaturated, essential fatty acids in the seeds of P. volubilis. However, although the seed biochemistry of P. volubilis is well established, only a few studies of P. huayllabambana, P. polyadenia, and P. conophora have been performed while no studies of the lipid composition of P. carolis-vegae exist. Nevertheless, the available data indicates that other large-seeded species of the genus have an oil composition that is similar to that of P. volubilis or perhaps even more nutritionally interesting.The genus P. volubilis are known to have a lipid content of 33–58%, although the majority of studies report approx. 45–50%, of which approximately 77.5–84.4% are polyunsaturated fatty acids (PUFA), comprised of 35.2–50.8% α-linolenic acid  and 33.4–41.0% linoleic acid . Of the remaining lipid fraction, 8.4–13.2% are monounsaturated fatty acids (MUFA), and 6.8–9.1% are saturated fatty acids (SFA) . In comparison, the oil contents of the Neotropical species P. huayllabambana and P. polyadenia are similar to that of P. volubilis  while the oil content of the Paleotropical P. conophora is somewhat greater (approx. 54%). The size of the PUFA fraction is comparable between the four species. However, both P. huayllabambana and P. conophora seed oil has a higher content of ALA than P. volubilis , while that of P. polyadenia is lower (approx. 35%) .The seeds of 22. The inherent essentiality of ALA has been debated24, however, as more studies accumulate, the consensus has become that this plant-derived ω-3 fatty acid has a distinct role, and studies indicate that it influences brain health, vascular function, and the condition of skin and hair24. Further, several studies have shown benefits of ALA intake, e.g., the MRFIT  study of 6250 men showed significant inverse associations between ALA and mortality from coronary heart disease, all cardiovascular diseases, and all-cause mortality25. Similarly, in the Lyon study—in which ALA content in the diet was a main difference between test and control diets—sudden death was reduced by 40% in the first year26. However, while the relevance of plant-derived ω-3 fatty acids is becoming clear, it may be complicated to incorporate larger amounts into the diet, and exploration of novel sources seems prudent.ALA is the precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have numerous documented health effects, including prevention of cardiovascular disease, a protective effect in mood disorders, and improved cognitive function in childrenPlukenetia seeds can be consumed whole, as ‘nuts’, or pressed for oil and the sensory acceptability of P. volubilis has been demonstrated to be good27. The high elevation ‘Mountain Sacha Inchi’ species complex, consisting of P. carolis-vegae, P. huayllabambana, and P. sylvestris, is sister to P. volubilis and all the species have larger seeds than P. volubilis4. The seeds of P. carolis-vegae are the largest in the group, and the available studies of the seed lipid composition of P. huayllabambana indicate that the ALA fraction of this species is greater than it is in the seed oil of P. volubilis17. Furthermore, a recent phylogenetic study suggests that P. huayllabambana is a hybrid between P. volubilis and P. sylvestris, the latter also being hypothesised to be the wild progenitor of P. carolis-vegae3. This relationship may show promise for a nutritionally interesting composition of the seed of P. carolis-vegae, and a study of the fatty acid composition of the seed oil of this species seems very relevant. Also, the current authors have demonstrated compatibility between P. carolis-vegae and P. volubilis, lending support to hybridisation events occurring within the species complex, and a study of the seed oil of the artificial hybrid P. volubilis × P. carolis-vegae may also aid in better understanding the nutritional qualities of the ‘Mountain Sacha Inchis’.P. carolis-vegae and P. huayllabambana using P. volubilis as a reference. Furthermore, the oil composition of the newly developed hybrid between P. volubilis and P. carolis-vegae will be studied. We hypothesise that P. carolis-vegae will have a similar lipid composition to P. huayllabambana, but with a higher fraction of ALA, and that P. volubilis × P. carolis-vegae will have a composition within the range of the parents’ lipid composition.The current study aims to analyse the oil composition of Plukenetia volubilis L. seeds were obtained from Joya de los Sachas, Orellana, Ecuador  and from Tarapoto, San Martín, Peru . Plukenetia huayllabambana Bussmann, C.Tellez & A.Glenn. seeds were collected in Chachapoyas, Amazonas, Peru (6° 28′ 56.9″ S 77°21′ 53.5″ W), and Plukenetia carolis-vegae Bussmann, Paniagua & C.Téllez seeds in Rodríguez de Mendoza, Amazonas, Peru (6° 23′ 45.8″ S 77° 34′ 11.0″ W). Hybrid seeds (‘P. volubilis × P. carolis-vegae’) were obtained from a controlled hybridisation using pollen of P. carolis-vegae from Rodríguez de Mendoza, Amazonas, Peru (6° 23′ 45.8″ S 77° 34′ 11.0″ W), to fertilise a plant of P. volubilis from Pampamonte, San Martín, Peru (6° 21′ 23.5″ S 76° 35′ 38.8″ W). Seeds were collected at full maturity as evaluated by capsule colour and dehiscence to ensure comparable stages of seed lipid synthesis. In order to best compare species with different ecological niches, care was taken to collect seeds from vigorous plants at least 2 years of age grown under suitable conditions. All collections were carried out in May and early June and from each species or cultivar an estimated 10 g of seeds were collected for oil extraction. All methods were performed in accordance with the relevant guidelines and regulations and permissions for the collection of plant material were obtained. Voucher specimens of the plant material will be deposited at University of Copenhagen (C) during 2022.The testae of the seeds was disrupted using a mortar and pestle and carefully removed, following which the seeds were milled using a coffee grinder.P. volubilis × P. carolis-vegae sample for which material was only available for two extractions. The samples were subjected to supercritical carbon dioxide  for 30 min at 500 bar and 60 °C at a flow rate of 2.5 L min−1.Supercritical fluid extraction was performed on a laboratory scale unit  using 10 mL extraction tubes. Oil from milled seeds (2 g) was extracted in triplicates, with exception of the 28. A BDS hypersil C18 column  was used and isocratic elution was performed with acetonitrile:2-propanol:CO2 (56:14:30) at a flow rate of 1.5 mL min−1. The column temperature was set to 40 °C, and the column back pressure was 200 bar. The TAGs were detected with UV detection at 210 nm, and with ELSD . The ELSD conditions were as follows: nebulizer temperature of 68 °C, evaporator temperature of 40 °C, and nitrogen pressure of 0.15 MPa. A 5 µL injection loop was used for analysing the TAG profile. For identification of the individual TAG composition preparative EFLC was applied in combination with GC-FAME analysis of each fraction. For the preparative EFLC a 200 µL injection loop was used and fractions collected every minute. Each fraction was then subjected to the FAME (fatty acid methyl esterification) procedure, and the fatty acid identified using a Supelco 37 Component FAME mix  and with spiking of individual fatty acid methyl ester standards .The triacylglycerol (TAG) profile of the oils were analysed by enhanced liquid chromatography (EFLC) on a SFE/HPLC hybrid system  according to the method of Buskov et al.29, with modifications as follows; 20 mg oil was added to 1 mL methanolic NaOH (0.5 M) with 200 µL internal standard  , and vortex mixed with 1.5 mL boron-trifluoride methanol (BF3) solution. The mixture was heated at 70 °C for 2 min, where after a saturated NaCl solution was added and the FAMEs extracted into cyclohexane (1 mL).The FAME procedure was performed according to the AOAC Official Method 969.33f 0.20 μm) was used. Supelco 37 Component FAME mix  was used as a standard. The temperature program was as follows: initial temperature of 60 °C hold 2 min, ramp to 200 °C hold 0 min, and ramp to 240 °C hold 7 min. Helium was used as the carrier gas with a flow of 2.477 mL min−1, the injection volume was 1 mL with a split ratio of 52:1, and cyclohexane was used as a solvent.The fatty acid profile was determined using gas chromatography (GC) with flame ionization detection (FID) using the Agilent Technologies GC System 7820A . A Supelco SP™-2380 capillary column  test followed by a Tukey HSD test was used to determine significant differences in the fatty acid composition of the different species and cultivars of P. volubilis is the most well studied in the genus, and the results from the two P. volubilis accessions from Ecuador and Peru in the current study are similar to previous results. The most abundant fatty acid in the seed oil of P. volubilis from Ecuador and Peru, respectively, is α-linolenic acid , followed by linoleic acid , oleic acid  and smaller amounts (< 5%) of palmitic (C16:0), stearic (C18:0), eicosanoic (C20:0), and eicosenoic acids  and chia  are known for their high ALA content, and according to Ciftci et al.32, linseed oil contains 58.2 ± 0.64% ALA, 15.3 ± 1.01% LA, and 18.1 ± 0.45% OA, while chia oil contains 59.76 ± 0.13% ALA, 20.37 ± 0.19% LA, and 10.53 ± 0.17% OA. In comparison, the common cooking oils olive (Olea europaea L.) and sunflower (Helianthus annuus L.) contain < 1% ALA, while rapeseed oil (Brassica napus L.) contains approx. 10% ALA35.Regardless of the compositional variation, the amount of ALA in the seed oil of Plukenetia huayllabambana is one of the more recently described species of Plukenetia2 and have very large seeds; estimated as 6627 mm3 per seed compared with the approx. 997 mm3 of P. volubilis4. According to a recently published revised classification of the Plukenetia genus, P. huayllabambana is a putative hybrid between P. volubilis and the newly described P. sylvestris, a large-seeded species of the high elevation species complex sister to P. volubilis3. Our analysis shows that seed oil from P. huayllabambana has a significantly higher content of ALA (56.6 ± 0.2%) than P. volubilis from both Ecuador and Peru , while the content of LA is also significantly lower , and Cai et al. (2012) observed that the ALA content of P. volubilis generally increased with higher altitude and decreasing temperatures30. However, studies of the oil composition of P. carolis-vegae cultivated at lower altitudes need to be conducted to assess whether the high ALA content is due to environment, genetics or both.The seed oil of ALA Fig. , which wP. carolis-vegae seed oil was 10.5 ± 0.0%, which was significantly higher than in both P. volubilis cultivars, P. huayllabambana, and the hybrid P. volubilis × P. carolis-vegae. The content of eicosenoic and palmitic acid in P. carolis-vegae seed oil  was found to be mostly similar to the content in the seed oil of the other analysed species. In contrast, the content of eicosanoic and stearic acid  was significantly lower than in both the P. volubilis cultivars and P. huayllabambana but similar to the levels in the hybrid. Overall, the most striking difference between the oils was the very high ALA content of P. carolis-vegae seed oil, a property which may be promising for further cultivation and domestication of the species.The amount of OA in P. volubilis × P. carolis-vegae hybrid seed oil is 46.8%, which is similar to that of the two P. volubilis cultivars, but lower than that of P. huayllabambana and P. carolis-vegae. Conversely, the LA content of the oil is 39.9%, which is significantly higher than all other samples except P. volubilis from Peru  and LnLnLn , while in P. huayllabambana they were LnLnLn (23.5 ± 0.8%) and LnLL (14.1 ± 0.8%). In all samples, TAGs composed of polyunsaturated Ln (ALA) and L (LA) constituted more than two-thirds of the total TAG molecules . Moreover, most of the identified TAGs (88. 5–95.9%) contained at least one residue of ALA.The most abundant TAG in the P. volubilis oil measured by Fanali et al.37, who identified LLnLn as the most abundant TAG, and found that > 80% of TAGs contained ALA. The predominant TAGs after LLnLn in P. volubilis were LnLL and LnLnLn37. Similarly, Chasquibol et al.15 found LLnLn to be the most prevalent TAG in both P. volubilis and P. huayllabambana.These results correspond well with the TAG composition in P. carolis-vegae × P. volubilis to 8.4% in P. huayllabambana. Similarly, the MUFA content ranges from 7.6% in P. carolis-vegae × P. volubilis to 11.1% in P. carolis-vegae  and the P. carolis-vegae × P. volubilis hybrid containing the lowest (46.8%) although it has the highest total amount of PUFA. The differences in the composition of the PUFA fraction might be a result of genetic differences between the species and cultivars in combination with environmental factors.Across all the examined species and cultivars, the SFA content is relatively low, ranging from 5.7% in gae Fig. . The rem39. Until recently, the molecular mechanisms underlying the synthesis of the very high PUFA content in P. volubilis had not been elucidated, although a few studies had been published41. However, in a study by Yang et al.39 two FAD genes named PvFAD2 and PvFAD3 were isolated from P. volubilis and demonstrated to catalyse the synthesis of LA and ALA, respectively, although the authors point out that their results do not fully explain the massive accumulation of PUFA in P. volubilis seeds. Nevertheless, the differences observed between the species and cultivars included in the current study may at least in part be caused by differences in the expression of PvFAD2 and PvFAD3. If so, P. carolis-vegae may have a relatively high expression of PvFAD3, leading to the synthesis of a very high amount of ALA in the seed oil, while, conversely, P. volubilis from Peru may have a lower expression of PvFAD3, yielding a lower amount of ALA in the seed oil. Furthermore, it can be speculated that the similar amounts of PUFA, but varying compositions of the PUFA fraction, observed in almost all the species and cultivars of the study could be an effect of LA being used as a substrate for PvFAD3 to produce ALA, reducing the amount of LA while increasing the amount of ALA.The common pathway of PUFA biosynthesis in plants is initiated in the plastid with the formation of acyl-chains by the fatty acid synthase (FAS) complex, generating C16:0 and C18:0 fatty acids. Desaturation ensues by the action of a stearoyl-acyl carrier protein desaturase (SAD) to form OA (C18:1), which is further desaturated to LA (C18:2) by fatty acid desaturase-2 (FAD2) in the endoplasmic reticulum, and the third double bond is introduced at the ω-3 position of LA by fatty acid desaturase-3 (FAD3), also in the endoplasmic reticulumP. carolis-vegae × P. volubilis has the highest PUFA fraction of all the studied species and cultivars of Plukenetia, although the ALA fraction is comparably small. This characteristic could be caused by a higher expression of genes early in the biosynthetic pathway of ALA synthesis, e.g. SAD, in combination with a lower expression of PvFAD3 compared to the other samples. However, it is also possible that a lower growing temperature would have induced activity of PvFAD3 and led to a higher accumulation of ALA relatively to LA; Yang et al.39 found that the activity of PvFAD3 was sensitive to temperature when expressed in yeast (Saccharomyces cerevisiae) cells, with low temperature (20 °C) significantly increasing biosynthesis of ALA. Accordingly, an analysis of the seed oil of all the ‘Mountain Sacha Inchis’, including the new species, P. sylvestris, and P. volubilis, grown at 20 and 30 °C might be useful in better understanding the very high accumulation of PUFA, primarily ALA in the large-seeded species of Plukenetia.Intriguingly, P. carolis-vegae and P. huayllabambana, the two cultivars of P. volubilis, and the newly developed hybrid between P. volubilis and P. carolis-vegae contains exceptionally high amounts of PUFA, at least 82%, and a remarkably high amount of ALA , at least 46%. It is of particular interest that the very large-seeded P. carolis-vegae has the highest ALA content in the lipid fraction, > 57%, followed by P. huayllabambana, P. volubilis, and P. volubilis × P. carolis-vegae. This underlines that P. carolis-vegae is a highly promising underutilised crop that may provide many benefits for farmers as well as consumers.The seed oil of the ‘Mountain Sacha Inchis’ Plukenetia carolis-vegae, but also P. huayllabambana and P. volubilis, has a huge potential for further domestication and industrialisation, due to the extraordinary nutritional qualities of the seed oil. Additionally, cultivation of the ‘Mountain Sacha Inchis’ may provide both nutritional and economic benefits for farmers and their communities. Finally, as demonstrated by the very high PUFA content of the P. volubilis × P. carolis-vegae hybrid, hybridisation breeding using ’Mountain Sacha Inchi’ germplasm might grant exciting opportunities.


In this study, the levels of indicator PCBs congeners and PAHs compounds were reported in cow milk from selected areas in Dhaka, Bangladesh, and the potential human health risks were assessed. A total of 100 milk samples were collected and analyzed using gas chromatography-tandem mass spectrometry (GC-MS/MS). Method validation was performed using recovery performance, linearity, limit of detection (LOD), and limit of quantification (LOQ) assays. PCBs congeners, including PCB No. 52 , PCB No. 101 , PCB No. 153 , and PCB No 209  were detected, whereas PCB No 28 , PCB No. 138 , and PCB No 180  were not detected in the analyzed milk samples. Among the 16 PAHs compounds, benzo (a) anthracene and chrysene were detected in milk samples. The Σ hazard risk index (HI) of all detected PCBs congeners was below the limit set by the European Food Safety Authority, which indicates limited health risks to animals and humans in the study area. However, the presence of PCBs and PAHs in milk samples from industrial areas may negatively affect human health, and further detailed studies are required to ensure public health safety.  However, human activities are primarily responsible for their massive discharge into the atmosphere. Polychlorinated biphenyls (PCBs), hexachlorocyclohexane (HCH), polybrominated diphenyl ethers (PBDEs), polyaromatic hydrocarbons (PAHs), pesticides, and heavy metals are anthropogenic inorganic and organic pollutants that are dispersed throughout the atmosphere, hydrosphere, and lithosphere. They may be transformed into other compounds that are even more toxic to flora and fauna PCBs are long-lasting contaminants that result from the defective combustion of organic and synthetic materials PAHs comprise hydrogen and carbon fused with two or more aromatic rings PAHs can contaminate foods from environmental pollution or during food preparation and processing. Most gas-phase PAHs compounds are adsorbed in the particulate matter and finally accumulate in water and soil with heavy PAHs, which enter the food chain through vegetation and plant foods. Therefore, grazing cows can uptake PAHs from the soil through their feed and accumulate them in animal-origin foods Several reports have demonstrated that products with high-fat content are important sources of human exposure to recognized toxins in the environment. Consequently, fatty foods, including meat, milk, and eggs, may contain significant amounts of POPs and other chemicals The impacts of PCBs and PAHs, PCBs and PBDEs are considered seriously because of their persistence nature in the environment Although the presence of PCBs and PAHs in fat-rich foods, such as milk, is a major health concern, not much work has been done so far in Bangladesh. This study aimed to determine the levels of PCBs and PAHs in cow milk from selected areas in Bangladesh and assess possible human health risks.22.1A total of 100 raw milk samples from 10 sampling sites were collected from the dairy firms of Mohammadpur and Hazaribag in Dhaka. The samples were collected between October 2020 and February 2021. Hazaribag is used in the leather industry. The site selection was based on industrial and agricultural activities. Many leather and tanning industries are located in Hazaribag. Mohammadpur is adjacent to the Turag River. The water of the Turag River is contaminated by industrial waste. Moreover, several waste dumping sites are located near both sampling areas, where the wastes are burned regularly. Milk samples were collected from dairy farms and local shops. Raw milk samples were used in this analysis. About 500 mL of milk samples were collected in plastic polythene bags, transported to the laboratory on ice box and stored at 4ºC and analyzed within 24 h of sample collection.2.2A cocktail standard mix of PCBs congeners including PCB No 28, PCB No 52, PCB No 101, PCB No. 138, PCB No. 153, PCB No.180, and PCB No. 209 (PCB standard solution 7) and a cocktail mix of 16 PAHs compounds containing Napthalene, 2-methylnapthalene, 1-methylnapthalene, Acenapthylene, Acenapthalene, Fluorene, Phenanthrene, Anthracene, Fluranthene, Pyrene, Benzo (A) Anthacene, Chrysene, Benzo (B) Fluoranthene, Benzo (K) Fluranthene, Benzo (A) Pyrene, Indeno  Pyrene and Dibenz  Anthracene brought from Sigma Aldrich . Acetonitrile  and other chemicals including n-hexane, magnesium sulfate, sodium acetate, and NaCl were purchased from Merck .2.3Sample extraction was performed using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method 2SO4 were loaded onto a glass chromatographic column, and n-hexane was used. The concentrated extracts were dissolved in 5 mL of n-hexane, loaded into the column, and eluted with 50 mL of n-hexane. Finally, the eluents were concentrated and dissolved in n-hexane  was prepared, 15 mL of the mixture was added, and the mixture was shaken for a few seconds. Subsequently, magnesium sulfate (6 g) and sodium acetate (1.5 g) were added to the mix 2.4PCBs congeners were analyzed using a gas chromatograph coupled with a MS . Analytical separations were performed using a Trace GOLD™ TG-5MS GC Column (0.25 mm × 0.25 µm X 0.25 m). Helium was used at a constant pressure (18.29 psi) at 1.6 mL/min flow rate. The injection port temperature was 250 °C, and the initial oven temperature was 60 °C, which was further increased to 220ºC at 5ºC/min heating ramp. The detector temperature was approximately 320 °C during the sample analysis. The sample injection volume was 2 µL.For the PAHs analysis, helium was used as the carrier gas at 1.8 mL/min flow rate. The injection volume was 1 µL in split-less mode. The ion source and quadrupole temperatures were 280 °C and 180 °C, respectively, whereas the injector and transfer line temperatures were 280 °C and 310 °C, respectively. The column temperature was initially set at 35 °C for 1 min, then increased to 200 °C at a ramp rate of 25 °C/min, and finally ramped to 310 °C at 8 °C/min and kept for 3.5 min. Both of PCBs and PAHs analyte detection was carried out using MS at 70 eV ionization energy in selected ion monitoring (SIM) acquisition mode.2.5Working solutions were prepared by diluting standard stock solutions with n-hexane. For the linearity test, working solutions of several dilutions in the range of 50–200 µg/L were prepared and injected into the GC-MS/MS system. The LOQ and LOD were determined using the signal-to-noise ratio. Method validation was performed using a recovery performance evaluation. The recovery percentage was determined using the following equation:Here, Pi is the recovery percentage, Si is the result of the control, and Ti is the percent recovery of the spiked samples.2.6Human exposure to chemicals in food is estimated using three different parameters: substance concentration in food (ng/kg lipid weight (l.w)), food consumption rate (kg), and population body weight (kg).The estimated daily intake (EDI) is determined through the following equation:In this case, EDI is the estimated daily intake (g day−1), DFC indicates daily food consumption (kg/day), FFC indicates the food chemical concentration present in the analyzed food (mg/kg), and BW indicates the body weight (kg) of consumers. In this analysis, an average body weight of 70 kg/per adult person was considered for calculating the dietary exposure. The per capita milk consumption per day in the Bangladeshi population is 27.31 g 2.7The following formula was used for HI calculation:EDI: estimated daily intake of PCBs; ADI: acceptable daily intake through milk consumption. HI > 1 is considered a potential health hazard, and HI< 1 is considered safe for public health 2.8Data were processed using mass spectrometry. The peak areas were considered for PCB quantification. Analytical data were collected and organized using Microsoft Excel. Mean concentration differences and standard deviations in milk samples, in accordance with the sampling sites, were determined using one-way ANOVA. Samples below the LOD values were considered to have a concentration of 0.0 for statistical analysis. Median, Interquartile range of detected analytes were calculated using Calculator Soup online software.33.1There are two common methods for the detection of PCBs in food samples: GC with an ionizing flame detector and MS 3.2The levels of PCBs in cow milk samples collected from local firms in Dhaka, Bangladesh, were determined using GC-MS/MS. In this study, 100 milk samples were collected. PCBs residues including PCB No 52 (0.63 ± 0.21 ng/g l.w), PCB No 101 (0.52 ± 0.22 ng/g l.w), PCB No 153 (4.75 ± 0.41 ng/g l.w), and PCB No 202 (5.35 ± 0.87 ng/g l.w) were detected, whereas PCB No 28, PCB No 138, and PCB No 180 were not detected or detected below the detection limit (<LOD). The mean values, median, Interquartile range of detected PCBs congeners and Percentage of Positive samples are shown in 3.3The mean values, median, interquartile range of detected PAHs analytes and Percentage of Positive samples of PAHs in the milk samples are presented in 3.4−6, 4.61 × 10−6, 4.20 × 10−5, and 4.73 × 10−5, respectively. From the risk analysis, it can be concluded that the milk samples from the study area were safe for consumers, as the HI values were far below the risk level. However, milk samples containing PCBs residues for a longer time may cause health hazards. The results obtained from the method validation were satisfactory for the analysis of PCBs and PAHs. The EDI and HI values of the PCBs are presented in Animals are continually exposed to environmental toxins and are able to collect high concentrations of pollutants in their tissues, particularly fat tissues Studies have reported that amyloidogenesis can be inhibited by polychlorinated biphenyls upon binding to transthyretin in the blood The metabolism of PAHs occurs through the cytochrome P450 peroxidase and aldo-keto reductase pathways. These pathways involve diol-epoxides and radical cations, which disrupt DNA and damage cells by binding to DNA and proteins. As a result, carcinogenic, mutagenic, immunosuppressive, and teratogenic damage occurs and tumors finally develop tumors PAHs are allocated throughout the environment and persist for a longer time Several PAHs compounds exist; however, only 16 PAHs compounds have been studied so far in most of the studies. These results indicate that environmental pollution plays an important role in the contamination of milk by PAHs 4This study investigated the presence of PCBs congeners and PAHs compounds in milk samples from several parts of Dhaka, Bangladesh using GC-MS/MS. The presence of PCBs in milk samples indicates the presence of environmental pollution. There are several other sources of PCBs contamination and there is a possibility of food contamination through PCBs congeners. The majority of PAHs compounds were not detected in milk samples. Very low amounts of benzo (a) anthracene and chrysene were detected in samples from industrial areas, where more human activities were observed. Therefore, proper detection and monitoring are required to ensure the safety of foods from animal sources. Detected values below the recommended levels indicate that the milk consumed by the population is free from toxicological risks. However, long-term exposure may result in significant exposure and cause a high contamination risk. Therefore, further studies are needed to establish specific MRL values for the Bangladeshi population.G.M.M. Anwarul Hasan: Conceptualization, Investigation, Methodology, Data curation, Formal analysis, Writing − original draft. Md. Aftab Ali Shaikh: Conceptualization, Funding acquisition, Project administration, Writing − review & editing. Mohammed A. Satter: Data curation, Formal analysis, Funding acquisition, Investigation, Resources, Software, Supervision, Validation. Md. Sabir Hossain: Methodology, Software, Supervision, Validation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.