Moringa oleifera seeds-removed ripened pods as alternative for papersheet production: antimicrobial activity and their phytoconstituents profile using HPLC

In the present study, and for the waste valorization, Moringa oleifera seeds-removed ripened pods (SRRP) were used for papersheet production and for the extraction of bioactive compounds. Fibers were characterized by SEM–EDX patterns, while the phytoconstituents in ethanol extract was analyzed by HPLC. The inhibition percentage of fungal mycelial growth (IFMG) of the treated Melia azedarach wood with M. oleifera SRRP extract at the concentrations of 10,000, 20,000, and 30,000 µg/mL against the growth of Rhizoctonia solani and Fusarium culmorum was calculated and compared with fluconazole (25 µg). The produced papersheet was treated with the ethanol extract (4000, 2000, and 1000 µg/mL) and assayed for its antibacterial activity against Agrobacterium tumefaciens, Erwinia amylovora, and Pectobacterium atrosepticum by measuring the inhibition zones and minimum inhibitory concentrations (MICs). According to chemical analysis of M. oleifera SRRP, benzene:alcohol extractives, holocellulose, lignin, and ash contents were 7.56, 64.94, 25.66 and 1.53%, respectively, while for the produced unbleached pulp, the screen pulp yield and the Kappa number were 39% and 25, respectively. The produced papersheet showed tensile index, tear index, burst index, and double fold number values of 58.8 N m/g, 3.38 mN m2/g, 3.86 kPa m2/g, and 10.66, respectively. SEM examination showed that the average fiber diameter was 16.39 µm, and the mass average of for elemental composition of C and O by EDX were, 44.21%, and 55.79%, respectively. The main phytoconstituents in the extract (mg/100 g extract) by HPLC were vanillic acid (5053.49), benzoic acid (262.98), naringenin (133.02), chlorogenic acid (66.16), and myricetin (56.27). After 14 days of incubation, M. oleifera SRRP extract-wood treated showed good IFMG against R. solani (36.88%) and F. culmorum (51.66%) compared to fluconazole, where it observed 42.96% and 53.70%, respectively. Moderate to significant antibacterial activity was found, where the minimum inhibitory concentration (MIC) values were 500, 650, and 250 µg/mL against the growth of A. tumefaciens, E. amylovora, and P. atrosepticum respectively, which were lower than the positive control used (Tobramycin 10 µg/disc). In conclusion, M. oleifera SRRP showed promising properties as a raw material for pulp and paper production as well as for the extraction of bioactive compounds.


Materials and methods
Plant material and extract preparation of Moringa oleifera seeds-removed ripened pods. This study is complied with relevant institutional, national, and international guidelines and legislation. This study does not contain any studies with human participants or animals performed by any of the authors, where Moringa oleifera Lam. seeds-removed ripened pods (SRRP) were collected from Alexandria, Egypt, 2020. The plant was identified at the Department of Forestry and Wood Technology, Faculty of Agriculture, Alexandria University and a sample was deposited (voucher number Zidan0077). The SRRPs were ground into powder and screened (size 40-60 mesh), and then 100 g of this powdered size were extracted with ethanol (200 mL) by soaking method for 3 days 25 , where every day it was agitated at least three times for 5 min, and it should be noted that every day the amount of ethanol was replaced with the another amount (200 mL), therefore we used 600 mL ethanol for three days extraction. The extracted material was filtrated using Whatman filter paper no. 1 to get rid of residues and the dissolved extract was concentrated by evaporating the solvent using the rotary evaporator.
The antifungal activity of wood treated with M. oleifera (SRRP) extract. Two fungi Fusarium culmorum (Acc# MH352452), and Rhizoctonia solani (Acc# MH352450), were used for the bioassay [26][27][28] . Melia azedarach wood specimens (2 × 1 × 0.5 cm), that autoclaved (121 °C for 20 min) and left to cool, were treated with M. oleifera SRRP extract at the concentrations of 10,000, 20,000, and 30,000 µg/mL. Each wood sample was received 100 µL from each concentration of M. oleifera SRRP extract. Petri dishes contained PDA media were inoculated with 5 mm-disc diameter of each fungus and the treated wood samples were put directly over the media at the opposite side of the fungus disc 29,30 . The treated wood samples were compared with control treatment (autoclaved-untreated). The percentage of fungal inhibition was calculated with the formula of the inhibition percentage of fungal mycelial growth (IFMG %) = [(T 0 − T 1 )/T 0 ] × 100, where T 0 and T 1 are the average diameters (mm) of fungal colonies under the control and experimental treatments, respectively, after insuring that the growth of fungi in control treatment, the measurement was done according to the previous works [28][29][30][31][32][33] .
The IFMG values were compared with the positive (25 µg of fluconazole) and negative (10% DMSO) controls 34  Chemical analysis of M. oleifera SRP and Kraft pulping. Moringa oleifera SRRP (Fig. 1a) was collected after the seeds were removed then cut into small pieces or flakes to be suitable for pulping (Fig. 1b). For chemical analysis, about 200 g of M. oleifera SRRP were ground into fine powder then screened to obtain the size 40-60 mesh fraction. Extractives content (alcohol and benzene), holocellulose, insoluble lignin content and Ash content were measured according to T204, T249, T222 om88, and T211, respectively.
For Kraft pulping, 200-g oven-dried pieces of M. oleifera SRRP were swelled for one day, filtrated, washed several time with hot water. Kraft pulping was conducted in stainless steel vessel with capacity 2 L under rotation in oil bath. The conditions used for pulping of M. oleifera SRRP were: active alkalinity (11%), temperature Examination of the produced paper sheets via scanning electron microscopy (SEM). The produced papersheets (Fig. 1d) from M. oleifera SRRP pulp were studied by scanning electron microscopy (SEM), attached with energy dispersive spectrometry (EDX), JFC-1100E ion sputtering device (model JEOL/MP, JSM-IT200 Series, Japan) with acceleration voltage of 20.00 kV to show the elemental compotation and diameter of the fibers from three points and the average was taken. The measurements were taken from three parts of the paper sheets 25,44-46 . In vitro antibacterial evaluation of treated-papersheets with the extract. Discs with approximate dimension of 1 × 1 cm were cut from the M. oleifera SRRP pulp paper treated with three concentrations (4000, 2000, and 1000 µg/mL) from M. oleifera SRRP extract as well as the control treatment (DMSO 10%) 25 . Three plants pathogenic bacteria Agrobacterium tumefaciens (acc# MG706145), Erwinia amylovora (acc#LN876573) and Pectobacterium atrosepticum (acc#MG706146), were used for the antibacterial activity and were previously www.nature.com/scientificreports/ identified through molecular identification [47][48][49][50][51] . The agar disc diffusion method was employed for antibacterial activity determination of the extract by recording the inhibition zone 52 . All tests were performed in triplicate. Also, micro-dilution method with serial concentrations of 32-1000 µg/mL was measured and compared with the control (Tobramycin 10 µg/disc) 26,53 .
Statistical analysis. Tensile index, burst index, tear index, double fold number, brightness and optical measurements from the tested papersheet produced from M. oleifera SRRP pulp paper were recorded as mean ± SD from three measurements. The measurements of antifungal and antibacterial activities were statistically analyzed with one way ANOVA using SAS system and comparisons among the means were recorded using LSD test at an alpha value of 0.05 54 .
Compliance with ethical standards. This study is complied with relevant institutional, national, and international guidelines and legislation. "This study does not contain any studies with human participants or animals performed by any of the authors".  63 , and flax plant (70%) 46 . While it was higher than those from Zea mays stalks (62.33%) and Sorghum bicolor stalks (63.40%) 67 , lotus leaf stalks (53.8%) 68 , and Posidonia oceanica (61.8%) 60 .
The unbleached M. oleifera SRRP pulp (Table 1) showed the following properties; Freeness (300 mL CSF), screen pulp yield (39%), Kappa number (25), and the residual alkali (13.4 g/L). Compared to other study, the Mechanical and optical properties of papersheets. Table 2  The tensile index value (58.8 N m/g) was higher than those reported from papersheet produced from rice straw pulps (38.0-55.2 N m/g) 87 , flax material (42.66 N m/g) 46 , and oil palm empty fruit bunches pulp (20.4 N m/g) 88 . While it was lower than from the papersheet produced from depithed Bagasse pulp (60 N m/g) 72 .

SEM-EDX examination of the produced papersheet.
To confirm the distribution, construction and fiber diameters of the produced papersheet from M. oleifera SRRP pulp, SEM-EDX technique was used. The images of SEM-EDX were taken from three places of the produced papersheet. The SEM images showed that the average fiber diameters was 18.52 µm (Fig. 2a), 12.66 µm (Fig. 2b) and 18.29 µm (Fig. 2c), and the whole average was 16.39 µm. Other study designed to evaluate the fiber characteristics of M. oleifera wood slivers to predict its suitability for pulp and paper production showed that the average fibre diameter was 61.31 µm 23 , while other study showed that the value was 15.01 µm, 15.04 µm, and 15.08 µm from stem-wood of 1, 3, 5 years old M. oleifera trees, respectively 24 , and 15.0 μm in width 55 .
Furthermore, most of failure zones and the increase in fiber deformations, which probably could be found in pulp fibers such as curl, kink, lumen collapse, dislocation, microcompression and twist 92,93 were shown in low amounts in M. oleifera SRRP papersheet.

HPLC analysis, antibacterial and antifungal activities and extract from M. oleifera SRRP.
For the antifungal activity, the visual observations of wood-treated with M. oleifera SRRP extract and inoculated with Rhizoctonia solani and Fusarium culmorum after 14 days from the inoculation are shown in Fig. 4. Wood-treated with the extract showed inhibition percentage of fungal mycelial growth (IFMG) ranged from 27.51 to 36.88% and from 22.11 to 51.66% against the growth of R. solani and F. culmorum, respectively (Table 4). Table 5 observes that M. oleifera SRRP extract at 4000 µg/mL showed antibacterial activity against the growth of Agrobacterium tumefaciens, Erwinia amylovora, and Pectobacterium atrosepticum, with inhibition zones values of 11 mm, 6.66 mm and 16.66 mm, respectively, after the incubation period (24 h) as shown in Fig. 5. The recorded MIC values 500, 650, and 250 µg/mL against the growth of A. tumefaciens, E. amylovora and P. atrosepticum, respectively, were lower than of the positive control (Tobramycin 10 µg/disc) 32-64 µg/mL.

Mechanical properties
Optical properties
Myricetin which found in the amount of 56.27 mg/100 g extract of M. oleifera SRRP, has been previously possessed potential antibacterial activities 107 , also myricetin and rutin were observed potent antifungal agents  www.nature.com/scientificreports/  www.nature.com/scientificreports/ www.nature.com/scientificreports/