Introduction

Moringa oleifera Lam. (family Moringaceae) is a fast-growing and drought-resistant tree, native to the Indian subcontinent with multipurpose uses1. Fruits of Moringa are three-sided pods with pendulous and linear shape, also, the pod generally has 250–450 mm long contains approximately 20 globular seeds2. From the literature survey, all the works are concentrated in how to use leaves, flowers, pods and roots of Moringa in different purposes3,4,5. Leaves and seeds of M. oleifera are promised as a first stage in the treatment for waste waters6,7,8 or for coagulant of primary treatment of paper mill effluent9. Acid activated from M. oleifera leaf was also prepared, which act as a good alternative adsorbent for dyes and heavy metal recoveries from aqueous solutions10. Petals of M. oleifera were used as a mediated green synthesis of gold nanoparticles11. Leaves and other parts from the tree were used as a source for antimicrobial and antioxidant agents as well as for pharmaceutical purposes12,13,14. In livestock application, leaves and seeds of M. oleifera are used for animal nutrition, where they have many nutritional compounds such as oils, carbohydrates, vitamins, fatty acids, amino acids, lipid, minerals and other chemical compounds5,15,16.

Several bioactive compounds were isolated and identified from different parts of Moringa (leaves, seeds, bark, flowers, pods, and root) and were summarized in the review articles of Chhikara et al.2 and Trigo et al.17. Quercetin, myricetin glycosides, caffeoylquinic acid, coumaroylquinic acid, hydroxybenzoic acid, kaempferol, glucotropaeolin, glucosinalbin, glucoraphanin, glucomoringin, glucoiberin, glucosinolates, apigenin, luteolin, lutein, luteoxanthin, zeaxanthin, b-carotene and isothiocyonates were identified as the main compounds in the extracts from moringa2,18,19. Phenolic compounds from M. oleifera seed, including gallic acid, ellagic acid and kaempferol were observed good antioxidant activity20,21.

For the production of pulp and paper from M. oleifera, there are little works from the literature, i.e., kraft pulping yield of M. oleifera and M. concenensis (M. concanensis) stems showed satisfactory strength properties for wrapping and writing papers compared to those of conventional raw materials22. Also, some investigations showed that the fiber characterizations such as fiber length and diameter of M. oleifera stem indicated that stem-wood from the middle and base was best suited for pulp and paper production23, while among the collected stems from 1, 3 and 5 year olds Moringa oleifera, the fiber characteristics from 5 year old M. oleifera stem-wood showed the best suited for the production of pulp and paper24.

To the best of our knowledge, this is the first work showing the value-added of M. oleifera seeds-removed ripened pods in the production of papersheet and as source for bioactive compounds for antibacterial and antifungal activities.

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 days25, where every day it was agitated at least three times for 5 min, and it should be noted that every day the amount of ethanol was replaced with the another amount (200 mL), therefore we used 600 mL ethanol for three days extraction. The extracted material was filtrated using Whatman filter paper no. 1 to get rid of residues and the dissolved extract was concentrated by evaporating the solvent using the rotary evaporator.

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 bioassay26,27,28. 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 disc29,30. 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 %) = [(T0 − T1)/T0] × 100, where T0 and T1 are the average diameters (mm) of fungal colonies under the control and experimental treatments, respectively, after insuring that the growth of fungi in control treatment, the measurement was done according to the previous works28,29,30,31,32,33. The IFMG values were compared with the positive (25 µg of fluconazole) and negative (10% DMSO) controls34.

HPLC analysis of extract

HPLC 1260 Infinity Agilent System (Agilent Technologies, Santa Clara, CA, USA) equipped with a Quaternary pump and a Zorbax Eclipse Plus C18 column (100 mm × 4.6 mm i.d.) operated at 30 °C was used to identify the phytochemical compounds in M. oleifera SRRP extract. Separation conditions can be found in previously published works4,27,35,36,37,38. The following standard phytochemical compounds with HPLC grade (Sigma-Aldrich, St. Louis, MO, USA) were used; catechol, p-hydroxy benzoic acid, caffeine, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, benzoic acid, rutin, ellagic acid, o-coumaric acid, salicylic acid, cinnamic acid, myricetin, quercitin, rosmarinic acid, naringenin and kaempferol.

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.

Figure 1
figure 1

Shows M. oleifera SRRP (a), the cut pieces of M. oleifera SRRP (b), pulp of M. oleifera SRRP (c) and the produced papersheet from the pulp (d).

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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 (160 °C), reaction time (35 min) and the liquor ratio (liquid to M. oleifera SRRP ratio of 10:1). The solid residue was defibrated, washes with hot and cold water till neutral pH, and the resulted pulp (Fig. 1c) screened in a valley flat screen having 0.25 mm slots. The screened unbleached pulp yield39, Kappa number of unbleached pulp40, the CSF Freeness of Pulp 41, and the Residual alkali42 were determined.

Papersheet forming (Fig. 1d) was carried out followed with our previous works, where the pulp with standard papersheet samples (200 cm2) with grammage of about 60 g/m2 were obtained43. Papersheets were made and tested for the strength properties according to TAPPI test methods T218 and T220. The papersheets were tested for tensile resistance T404, tear strength T414, bursting strength T405 and double fold T423. Analysis of physical strength of pulp was performed according to TAPPI standard methods with sheets standard 60 g/m2. All the experimental works were performed in triplicate.

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 sheets25,44,45,46.

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 identified through molecular identification47,48,49,50,51. The agar disc diffusion method was employed for antibacterial activity determination of the extract by recording the inhibition zone52. All tests were performed in triplicate. Also, micro-dilution method with serial concentrations of 32–1000 µg/mL was measured and compared with the control (Tobramycin 10 µg/disc)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 0.0554.

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”.

Results and discussion

Chemical characterization of M. oleifera SRRP and unbleached pulp properties

Chemical characteristics of M. oleifera SRRP and the produced unbleached pulp are shown in Table 1. The level of holocellulose content in M. oleifera SRRP is 64.94%, which indicates that it would be good sources of cellulose and hemicellulose. Furthermore, this content is well-compared with those reported by other studies, where the holocellulose content in M. oleifera stem was 65.5%55. While it was lower than those from other non-woody materials, i.e., Sorghum bicolor stalks (71.0%)56, Musa sapientum (73.43%), M. paradisiaca (72.60%) and Tithonia diversifolia (71.60%)57, bamboo (70.50%)58, Tunisian Alfa stems (68.2%)59, Date palm rachis (74.8%)60,61, Hesperaloe funifera (76.5%)62, Cotton stalks (72.9%)63, Canola straw (77.5%)64, Luffa cylindrica (83.0%)65, Hibiscus cannabinus (81.1%)66, Arundo donax (70.2%)63, and flax plant (70%)46. While it was higher than those from Zea mays stalks (62.33%) and Sorghum bicolor stalks (63.40%)67, lotus leaf stalks (53.8%)68, and Posidonia oceanica (61.8%)60.

Table 1 Chemical composition of M. oleifera SRRP and unbleached pulp.
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Comparing to the woody materials, holocellulose content in M. oleifera SRRP was lower than the amount presented in Paulownia elongota wood (75.74%)69, Pinus pinaster wood (69.6%)63, Albizia lebbeck wood (78.60%)70, Eucalyptus globulus wood (80.5%)63, Acer rubrum wood (67.4%)71, Leucaena diversifolia wood (77.9%)63, and depithed Bagasse (72.38%)72. While it was higher than from those of Prosopis alba wood (63.6%)63, E. camaldulensis wood (56%) and Meryta sinclairii wood-branch (61%)25, and woods from Bougainvillea spectabilis (54.56%), Ficus altissima (54.73%), and F. elastica (53.37%)67.

Lignin content (25.66%) in M. oleifera SRRRP was lower than from the reported in M. oleifera stem (20.5%)55. While it was equal to those found in lotus leaf stalks (25.4%)68, and were higher than those from rice husks (21.98%)73, rice hulls (20.44–23.33%)74, sugar beet (17.67%)75, stalks of Zea mays (19.9–20.1%)76, Sweet sorghum (21%)77, Corn stover (19%)78, Tall fescue (14.0%), and Miscanthus giganteus (17.8%)79, bamboo (24.5%)58, H. funifera (7.3%)62, Cotton stalks (21.4%)63, Canola straw (20.0%)64, Luffa cylindrica (15.2%)65, Kenaf (12.7%)66, Wheat straw (19.64%)80, A. donax (22.3%)63, flax plant (6.8%)46, depithed Bagasse (20.03%)72, Bagasse (23.33%)81, Cynara cardunculus stalks (16–19%)82,83 and Miscanthus × giganteus stalks (13%)84. While it was lower than amount from Nut shells (30–40%)85.

The content of lignin from M. oleifera SRRP was in the range of hardwood species (25–35%)86, i.e., in Albizia lebbeck wood (25.14%)70 and lower than those from Date palm rachis (27.2%)49,61, and Posidonia oceanica (29.8%)60. Compared to woody plant materials, it was lower than those from Pinus pinaster (26.2%)63, Acer rubruma (26.0%)71, and E. camaldulensis (27%)25, and higher than those from E. globulus (20.0%)63, L. diversifolia (19.1%)63, P. alba (19.3%)63, and M. sinclairii (23%)25.

The ash content in M. oleifera SRRP (1.53%) was lower than the amount in stem (3.5%)55, while the Alcohol-benzene solubility (7.56%) was higher from the measured in the stem (3.16%)55.

The 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 screened yield from unbleached pulp of M. oleifera stem was 38.2–40.29%, Freeness mL, CSF (650), and Kappa number (16.2–21.7)55.

Mechanical and optical properties of papersheets

Table 2 shows the mechanical and optical properties of the produced papersheet from M. oleifera SRRP pulp, where the tensile index (58.8 N m/g), tear index (3.38 mN m2/g), burst index (3.86 kPa m2/g), double fold number (10.66), brightness (32%) and opacity (67%).

Table 2 Mechanical and physical properties of the produced papersheet from M. oleifera SRRP pulp.
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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. The tear index value (3.38 mN m2/g) was lower than from papersheets manufactured from pulps of rice straw (6.49–7.49 mN m2/g)87, depithed Bagasse (5.0 mN m2/g)72, flax plant (4.33 mN m2/g)46 and palm oil empty fruit bunches (7.20 mN m2/g)89, while it was partially equal to the measured from wheat straw (3.86 mN m2/g)90 and higher than of sunflower stems (2.04 mN m2/g)91.

The burst index value (3.86 kPa m2/g) was in the range of the value reported from papersheets manufactured from pulps of rice straw (2.43–5.34 kPa m2/g)87, but lower than from depithed Bagasse (4.8 kPa m2/g)72. Double fold number (10.66) was lower than the value reported from the papersheets derived from pulps of rice straw (35–173)87, and depithed Bagasse (26–42)72.

Tensile, burst, and tear indices from papersheets produced from refined unbleached Kraft pulp from M. oleifera stem were 48.7 N m/g, 3.56 kPa m2/g, and 5.8 mN m2/g, respectively55. The unbleached pulp brightness of M. oleifera SRRP (32%) was higher than the reported from unbleached stem pulp (25.4–29.5%)55.

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 µm23, 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, respectively24, and 15.0 μm in width55.

Figure 2
figure 2

Shows the SEM–EDX measurements of papersheets from M. oleifera SRRP pulp paper at three points (a), (b) and (c). C Curl, T Twist, M Microcompression.

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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 twist92,93 were shown in low amounts in M. oleifera SRRP papersheet.

Elemental composition by EDX showed that the mass (%) of C and O is 44.04%, 55.96% (Fig. 2 Spc_001), 43.29%, 56.71% (Fig. 2 Spc_002), and 45.29%, 54.71% (Fig. 2 Spc_003), and the mass average was 44.21 ± 1.01%, and 55.79 ± 1.01%, respectively.

HPLC analysis, antibacterial and antifungal activities and extract from M. oleifera SRRP

Figure 3 shows the HPLC chromatogram of the polyphenolic compounds in the extract and the identified compounds is presented in Table 3, where the main compounds were vanillic acid (5053.49 mg/100 g extract), benzoic acid (262.98 mg/100 g extract), naringenin (133.02 mg/100 g extract), chlorogenic acid (66.16 mg/100 g extract), and myricetin (56.27 mg/100 g extract).

Figure 3
figure 3

HPLC fingerprint of the identified phytoconstituents in M. oleifera SRRP extract.

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Table 3 Phytoconstituents profile of M. oleifera SRRP extract.
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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).

Figure 4
figure 4

Visual observation after 14 days of the treated wood with M. oleifera SRRP extract and inoculated with two fungi (Rhizoctonia solani and Fusarium culmorum).

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Table 4 Antifungal activity of wood-treated M. oleifera SRRP extract.
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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.

Table 5 Antibacterial activity of extract from M. oleifera SRRP.
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Figure 5
figure 5

Antibacterial activity of treated papersheet discs with M. oleifera SRRP extract against (Ag) Agrobacterium tumefaciens; (Ea) Erwinia amylovora; (PA) Pectobacterium atrosepticum. c: Control; 1: Extract concentration 1000 µg/mL; 2: Extract concentration 2000 µg/mL; 3: Extract concentration 4000 µg/mL.

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It is important to note that a MIC value between 100 and 200 μg/mL was considered as positive for plant extracts94,95,96,97,98. However, the activity of plant extracts have been classified as significant (MIC < 100 μg/mL), moderate (100 < MIC ≤ 625 μg/mL) or weak (MIC > 625 μg/mL)99,100. In addition, Tamokou et al.101 proposed new threshold values of MIC for extracts as follow; highly active (MIC < 100 μg/mL), significantly active (100 ≤ MIC ≤ 512 μg/mL), moderately active (512 < MIC ≤ 2048 μg/mL), low activity (MIC > 2048 μg/mL), and not active (MIC > 10 mg/mL). According to these classifications, the activities M. oleifera SRRP extract were moderate to significant against A. tumefaciens and P. atrosepticum and weak to moderate against E. amylovora.

Total polyphenols (13.7 g/100 g extract dry weight) and total flavonoids (69.0 g/100 g extract dry weight) were reported from the pods2,102. Several phytochemical compounds were identified in different parts of M. oleifera including quercetin, ellagic acid, gallic acid and kaempferol103.

Revealed to the concentration used, Salvadora persica root-bark acetone extract showed inhibition zones (IZs) against A. tumefaciens (13.6–18.6 mm), P. atrosepticum (15.3–23 mm)51. Chloroform leaf extracts from Lantana camara Duranta plumieri variegata and Citharexylum spinosum showed IZs with the range of 8.3–24.3 mm, 8–13.6 mm, 8–11.6 mm, against A. tumefaciens, and 6.6–9.6 mm, 0–9.3 mm, and 9.6–13.6 mm against P. atrosepticum, respectively50. Callistemon viminalis flowers acetone extract observed IZ value 15.0 mm against the growth of A. tumefaciens49.

Moringa oleifera SRRP extract-treated wood showed potential antifungal activity against F. culmorum (IFMG 36.88% at concentration 30,000 µg/mL) and R. solani (IFMG 51.66% at concentration 30,000 µg/mL). Also, the present results showed that the FMIP against F. culmorum was lower than the standard biofungicide Fluconazole (25 μg), which observed IFMG 53.70% and higher than Fluconazole (42.96% against R. solani) when applied to wood samples34. Previously, different parts of M. oleifera plant extracts have been observed to inhibit some phytopathogenic fungi including Alternata burnsi, Aspergillus niger, A. paracitic, A. flavus, Candida Albicans, F. oxysporum and Trichoderma harzanium104. Comparing to other natural extracts applied to wood samples as biofungicide preservatives, i.e., Haplophyllum tuberculatum whole plant extract with its main compounds resveratrol, kaempferol, myricetin, rutin, quercetin, and rosmarinic acid showed potential antifungal activity against F. culmorum and R. solani when applied to Melia azedarach wood34,105. The extracts from Coccoloba uvifera with its main compounds of gallic, benzoic, ellagic, and o-coumaric acids applied to Pinus roxburghii wood observed good activity against R. solani, Botrytis cinerea, and F. culmorum37. Flower extract from Acacia saligna-treated M. azedarach wood, with the presence of quercetin, naringenin, benzoic acid, o-coumaric acid, caffeine and kaempferol compounds observed antifungal activity against F. culmorum, R. solani, and Penicillium chrysogenum26. An antimicrobial potential activities against R. solani, F. culmorum and A. tumefaciens, were observed as wood-treated with Musa paradisiaca peel extract, where the HPLC analysis of the extract identified gallic acid, ellagic acid, naringenin, rutin, and myricetin as main compounds27. Furthermore, salicylic acid, rutin, vanillic acid and myricetin were found in Withania somnifera fruit extract that showed good wood-biofungicide activity against F. culmorum and R. solani wood-bactericide against A. tumefaciens, E. amylovora, and Pseudomonas cichorii106.

Myricetin which found in the amount of 56.27 mg/100 g extract of M. oleifera SRRP, has been previously possessed potential antibacterial activities107, also myricetin and rutin were observed potent antifungal agents against Candida albicans and C. parapsilosis108. A. flavus and A. parasiticus were completely inhibited in terms of their growth and the production aflatoxin by vanillic and caffeic acids at 0.2 mg/mL109. Also, phenolic compounds of Stenoloma chusanum extract including vanillic acid showed potential antifungal activity110.

Conclusion

As from the present study and commercially, moringa, the fast growing with multipurpose uses, and after obtaining the ripened seed, the seeds-removed pods have been shown some important properties. It acts as a raw material for the production of pulp and paper due to limited wood resources, where the mechanical and physical properties of the produced papersheet were comparable with those reported from the literature from woody and non-woody materials. Also, from the HPLC analysis of phytoconstituents profile, some important phenolic compounds vanillic, benzoic, syringic, and ferulic acids and flavonoid compounds myricetin, naringenin and kaempferol were identified. This study showed the maximizing the utilization of moringa residues in the pulp industry and the production of bioactive chemicals.