Stimulating action of sodium nitroprusside and vinasse on salicin and direct regeneration in Salix Safsaf Forssk

Abstract

The present study aimed to enhance salicin and direct regeneration in willow (Salix safsaf Forssk) using the sodium nitroprusside (SNP) regulation of nitric oxide (NO) and vinasse for its nutrition effect in culture medium. Internodes of Salix safsaf were cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) (0.25 mg L⁻¹) and different concentrations of SNP (0, 5, 10, 15, and 20 mg L⁻¹) or vinasse (0, 5, 10, and 20%) to examine shoot regeneration, antioxidant defense enzyme activity, total phenolic compounds, flavonoids, and salicine contents. The reported data revealed that application of SNP at 15 mg L⁻¹ and vinasse at 10% induced a significant effect in vitro Salix safsaf shoot regeneration. To confirm that, nitric oxide is required for auxin-mediated activation of cell division in a dose-dependent manner. A concentration of 15 mg L⁻¹ SNP promotes regeneration and salicin accumulation (3162.16 mg/100 g) during signaling action. On the other hand, the cross talk effect of nitric oxide and vinasse combination in Salix safsaf significantly induced a synergistic effect on direct propagation more than vinasse alone. SNP significantly stimulates salicylate accumulation in a dose-dependent manner, but the data on the association of vinasse and SNP on salicylate up-regulation showed a significant reduction in salicin accumulation when SNP was combined with 10% vinasse, which directly affected the signaling action of SNP as secondary product stimulators. Vinasse’s phenolic compounds affect directly on the reduction activity of SNP to suppress its signaling action, or indirectly by inhibiting the sequence cascade of the SNP signaling transduction process to decrease the accumulation of salicin contents. Data confirmed that vinasse and SNP stimulated the antioxidant enzymes activity throw quenching the stimulated reactive oxygen species that produced via SNP. Results show that modified media with SNP administration at 15 mg L⁻¹ and the combination of vinasse at 10% and SNP at 15 mg L⁻¹ are recommended for modifying tissue culture media for induced direct regeneration and salicin accumulation in tissue culture applications, which will be very useful for commercial salicin overproduction as a biological active ingredient in willows.

Introduction

Willow has been utilized in traditional medicine for more than 3500 years. The active ingredient in willow bark is salicin, the base for the production of aspirin, which reduces the risk of cardiovascular disease (Desborough and Keeling 2017). There are many species of the genus Salix. It is used in many traditional treatments, and also considered the source of salicylic acid that is stored in abundance just under the tree bark. About 32 novel compounds have been identified in Salix species. The active compounds of the willow leaf extracts (salicin is related to salicylic acid) were shown to be anti-leukemia agents (El-Shemy et al. 2003).

Biotechnological methods (e.g., micropropagation, callus cultures, and bioreactors) can yield a large amount of clones, which can decrease costs (Gago et al. 2021). Mainly, buds or shoots are used for in vitro propagation (Peternel et al. 2009). Contamination, browning, and death of explants are common problems in the effective micropropagation of woody plants. The growth of explants is affected by the physiological phase of donor plants and by the season. Modified MS medium supplemented by cytokinin and auxin is extensively used for the shoot propagation of woody plants to induce new plants (Dobránszki and Teixeira da Sliva 2010). A medium containing benzyl adenine (BA) has proven successful for hybridization (Agrawal and Gebhardt 1994). Most of the willow species have the capability for vegetative clonal reproduction and biomass productivity, but their production may be expensive (Tharakan et al. 2005; Yoon et al. 2021).

Therefore, using a different, less expensive growth medium, such as vinasse (an industrial by-product of sugarcane), which is rich in carbon sources and contains active components (phenols and flavonoids), is beneficial. According to Yousef et al. (2024), sugarcane by-products are effective as economic culture media rich in carbohydrates and amino acids. In the ethanol industry, vinasse is taken out of the base of distillation columns. It is a liquid residue with a dark brown appearance. For every liter of alcohol produced, 12–15 l are created on average (Nair and Taherzadeh 2016). Vinasse, on the other hand, contains macro- and micronutrients that are thought to be crucial for crop development (Carpanez et al. 2022). Sodium nitroprusside (SNP) is used in the treatment of acute hypertension as a potent vasodilator (Holme and Sharman 2024). Nitric oxide (NO), a highly reactive gas that plays a central role in signal transduction in plant stress response and regulates a wide variety of biological functions that promote plant growth and development, retard senescence, and seed germination, has also been recognized as a phytohormone (Leterrier et al. 2012; Arc et al. 2013). The regulatory mode of action of NO is probably based on its chemical nature as a free radical. It reacts mainly with metals and other free radicals in the microenvironment where it is produced (Thomas 2015). The oxidative status generated by reactive oxygen species (ROS) in stressed plants seems to be alleviated by NO through the improvement of the antioxidant capacity, thus contributing to redox homeostasis (Correa-Aragunde et al. 2015). Nitric oxide has the potential to be a useful tool in optimizing the output and quality of horticulture crop species, since it controls plant growth, improves nutrient uptake, and activates stress tolerance mechanisms in most plants (Sun et al. 2021). The role of signaling and antioxidant molecules in the defense response during stresses increases salicylic acid (SA) and nitric oxide, which may increase the production of ROS. ROS can also injure the plant cell, but they can be controlled by oxidized glutathione and ascorbate (Nagai et al. 2020). Plants have an antioxidant system against oxidative stress, which is composed of ROS-scavenging enzymes such as superoxide dismutase, ascorbate peroxidise, and catalase (Ali et al. 2024). Some studies have indicated that plants produce SA from cinnamate created by phenylalanine ammonia lyase (PAL). PAL is a key regulator in the phenol synthesis pathway and is induced under a variety of biotic and abiotic stress conditions (Dong et al. 2012).

This work investigated the effect of SNP, vinasse, and its combination, which were added to MS media for direct regeneration induction and salicin up-regulation in willow (Salix safsaf Forssk) through the regulatory effect of nitric oxide. In addition to its effects on antioxidant defense enzyme activity and PAL enzyme activity, it also increase the accumulation of total phenolic and flavonoid compounds.

Materials and methods

This study was carried out in the Center Tissue Culture Laboratory, Agricultural Botany Dept., Faculty of Agriculture, Cairo University. Plant material was obtained from the uppermost branches of willow trees (Salix safsaf Forssk) grown in Orman Botanic Garden, Giza Governorate, Egypt.

Plant tissue culture method

Explants (internodes) from the uppermost branches of willow were sterilized by flooding in ethyl alcohol 70% for 1 min. These were then soaked in 0.1% (w/v) mercuric chloride (HgCl₂) solution for 3 min, rinsed in sterile distilled water (15 min), and then cultured on MS medium (Murashige and Skoog 1962) supplemented with BA (0.25 mg L⁻¹) (Mohamed et al. 2015)., 3% sucrose, 0.2% gel right agar, and different concentrations of vinasse (0, 5, 10, 15, 20%), or sodium nitroprusside (SNP) (0, 5, 10, 15, 20) mg L⁻¹. Another culture was supplemented with the best treatment 15 mg L⁻¹ SNP and different concentrations of vinasse (5, 10, 15%). All pH media were adjusted to 5.7–5.8. All culture media was sterilized by autoclaving at 120 °C for 20 min, and incubated under controlled conditions of temperature 24 ± 2 °C with 16 h of photoperiod under cool white fluorescent light. The intensity of illumination was 40.5 μmol m⁻² s⁻¹. At the end of the culture experiment (5 weeks later), data were recorded for salix safsaf plantlets and the following parameters were estimated:

$$\text{Regeneration}\%=\left(\text{no}.\text{of explants regenerated shoots}/\text{treatment}\right)÷(\text{no}.\text{of explants}/\text{treatment}).$$

Shoot length (cm), number of brunches, fresh and dry weight (g).

Vinasse chemical analysis

Crude protein, total lipids, total nitrogen, and dry matter were measured using the procedures outlined by A.O.A.C. (1990). The Rosein (1957) method was used to determine the total amino acids. The phenol–sulfuric acid method was used to determine the total amount of hydrolyzable carbohydrates, as per Dubois et al. (1956). Colorimetric analysis was used to quantify phenolic chemicals using the procedures outlined by Arnaldos et al. (2001).

Plant biochemical analysis

Determination of enzymes activities

Protein was determined in shoot samples according to Bradford (1976) with bovine serum albumin as standard.

Polyphenol oxidase activity was determined spectrophotometrically at 400 nm using chlorogenic acid as substrate according to González et al. (1999).

Catalase activity was assayed spectrophotometrically at 510 nm according to Aebi (1984) and Fossati (1980).

Glutathione peroxidase activity was measured according to Paglia and Valentine (1967) by recording the decrease in absorbance at 340 nm per min.

Phenylalanine ammonia-lyase (PAL) activity was determined using a modified method of Cahill and McComb (1992).

Determination of the total phenols and flavonoids content

The content of total phenols was determined using a Folin–Ciocalteu method described by Singleton (1974). Flavonoids were estimated using the aluminum chloride colorimetric method of Zhishen et al. (1999).

Determination of salicin content by HPLC

Standard salicin was purchased from Sigma-Aldrich (China). 2 g of powdered shoot sample was extracted with 70% ethanol for 30 min on a water bath at 80 °C. After extraction, the mixture was centrifuged at 4000 rpm. To study the presence of salicin, the mixture was mixed together with 2 M hydrochloric acid, heated at 80 °C for 60 min on a water bath, and centrifuged at 4000 rpm. The mixture was diluted with distilled water in a 10 ml volumetric flask and purified using a 0.45 µm filter (Toiu et al. 2011). Agilent 1260 infinity HPLC Series (Agilent, USA), equipped with a Quaternary pump, a Zorbax Eclipse plus C18 column 150 mm × 406 min i.d., and 5 µm particles (Agilent technologies, USA), operated at 45 ºC were used. The mobile phase was HPLC grade water with 0.1% (v/v) orthophosphoric acid and acetonitrile (90:10), the flow rate was 1 ml min⁻¹, and the injection volume was 20 µL. Detection: florescence detector. Environmental conditions: temperature: 20 °C and humidity: 30%.

Statistical analysis

Experiments were submitted in complete randomized block design; 20 internodes were examined for each treatment in three replicates. The analysis of variance (ANOVA) was conducted and the means of the treatments were compared utilizing least significant difference test (L.S.D) at 5% level as termed by Snedecor and Cochran (1980).

Results and discussion

Chemical analysis of vinasse

Data in Table 1 reported the chemical analysis of vinasse, which reflected a significant added value of vinasse administration in tissue culture medium for commercial production strategies in Salix plant propagation. The data reported that vinasse is a side product rich in crude protein (20.75%), amino acids (16.43%), total carbohydrates (4.16%), total lipids (2.18%), and total phenols (4.12%). The chemical composition of sugarcane vinasse differ depends on the raw materials, which are affected by the preparation, fermentation system, and types of yeast according to Arnoux and Michelot (1988).

Table 1.

Chemical analysis of vinasse

Elements	Concentration (%)
Crude protein	20.75
Total amino acids	16.43
Total carbohydrates	4.16
Total lipids	2.18
Total phenols	4.12

Effect of vinasse on direct multiple shoot induction in willow

The percentage of direct shoot regeneration, shoot length (cm), fresh weight (g), and dry weight (g) were recorded after 5 weeks of incubation. Vinasse administration significantly promotes the growth parameters. Data in Table 2 explain the internodes cultured in media containing different concentrations of vinasse (0, 5, 10, 15, and 20%). The highest percentage of regeneration capacity (95%), the highest shoot length (4.00 cm), and fresh and dry weight (3.2 and 0.35 g, respectively) were recorded with 10% vinasse (Fig. 1). The data suggest that vinasse contains a large amount of nutrients supporting tissue culture techniques, but at a low dose of 10%. The reported data was in agreement with that of Gollo et al. (2016), who reported that a low concentration of vinasse can be added to plant tissue cultures, and the mineral nutrients it contains can help plants grow in vitro. Nonetheless, to improve vinasse efficiency, some nutrients need to be added.

Table 2.

Effect of supplementation of medium with different concentrations of vinasse on direct Salix safsaf Forssk shoot regeneration

Vinasse (%)	Regeneration (%)	Shoot length (cm)	Branches number	Fresh weight (g)	Dry weight (g)
0	45 ± 1.15d	1.90 ± 0.17b	0	1.60 ± 0.06c	0.16 ± 0.01b
5	80 ± 1.15b	3.25 ± 0.14b	0	2.00 ± 0.12b	0.18 ± 0.01b
10	95 ± 1.15a	4.00 ± 0.23a	2	3.20 ± 0.12a	0.35 ± 0.02a
15	75 ± 1.15c	3.00 ± 0.05b	0	2.30 ± 0.17b	0.21 ± 0.02b
20	0	0	0	0	0
LSD (5%)	3.25	0.462	0.245	0.345	0.056

Data are displayed as mean values ± standard deviation; LSD refers to the least significant difference test. In each column, the different letters mean significant difference at p ≤ 0.05

Fig.1.

Direct shoot regeneration from the stem of Salix safsaf cultured on MS medium (control) and 10% of vinasse

Also, with the administration of a high dose of vinasse (20%) to Salix safsaf Forssk, all growth parameters are suppressed. This constitutes a dose-dependent supplement to prevent the high toxicity of ions such as sulfate, salt, and chloride, as well as the very toxic effects of phenolic compounds (Gollo et al. 2016).

This toxicity could be due to the presence of a high phenolic content in vinasse (4.12%), which is produced by the catabolism of lignin originating from sugarcane. It could increase the browning of internodes. So, vinasse is a dose-dependent supplement.

Effect of different applications of SNP oin Salix safsaf Forssk culture

Direct multiple shoots induction

To study the plant responses (Salix safsaf Forssk) to NO, we designed an experiment to study the effect of different concentrations of SNP as a donor for NO on MS medium supplemented with BA (0.25 mg L⁻¹). We suggested using MS medium with BA as a control in agreement with Khan et al. (2011), who indicated that basal medium without any growth regulator was unsuccessful in producing shoots from cultured nodal explants. Generally, a cytokinin is important for in vitro shoot induction.

The data in Table 3 explains the effect of different concentrations of SNP (0, 5, 10, 15, 20 mg L⁻¹). The administration of SNP (15 mg L⁻¹) significantly promoted direct propagation in Salix safsaf Forssk. We recorded 100% with shoot regeneration, mean shoot length was 3.50 cm, and fresh and dry weight were 3.50 g and 0.35 g, respectively, after 5 weeks of culture, compared with the other treatments and control while the concentration of 20 mg L⁻¹ suppressed all growth parameters. NO has been involved in many physiological processes, such as germination, flowering, or leaf senescence, as a plant response to environmental stresses (Mur et al. 2013). We found that NO positively increased shoot regeneration, according to Paris et al. (2007). NO is involved in the regeneration of injured Solanum tuberosum leaves, and it may also play a role in enhancing shoot numbers and the elongation of shoots (Xu et al. 2009). SNP is used as an NO donor to promote the proliferation and regeneration in Malus hupehensis (Kazemi et al. 2019). The various roles of NO in plant biology make NO signaling a promising target for stimulating and promoting plant growth and development, enhancing mineral nutrient acquisition, delaying postharvest fruit senescence, and increasing resistance to biotic and abiotic stresses (Sun et al. 2021). These results are in agreement with Otvos et al. (2005), who reported that NO is a demand for auxin activation of cell division and embryogenic cell formation in alfalfa cell cultures by exogenous treatment with NO donors. The effects of NO on the cytokinin response pathway in plants have been reported, and the treatment of NO donors imitates cytokinin action (Scherer and Holk 2000). Moreover, cytokinins in a dose-dependent manner initiated the production of NO, which was rapidly created within 3 min in Arabidopsis, parsley, and tobacco cell cultures (Tun et al. 2001). These results implied that NO may mediate cytokinin signaling.

Table 3.

Effect of supplementation of MS medium with 0.25 mg L⁻¹ BA and different concentrations of SNP administration on direct Salix safsaf Forssk shoot regeneration

SNP (mg L−1)	Regeneration (%)	Mean shoot length (cm)	Branches number	Fresh weight (g)	Dry weight (g)
Control	45 ± 2.89d	1.90 ± 0.06c	0	1.60 ± 0.06d	0.16 ± 0.01d
5	77 ± 1.15d	2.00 ± 0/06b	2 ± 0.12	2.50 ± 0.17c	0.23 ± 0.02c
10	84 ± 0.58b	3.16 ± 0.03b	0	2.80 ± 0.17b	0.26 ± 0.01b
15	100 ± 0.33a	3.50 ± 0.12a	0	3.50 ± 0.12a	0.35 ± 0.01a
20	70 ± 1.15d	2.16 ± 0.0c	2 ± 0.12	1.60 ± 0.12d	0.16 ± 0.01d
LSD (5%)	4.767	0.205	0.005	0.422	0.038

Data are displayed as mean values ± standard deviation; LSD refers to the least significant difference test. In each column, the different letters mean significant difference at p ≤ 0.05

Enzymes activity, total phenols, and total flavonoids

The administration of different SNP concentrations significantly stimulated the activity of polyphenole oxidase, catalase, and glutathione peroxidase enzymes. Data in Fig. 2 show that the application with 15 mg L⁻¹ SNP significantly resulted in the highest increase in all enzymes activity (27.31 ΔAbs.min⁻¹.μg⁻¹ proteins, 2.84 U g⁻¹, and 77.8 U g⁻¹), respectively, compared with other treatments and control. Our results revealed that the administration of SNP stimulates the antioxidant activity against oxidative stress triggered during the signaling action of SNP.

Fig. 2.

Effect of different concentrations of SNP on polyphenol oxidase (a), glutathione peroxidase and catalase activities (c), PAL activity (b), total phenolic compounds, and total flavonoids and phenolic compounds (d) in Salix safsaf Forssk

Exogenous NO stimulates antioxidant activity, in harmony with Sehar et al. (2019), who found that SNP and glucose application increased glutathione (GSH) content and the activity of antioxidant enzymes. So, NO is important in the detoxification of H₂O₂ via modulation of antioxidant enzymes such as catalase and ascorbate peroxidase and the maintenance of cell redox couples (Tiwari et al. 2008). Overall, NO induced the biosynthetic pathway of GSH in plant cells and tolerance against oxidative stress (Xiong et al. 2010).

Similarly, some studies discussed ROS and NO signaling action using exogenous donors of NO that enhance the generation of H₂O₂ (Yamasaki et al. 2016). Also, Delledonne et al. (1998) found the participation of NO and ROS in apoptosis during the examination of acute and systemic plant responses to pathogen infection. In addition, SNP excites ROS production in sequence, which prompts the cells to immediately defend against ROS through direct and indirect antioxidant defenses.

There is a significant representation of PAL activity among NO-responsive co-regulations. Data in Fig. 2 show that, the application with 15 mg L⁻¹ SNP resulted in the highest PAL activity (3.17 µmol.mg⁻¹.h⁻¹) compared with other treatments and control. After that, total phenol (0.91 mg g⁻¹) and total flavonoid (0.55 mg g⁻¹) contents were detected to show significant results compared with other treatments and control. The administration of SNP stimulated PAL activity, which in sequence directly increased the accumulation of total penolic compounds and flavonoids. In this work, data revealed that NO stimulated the PAL activity that, in sequence, directly increased the accumulation of total penolic compounds and flavonoids. These data were in accordance with those of Morkunas et al. (2013), who reported a very high level of expression of the isoflavonoid biosynthesis pathway in oxysporum-inoculated embryo axes pre-treated with SNP. Similar results were also found in Russula griseocarnosa, where NO fumigation of freshly harvested fruiting bodies stimulated the activities of PAL and chalcone syntheses, leading to the accumulation of phenolics such as qucertin (Dong et al. 2012). PAL is a key regulator in the phenol synthesis pathway, and it is induced under a variety of biotic and abiotic stress conditions. This was in accordance with Mur et al. (2013), who found that NO was linked to flowering, germination, or leaf senescence as a response to several stresses.

Effect of the combination between SNP and vinasse in Salix safsaf Forssk culture

Direct multiple shoot induction

The internodes were cultured in media containing 0.25 mg/L BA, 15 mg/L SNP, and different concentrations of vinasse (0, 5, 10, and 15%). Data in Table 4 show that the highest regeneration percentage was recorded at 100%, with shoot length 3.80 cm, and fresh and dry weights 3.57 and 0.33 g, respectively, at the concentration of 10% vinasse and 15 mg L⁻¹ SNP (Fig. 3). This effect could be due to the cross talk effect of nitric oxide and vinasse in the plant, which significantly induced direct propagation more than vinasse without SNP, supporting the synergetic effect of the combination on the growth parameters of the plant. That could be due to the synergetic effect of SNP as a signaling agent and vinasse as a source of carbohydrates for metabolism necessary for propagation and growth under SNP induction molecules.

Table 4.

Effect of cross talk supplementation of MS medium, 15 mg L⁻¹SNP, and different concentrations of vinasse on direct shoot regeneration in Salix safsaf Forssk

Treatment	Regeneration (%)	Shoot length (cm)	Branches number	Fresh weight (g)	Dry weight (g)
15 mg L−1SNP	100 ± 0.3a	3.5 ± 0.1a	0	3.84 ± 0.1a	0.4 ± 0.06a
15 mg L−1SNP +  5% vinasse	80 ± 1.7b	3.55 ± - 0.02a	0	2.15 ± 0.02b	0.22 ± 0.01b
15 mg L−1SNP +  10% vinasse	100 ± 0.3a	3.8 ± 0.1a	0	3.57 ± 0.1a	0.33 ± 0.02a
15 mg L−1SNP +  15% vinasse	60 ± 3.5c	1.5 ± 0.1b	0	1 ± 0.05c	0.1 ± 0c
LSD (5%)	6.33	0.329	0.438	0.299	0.101

Data are displayed as mean values ± standard deviation; LSD refers to the least significant difference test. In each column, the different letters mean significant difference at p ≤ 0.05

Fig.3.

Direct shoot regeneration from the stem of Salix safsaf cultured on a medium supplemented with 15 mg L⁻¹ of SNP and 10% of vinasse

Enzyme activity, total phenols, and total flavonoids

The administration of 15 mg L⁻¹ SNP with different vinasse concentrations (0, 5, 10, and 15%) significantly reduced the enzyme activities (Fig. 4) when the application with 5% vinasse reported the lowest enzymes activities compared with other treatments and control where the application with 15% vinasse recorded the highest enzymes activities. The stimulation action of SNP for antioxidant enzymes was reduced by the vinasse combination when reacted, and quenching stimulated ROS via SNP, due to the ROS scavenging activities of vinasse phenolic compounds, which in sequence reduced the amounts of hydrogen peroxide, other ROS, catalase, and peroxidase enzymes activities, as reported by Langseth (1995). The study also proved the non-enzymatic scavenging activity of phenolic compounds against ROS scavenging activity. Enzymatic defense is a system of enzymes that decreases the concentration of ROS.

Fig. 4.

Effect of different combinations of SNP and vinasse on antioxidant defense enzyme activity (a, c), PAL activity (b), total phenolic compounds, and total flavonoid contents (d) in Salix safsaf Forssk. (T₁: SNP at 15 mg L⁻¹, T₂: 5% Vinasse + SNP at 15 mg L⁻¹, T₃: 10% Vinasse + SNP at 15 mg L⁻¹, and T₄: 15%Vinasse + SNP at 15 mg L⁻¹)

But at the high concentration of vinasse (15%), it synergistically reduces oxidative stress and increases the activity of antioxidant enzymes. Data, in agreement with many reports, showed the phenolic compounds in vinasse could be oxidized by ROS and transformed into oxidative molecules that increase oxidative stress and antioxidant enzymes. Phenolic antioxidants can act as prooxidants in the presence of oxygen molecules (Blokhina et al. 2003). Vinasse induced the formation of reactive oxygen species (ROS). Prooxidant activity can be shown in small polyphenols that are simply oxidized, such as quercetin and gallic acid. In contrast, hydroxycinnamic acids show prooxidant activity and cause DNA damage (Zheng et al. 2008).

Data in Fig. 4 showed that the administration of 15 mg L^–1 SNP with different vinasse concentrations (5, 10, and 15%) significantly affected the PAL activity, total phenols, and total flavonoids. The application with 15% vinasse resulted in the highest PAL activity and total phenols and total flavonoids contents compared with other treatments. The administration of vinasse combined with 15 mg L^–1 SNP significantly suppressed the PAL activity, either directly by inactivating the enzyme reaction or indirectly by reacting and suppressing the SNP signaling action. Also, the high content of vinasse phenolic compounds increases the content of phenolics and flavonoids. Vinasse is an antioxidant against H₂O₂ and other ROS that suppress the signaling action of SNP and generate ROS to activate PAL activity and phenolic compound accumulation, which is in accordance with Baiano (2014).

According to Kong et al. (2015), several compounds such as quercetin and gallic, cafeic, chlorogenic, ferulic, vanillic, and syringic acids have been isolated from different substances from sugarcane processing. Molina-Cortés et al. (2019) found that vinasse has better antioxidant properties than molasses.

Salicin content

The active ingredient salicylate derivatives produced in Salix shoots in vitro are shown in Table 5. Data revealed that the administration of SNP as a signaling molecule strongly accumulated salicin content at a concentration of 15 mg L⁻¹, which induced the synthesis and accumulation of salicin and recorded the highest accumulation against the combination of vinasse with SNP and control treatments. It could be due to the modulating effect of the activation of defense genes to produce different compounds as secondary products against stress through the accumulation of ROS and other signaling molecules in the network. It could be due to promoting salicylic acid accumulation from cinnamate produced by the activity of phenylalanine ammonia lyase (PAL) that had been activated via SNP administration. Administration of SNP stimulates the synthesis of secondary products, which increase the salicine accumulation, as confirmed by Morkunas et al. (2013), who found that stress caused by the action of SNP causes a very high post-infection accumulation of free isoflavone aglycones.

Table 5.

Effect of supplementation of MS medium with vinasse and SNP on salicin content in Salix shoots grown in vitro

Vinasse (%)	SNP (mg L−1)	Salicin (mg/100 g)
0	0	461.9
0	15	3162.16
10	15	483.13

Also, data showed a significant reduction in salicin accumulation when SNP was combined with 10% vinasse, which directly affected the signaling action of SNP to stimulate the production of secondary products as defense compounds. The reducing power of vinasse components (phenolic compounds) allow the direct reaction with ROS or NO and suppress its signaling action, by inhibiting the sequence cascade of the NO signaling transduction process, and decrease the accumulation of salicin contents in Salix shoots grown in vitro. Many reviews reported that vinasse has a high content of phenolic compounds that work as direct antioxidants against ROS formation via SNP and signal transduction of salicin accumulation (Gollo et al. 2016).

Conclusion

Cross talk effect of vinasse and SNP in Salix safsaf Forssk significantly induced direct propagation more than vinasse without SNP, supporting the synergistic effect of the combination of vinasse (10%) with SNP (15 mg L⁻¹) on growth parameters. Data showed a significant increase in salicin accumulation with this combination between vinasse and SNP. There is a direct effect of the signaling action of SNP as secondary products stimulators throw the reducing power of vinasse components that could react directly with SNP and suppress its signaling action or indirectly by inhibition of sequence cascade of SNP signaling transduction process that decrease the accumulation of salicin contents but the amount of salicin still higher than the untreated plants. Further, this optimized protocol is a simple, rapid, and economic in vitro system reported for salix safsaf in vitro regeneration and salicin overproduction. Thus, the optimized tissue culture technique proposed in the study using 15 mg L⁻¹ SNP provides a valuable recommendation for commercial utilization and will have direct application in the conservation of this valuable salicin-active pharmacophore that competitively inhibits cyclooxygenase.

Author contributions

RS Yousef and OK Ahmed designed the research and performed chemical analysis; ZK Taha contributed plant material, performed tissue culture experiment, and collected and analyzed the data. RS Yousef and OK Ahmed wrote the manuscript. All authors reviewed the manuscript before submission.

Funding

This research did not receive any external funding.

Data availability

All data generated and analyzed during this study are included in this published article.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Not applicable.

Consent to participate

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Consent to publish

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