Introduction

Gastric ulcer is a harsh medical condition that targets the gastrointestinal tract. It affects roughly 5–10% of the global population. The primary etiology for gastric ulcer points out the imbalance between the gastro-protective endogenous factors and harmful exogenous factors in favour of the latter ones. The defensive factors involve blood flow, cell renewal, prostaglandins (PGs), endogenous antioxidants and cell proliferation. One of the vital pathways in managing cell renewal and proliferation is the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad3) signaling pathway (Bakar et al. 2022; El-Ashmawy et al. 2016a; Shaofang et al. 2018).

On the other hand, the harmful factors include drugs, especially non-steroidal anti-inflammatory drugs (NSAIDs), excessive alcohol consumption, stress, and infections with viruses or Helicobacter pylori (Tarnawski and Ahluwalia 2021). Ethanol is widely used to induce gastric injury/ulcers in rodents for the sake of screening the potential anti-ulcer effect of some natural compounds/metabolites. Ethanol can induce gastric injury either directly by affecting the gastric mucus layer, causing cell injury or indirectly by triggering internal inflammation and free radicals (Aziz et al. 2019; Shams and Eissa 2022).

Currently, H2 blockers, as well as proton pump inhibitors (PPIs; omeprazole) are widely prescribed therapeutic options for gastric ulcer treatment. Although it is a widespread therapy, omeprazole use comes with a high financial burden and undesirable adverse effects (Ren et al. 2021). Thus, it becomes necessary to seek natural substituents with no reported side effects and low prices (Bakar et al. 2022). Medical plants provide beneficial secondary metabolites that can be used in industrial or medicinal applications. Among these plants is the Lotus genus, which belongs to the family Leguminosae and is used as a prophylactic treatment for sexually transmitted disorders (El Mousallami et al. 2002). L. hebranicus is distributed in Egypt, Eritrea, Sinai, and Sudan according to Royal Botanic Gardens, Kew Plants of the World Online. The previous phytochemical investigations of the genus Lotus revealed the presence of flavonoids and their glycosides as the major secondary metabolites. Roots, specifically, are usually rich in isoflavone derivatives (Abdel-Kader et al. 2006; El Mousallami et al. 2002). Unfortunately, plant cultivation is often time-consuming, and metabolite extraction from plant roots destroys plant growth (Rekha and Thiruvengadam 2017). The in vitro true root culture can be used year-round regardless of planting season and can examine and manipulate the crucial components under controlled conditions (White 1934). In some legume plants, a unique in vitro root-growing system has been reported to have a long lifespan of more than 5 years from inception besides the advantage of affording rapid growth. Root cultures are a desirable method for providing raw materials for the production of useful secondary metabolites because they often grow quickly, are genetically stable, and frequently, but not always, simulate the biochemical profiles of plant roots (Flores and Curtis 1992; Akashi et al. 1998).Interestingly, true root cultures cannot be obtained with all plant species, arch are semi-dormant excised roots kept alive on the nutrient medium or hairy roots transformed with Agrobacterium rhizogenes (Akashi et al. 2003).

Thus, this current study is the first of a kind that aims to investigate the use of in vitro-grown true roots cultures of L. hebranicus to provide raw materials as an alternative technology for producing important secondary metabolites instead of consuming natural resources. In addition to evaluating the potential of whole plant and in vitro true roots cultures to provide a gastro-protective effect against an ethanol-induced gastric ulcer in mice.

Materials and methods

In vitro study

Collection of plant material and induction of root culture

Seeds of L. hebranicus Hochst. ex Brand were obtained from Al-Azhar Garden from Al-Azhar University at Cairo, Egypt. The identity of the plant was confirmed by prof. Monier Abd EL-Ghani Professor of plant taxonomy and flora, Faculty of Science, Cairo University. A voucher sample (44-208-586-69) was preserved at the flora of Cairo University.

For induction of true root culture, L. hebranicus seeds were surface sterilized to prevent contamination during growth by commercial Clorox® (Clorox Co., Oakland, CA; 5.25% sodium hypochlorite) in the concentration of 20% (v/v) for 20 min. Surface-sterilized seeds were cultured on a solid basal MS medium (Murashige and Skoog 1962) (Duchefa Biochemie, Haarlem, The Netherlands), free of plant growth regulators and incubated for growth at 26 ± 2 °C and a 16-h photoperiod. The germination results were taken after 4 weeks from culturing. The length of the root was 4–5 cm. For the establishment of root cultures, the In vitro germinated plantlets were carefully removed from cultured jars on filter paper using sterile tools (scalpels and forceps). Roots were excised with a 3–4 cm length and separately cultured on MS basal medium free of plant growth regulators for true root culture. All cultured roots were incubated in complete darkness for growth and subcultured 5 times every 4 weeks.

Growth kinetics of true root culture

After the fifth subculture, the characteristics of the root cultures were examined. Secondary root tips were transplanted to the MS basal medium free of plant growth regulators every 4 weeks for five consecutive subcultures to obtain enough root cultures for the subsequent experiments. For 40 days, the data on the dynamics of callus biomass growth were recorded every 5 days. To determine the fresh weight (FW), true root cultures were carefully removed from culture jars and put on filter paper to get rid of the leftover nutrient medium then it was weighed. And to determine the dry weight (DW), the fresh true root were dried at 45 °C until a consistent weight, the dried roots were powdered in a mortar and kept at 4 °C for later use. The growth index (I) was derived using the formula below (Abou El-Dis et al. 2021)

$${\text{Growth}}\,{\text{index}}\left( {\text{I}} \right) = \frac{{\left( {{\text{M}}_{{{\text{max}}}} - {\text{M}}_{0} } \right)}}{{{\text{M}}_{0} }}$$

M0 represents the initial mass of root cultures (mg); Mmax means the root culture mass at the end of the growing cycle (mg).

True root cultures obtained from the progressive deceleration phase, which lasted from the 20th day until the 40th day were used for chemical analysis, and whole plant powder was used as a control for comparison.

Extract preparation and phytochemical analysis

Liquid chromatography-mass spectrometry (LC-ESI-MS/MS) analysis of L. hebranicus extract was performed adopting previously reported methods (Attallah et al. 2022a; Elmongy et al. 2022). Negative electrospray ionization modes were utilized to detect the various phyto-constituents of the L. hebranicus extract. Targeted constituents were determined by comparing LC/MS data with previously published literature and reference databases (Attallah et al. 2021). PeakView™ software compared retention time and m/z values obtained by MS1 and MS2 (Tsugawa et al. 2015).

In vivo study

Chemicals and kits

Ethanol was purchased from IsoChem Co., Egypt. Omeprazole (Catalogue No. FO26561) was bought from Carbosynth LLC, USA. Malondialdehyde (MDA), glutathione (GSH) and Superoxide dismutase (SOD) colorimetric assay kits were obtained from Biodiagnostic (Cairo, Egypt). ELISA kits for PGE2 and IL-18 were obtained from Bioneovan Co. Ltd, Beijing, China. Life Technologies, USA was the source of TRIzol reagent (15,596,026). Qiagen, USA was the source from which QuantiTects Reverse transcription kit was purchased. Thermo Fisher Scientific, USA was the manufacturer of Maxima SYBR Green/qPCR Master Mix. ABclonal Technology, Woburn, USA was the source from which TNF-α and NF-ҡB antibodies were purchased for the immune staining process.

Animals

Swiss Albino mice (Forty-five male, 20–25 g) were purchased from the National Research Center (Giza, Egypt). Animals were housed for 14 days, with diet and clean water (freely accessed). The animals were handled pursuant to the Faculty of Pharmacy, Tanta University’s research ethics committee guidelines (TP/RE/2/23p-007).

Experimental design

Mice received absolute ethanol (1 mL/100 g, orally) as a single dose for ulcer induction (Azmatullah et al. 2022). At random, mice were separated, into 9 equal groups (six mice each) as illustrated in Table 1. Doses were chosen based on earlier studies on the Lotus genus as well as pilot investigation (Baali et al. 2020). Extracts were dissolved in Phosphate buffered saline as the vehicle.

Table 1 List of all tested animal groups
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The vehicle (PBS) was given (7 successive days) to both Group I and II, while group III was given 10 mg/kg omeprazole, groups IV and V were given 100 and 200 mg/kg Lotus plant, groups VI and VII were given 100 and 200 mg/kg Lotus root, while groups VIII and IX received (orally for 7 successive days) 200 mg/kg of plant and 200 mg/kg root, respectively. On the last day of experiment (day 7), after the last dosing, all mice were deprived from diet for 24 h. Also, accessibility to water was prohibited for 2 h before ulcer induction. Sixty minutes after vehicle (PBS, for group I, VIII, IX)/ethanol (for group II, III, IV, V, VI, VII) dosing, all animals were euthanized, and opened for extraction of the stomach (for the biochemical measurements, histological as well as immunohistochemical investigations).

Measurement of MDA, GSH and SOD

Tissue homogenate was prepared (250 mg in PBS, pH 7.5) and centrifuged at 4 °C (4000 rpm/15 min). According to the manufacturer’s protocol, the supernatant was used for the colorimetric measurements. With the usage of UV–VIS spectrophotometer (Unico, USA), the absorbance of each protein sample (MDA, SOD, GSH) was measured at 450 nm, 560 nm and 534 nm wavelengths, respectively. Following the attached formula, the three proteins’ concentrations were calculated.

$${\text{MDA level}}\left( {{\text{nmol}}/{\text{g tissues}}} \right) = \frac{{{\text{A sample}}}}{{{\text{A standard}}}} \times \frac{{10}}{{{\text{weight of tissues}}\left( {\text{g}} \right)}}$$

SOD activity= (U/g tissues) = % inhibition × 13.75 × (1/g tissues used).

where: \({\text{Percent inhibition}} = \frac{{\Delta {\text{A control }} - \Delta {\text{A sample}}}}{{\Delta {\text{A control}}}} \times 100\)

$$GSH\,concentration (mg/g\,tissues)=\frac{A\,sample \times 66.66}{weight\,of\,tissues\,used \left(g\right)}$$

qRT-PCR measurement of Smad3 and TGF-β

The relative gene expression of Smad3 and TGF-β was measured using qRT-PCR technique. The primer sequences are revealed in Table S1, indicating GADPH as the housekeeping gene. To conduct measurement, first, total RNA was extracted followed by reverse transcription into DNA. Then, the primers, in presence of Syber green master mix, were used for the cDNA amplicons amplification step. Using the previously mentioned Livak method (Schmittgen and Livak 2008), the gene expression’s fold change was calculated.

Determination of PGE2, IL-18, levels

The IL-18 and PGE2 levels were measured using ELISA kits. The supernatant of each tissue homogenate was added to wells pre-coated with specific antibodies, then, biotin and streptavidin-horseradish peroxidase-labeled antibodies, chromogens and stop solution were added to give the final measurable yellow color.

Histopathology

Tissues were put in freshly prepared formalin (10%), for fixation, then implanted in paraffin. For sampling, Sect. (5-µm thick) were taken with microtome (RM2135 Leica, Germany), mounted on the slides, and stained with Hematoxylin and Eosin (H&E) solution. Later, a pathologist evaluated the sections under a light microscope (in a blinded manner) (Selim et al. 2023; El-Ashmawy et al. 2016b).

Immunohistochemical studies

Sections were sliced, dewaxed, dehydrated, and then rehydrated with water. After that, sections were put in citrate buffer, boiled and incubated in H2O2, and rinsed 3 times in phosphate-buffered saline, followed by staining with antibodies for TNF-α and NFҡB (1:200, Ebioscience). The staining intensity was evaluated using Image J software (Attallah et al. 2022b).

The semiquantitative scoring for [TNFα, NF-ҡB] was applied, based on the percentage of TNF α cytoplasmic and NF-кB nuclear with or without cytoplasmic positivity as well as stained cells of gastric glands and the presence of inflammatory cells. The scoring was as follows: 0: no staining or less than 5%, 1 weak: 6–25%, 2 moderate: 26–50% and 3 strong staining: more than 50% (Samuhasaneeto et al. 2009).

Statistical analysis

Statistical Package for Social Science (SPSS) version 25 was used for the statistical analysis, where a one-way analysis of variance (ANOVA) was used to calculate the differences between groups. Data were plotted using Excel 2013, and p < 0.05 represents a significant difference.

Results and discussion

Establishment of root cultures

For in vitro germination of L. hebranicus seeds, the seeds are surface sterilized using a concentration of 1.05 NaOCl for 20 min. Sterilized seeds were cultured on a solid basal MS medium a 100% survival percentage was attained without contamination. In vitro germination percentage of L. hebranicus Hochst. ex Brand seeds were 100%, and there was intermittent germination, which took between 4 and 10 days. After 4 weeks, the roots were separated in order to achieve shoot independence fully, and they were subcultured 5 times every 4 weeks. The initial true root culture biomass was 0.5 g/jar. Growth index (I) for the biomass of true root cultures in the first, second, third, fourth, and fifth subcultures the biomass of true root cultures was obtained (4.54, 4.72, 4.92, 5.1, and 5.22 times from the initial biomass respectively. According to our findings, there are no significant differences in the increase in true root culture biomass between the first subculture and the fifth subculture (Fig. 1).

Fig. 1
figure 1

Effect of solid basal MS medium free of plant growth regulators on true root cultures fresh weight (g/jar) of Lotus herbanicus Hochst. ex Brand for five successive sub-cultures interval between every sub-culture was 4 weeks. Values followed by the same lower-case letter do not different significant at p ≤ 0.05.

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True root cultures are a desirable way to provide plant raw materials for the production of useful secondary metabolites because they are characterized by rapid growth, genetic stability, and they exhibit sustained growth for a long period of time completely independent of shoots, and they often mimic the biochemical content of the roots of the whole plant (Flores and Curtis 1992). In vitro-grown true root cultures of L. hebranicus, are a system that starts from the primary root to obtain large amounts from dried root cultures that can be used in chemical analyses, the cultivation is carried out on a culture medium without the need for external growth regulators this is in agreement with (Akashi et al. 2003). In addition, the fresh biomass of true root cultures was gradually increased by sub-culturing up to fifth subcultures without significant differences.

Growth kinetics of true root culture

The development curve of L. hebranicus Hochst. ex. Brand true root cultures, in the fifth subculture have a sigmoidal shape, with the lag, exponential, linear, and deceleration as seen in Fig. 2. From the start of the culture until the 5th day was the Lag phase. The growth activity of culture increased, indicating that the growth transition to the exponential phase persisted through the 10th day. The linear phase was noted between days 10th and 20th, after which the true root cultures growth slowed down gradually, and deceleration of growth occurred next. After the progressive deceleration phase, which lasted for 20 days to the 40th growth which lasted up to 40 days the stationary phase did not appear in the true root cultures growth curve of L. hebranicus Hochst. ex Brand until the 40th day. However, in the last periods, the true roots began to swell, turning part into callus and the other part into somatic embryogenesis (Fig. 3D). Further studies could be performed on the regeneration of L. hebranicus Hochst. ex Brand using callus and root cultures.

Fig. 2
figure 2

True root cultures growth curve of L. herbanicus Hochst. ex Brand for 40 days, cultivated in the dark, on solid basal MS medium free of plant growth regulators

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Fig. 3
figure 3

in vitro production of true root cultures. A source material, B true root cultures 10 days old, C true root cultures 20 days old, D true root cultures 40 days old

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Since most root cultures used in physiological and biotechnological experiments are either hairy root cultures transformed with A. rhizogenes or semi-dormant roots cut that remain alive in a nutrient medium, the production of in vitro true root cultures completely independent of shoots is considered an important alternative to the use of natural resources (Akashi et al. 2003). The growth curve is considered as one of the major characteristics of growth, which helps to estimate the processes of growth activity at various stages of cultivation and determine the optimal time for sub-culturing (Abbade et al. 2010; Benítez-García et al. 2014). In our study, the results showed that true root cultures must be transferred to a new medium before the deceleration phase (every 20 days). Previous studies indicated that the accumulation of secondary metabolites begins to increase during the stages of the deceleration phase as well as during cell differentiation, where the genetic makeup of somatic cells is totipotent (Heydari et al. 2020; Ramawat 2021).

LC-ESI-MS/MS analysis

A total of 22 compounds were tentatively identified in L. hebranicus plant extract using LC-ESI-MS/MS in negative mode and 21 compounds in root extract. The main substances are amino or fatty acids, carboxylic acids, alkaloids, and flavonoid derivatives agreed with previous study (Suzuki et al. 2008). The metabolite profile of the L. hebranicus plant and root extracts are presented in Tables 2 and 3. While Figures S1 and S2 showed the total ion chromatograph of the plant and root, respectively.

Table 2 List of tentatively identified metabolites in Lotus herbanicus plant extract analysed by LC-ESI-MS/MS
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Table 3 List of tentatively identified metabolites in Lotus hebranicus in vitro true root extract analysed by LC-ESI-MS/MS
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Gastro-protective effect of L. hebranicus

Impact of L. hebranicus on gastro-protective mediators

In Fig. 4A, the gastric content of PGE2 was substantially decreased (p < 0.05) in group II (61.6% decrease) compared with group (I) Omeprazole pre-treatment increased, significantly, (p < 0.05), the PGE2 content (2.19, 2.43-fold increase) relative to group (II) While pre-treatment with either L. hebranicus plant (100 mg/kg, 200 mg/g) or true root (100 mg/kg and 200 mg/kg) improved the PGE2 level, significantly, (1.4-, 1.84-, 1.82- and 2.07-fold increase), respectively, compared to group II.

Fig. 4
figure 4

Impact of different pre-treatments. A PGE2 Conc. B GSH Conc. C SOD activity. Data are revealed as mean ± standard deviation (SD). a: represents significant (p < 0.05) against group I, b means significant (p < 0.05) against group II. (n = 6)

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Prostaglandin E2 is one of the mediators that protect the gastric mucosa and manage the healing process; by providing survival mediators and a high blood supply (Yu et al. 2020). Thus, any inadequacy in the PGE2 level could result in gastric injury and even ulcer, as shown in the current study of mice receiving only ethanol (group II). However, groups pre-treated with L. hebranicus (IV, V, VI and VII) revealed a remarkable increase in the PGE2 values. Abood et al. 2014, reported that PGE2 leads to an inhibition of TNF-α levels, which was proven by our study and might be considered as an additional mechanism through which PGE2 could mediate its gastro-protective role. It is worth mentioning that, to our best knowledge, there is no previous work that studied the effect of lotus species on the PGE2 level in gastric ulceration.

In Fig. 4B, a significant decrease (p < 0.05) was noticed in GSH levels of group II (70.3% decrease) relative to group (I) On the other hand, different pre-treatments showed a significant increase (p < 0.05) in the GSH values (3.11, 1.4-, 1.76-, 1.79- and 2.3-fold increase, respectively) relative to group (II) Interestingly, gastric GSH levels in groups VIII and IX were not significant (p > 0.05) from those from group I.

Similar to the ethanol effect on gastric GSH, Herein, in group II, ethanol dose resulted in a significant decrease (p < 0.05) in the SOD activity (71.05% decrease) when compared to group I. On the other side, pre-treatments raised, markedly, the activity (1.48-, 1.48-, 1.85- and 2.4-fold, respectively) when compared with group II (Fig. 4C).

GSH and SOD are major mediators that protect cells from injury through halting free radicals harm and lipid peroxidation progression (Vickers 2017). As a key player mediating gastric ulcers induced by ethanol, oxidative stress consumes both SOD and GSH levels. As a result, both gastric SOD and GSH are suppressed (Ahmed Amar et al. 2019). As mentioned previously, in line with our findings, extract from L. hebranicus displayed a booster effect on SOD enzyme activity (Ali et al. 2021). To our best knowledge, there was no reported study was conducted on L. hebranicus and antioxidant mediators in gastric ulcers.

Impact of L. hebranicus on inflammatory mediators MDA and IL-18

In Fig. 5A, MDA values were substantially improved (p < 0.05) (sixfold) in group II, compared to group (I) While omeprazole pre-treatment inhibited the gastric MDA concentrations (p < 0.05) (79% decrease) relative to group (II) Furthermore, pretreating mice with L. hebranicus plant and root (100 and 200 mg/kg, for each) showed a remarkable decrease (37.6, 50.73, 49.3, and 70.7%), respectively, in comparison with the ethanol group.

Fig. 5
figure 5

Impact of different pre-treatments on A MDA Conc, B IL-18 Conc. Data are plotted as mean ± SD. a: represents significant (p < 0.05) against group I, b: significant (p < 0.05) against group II. (n = 6)

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In Fig. 5B, IL-18 concentration was increased (p < 0.05) in group II (2.81-fold increase) compared with group I. On the contrary, relative to group II, groups III, IV, V, VI and VII revealed remarkably decreased/partially corrected IL-18 levels (66.8, 27.8, 43.13, 43.5, and 52.9, respectively).

Malondialdehyde is a well-known mediator of cell injury, where it indicates the degree of lipid peroxidation, where it consumes the antioxidants including GSH and SOD within the tissue (Selim et al. 2023). In agreement with Selim 2023, the current study revealed that ethanol ingestion markedly inhibited (p < 0.05) both GSH and SOD and triggered MDA levels. On the other side, omeprazole or L. hebranicus plant/root pre-dosing lessened the oxidative stress accompanied MDA levels and improved (p < 0.05) both SOD and GSH.

Impact of L. hebranicus on gene expression of Smad3 and TGF-β

By comparing the obtained data from the ulcer group to those from the normal one, it was clear that the alcohol ingestion led to a significant decrease in the gene expression of both Smad3 and TGF-β (63.12 and 65.9% decrease), respectively. On the other hand, omeprazole as a pre-treatment substantially increased the aforementioned genes (2.55- and 2.12-fold increase, respectively). Likewise, pre-treating mice with Lotus plant and root different concentrations increased significantly (1.3, 161, 1.71, 2.108, respectively) Smad3 and TGF-β (1.2, 1.56, 1.5, 1.98, respectively) gene expression when compared with ulcer group (p < 0.05) (Fig. 6A and B).

Fig. 6
figure 6

Impact of different pre-treatments on the gene expression of A Smad3, B TFG-B. Data are plotted as mean ± SD. a: represents significant (p < 0.05) against group I, b: significant (p < 0.05) against group II. (n = 3)

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Smad3 and TGF-β are pivotal mediators that facilitate both cell division and growth, which is considered one of the most critical mechanisms involved in ulcer healing, for it affords the new cells that replace any damaged ones and complete the ulcer healing (Li et al. 2015; Milani and Calabrò 2001; Tanigawa et al. 2005). The Smad3 is strongly bonded to TGF-β, as the latter was found to activate the expression of Smad3 and mediate its action. Also, TGF-β modulates the activity of vascular endothelial growth factor as an essential factor for cell proliferation and regeneration (Walton et al. 2017). Interestingly, both PGE2 and TGF-β were positively correlated and overexpressed in the phase of wound healing (Su et al. 2010).

Supported by an earlier study (Abood et al. 2014), the present findings revealed that ethanol repressed TGF-β expression. However, Lotus extract powerfully counteracted the ethanol effect and improved the gene expression of both Smad3 and TGF-β, with the root extract showing the upper hand over the plant extract.

It is worthy to state that our study is the first to combine PGE2, TGF-β and Smad3 in ulcer protective potentiality of natural products.

Impact of L. hebranicus on histopathological examination

The stomach sections stained with H&E were illustrated in Fig. 7. As illustrated, In the present study, L. hebranicus plant and root and omeprazole showed a prophylactic impact in groups IV, V, VI, VII and III, as they exhibited moderate to mild injured or nearly normal gastric mucosal, respectively.

Fig. 7
figure 7

Stained stomach sections (H&E) (× 100). A Group I: showed healthy mucosa with mucin-secreting cells (red arrows), some parietal cells (blue arrows) and submucosa (black arrow). B Group II: showed ulcers (red arrow), acute inflammatory cells (black arrow) and mild dysplastic glands (blue arrows). C Group III: showed intact mucosa (blue arrows) with absence of dysplasia and acute inflammatory cells infiltrates (mild) (red arrows) and mild congested blood vessels (black arrow). D Group IV (100 mg/kg Lotus plant + ethanol) showed typical glands (black arrows), partially injured mucosa (blue arrow) in addition to infiltration of inflammatory cells (moderate) (red arrows) as well as congested vessels (green arrows). E Group V [200 mg/kg Lotus plant + ethanol) showed intact mucosa (blue arrow), with mild to moderate acute inflammatory cells infiltrate (black arrows) and congested vessels (red arrows). F Group VI (100 mg/kg Lotus root) showed intact mucosa (red arrow), with mild submucosal acute inflammatory cells infiltrate (blue arrow) and few congested vessels (black arrow). G Group VII (200 mg/kg Lotus root ethanol) showed intact mucosa (red arrows) with acute infiltrates of inflammatory cells (few) (blue arrow). H Group VIII (200 mg/kg Lotus plant) revealed submucosa (blue arrow), normal mucosa, and mucin secreting cells (red arrow), and musculosa (black arrow). I Group IX (200 mg/kg Lotus root) showed normal submucosa (blue arrow), normal thickness of the gastric wall and glands with mucin secreting cells (red arrow), and musculosa (black arrow). (Color figure online)

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Impact of L. hebranicus on immuno-histochemical examination

The TNF-α and NF-ҡB immune-staining of the gastric tissues of different groups is shown in Figs. 8 and 9. As illustrated, Group I showed positive TNF-α expression with score 0. While, Group II showed strong TNF-α expression with score 3. On the contrary, Group III showed weak expression (score 1), and groups IV and V showed moderate expression with score 2. (F) Groups VI and VII manifested weak TNF-α expression (score 1). Group VIII and IX revealed score of 0.

Fig. 8
figure 8

TNF-α immune-stained gastric sections [× 100]. A Group I showed positive TNFα expression in less than 5% of cells (score 0). B Group II showed strong TNFα expression in more than 50% of cells (score 3). C Group III showed weak TNFα expression (score 1). D Group IV (100 mg/kg Lotus plant + ethanol) showed moderate TNFα expression (score 2). E Group V [200 mg/kg Lotus plant + ethanol) showed moderate TNFα expression (score 2). F Group VI (100 mg/kg Lotus root) showed weak TNFα expression (score 1). G Group VII (200 mg/kg Lotus root + ethanol) showed weak TNFα expression (score 1). H Group VIII (200 mg/kg Lotus plant) showed positive TNFα expression in less than 5% of cells (score 0). I Group IX (200 mg/kg Lotus root + ethanol) showed positive TNFα expression in less than 5% of cells (score 0)

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Fig. 9
figure 9

NF-ҡB immune-stained sections. [× 100]. A Group I showed positive NF-ҡB expression in less than 5% of cells (score 0). B Group II showed strong NF-ҡB expression in more than 50% of cells (score 3). C Group III showed weak NF-ҡB expression (score 1). D Group IV (100 mg/kg Lotus plant + ethanol) showed moderate NF-ҡB expression (score 2). E Group V [200 mg/kg Lotus plant + ethanol) showed moderate NF-ҡB expression (score 2). F Group VI (100 mg/kg Lotus root) showed weak NF-ҡB expression (score 1). G Group VII (200 mg/kg Lotus root + ethanol) showed weak NF-ҡB expression (score 1). H Group VIII (200 mg/kg Lotus plant) showed positive NF-ҡB expression in less than 5% of cells (score 0). I Group IX (200 mg/kg Lotus root + ethanol) showed positive NF-ҡB expression in less than 5% of cells (score 0)

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Similarly, for NF-ҡB immune expression, Group I showed positive NF-ҡr expression with score 0. While, Group II showed strong NF-ҡB expression with score 3. On the contrary, Group III showed weak expression (score 1), Groups IV and V showed moderate expression with score 2. (F) Groups VI and VII manifested weak NF-ҡe expression (score 1). Group VIII and IX revealed score of 0.

As ethanol is considered one of the ultimate injurious agents attacking the gastric mucosa either directly or indirectly, it was the best candidate to study the gastric ulcer model and the potentialities of new anti-ulcerogenic agents (Wang et al. 2012). Herein, ethanol ingestion exhibited an aggressive tissue injury in the stomach wall, evident in the histopathological findings that showed inflammatory cell infiltration and deep tissue injury. These findings were in alignment with previous reports (Kan et al. 2017; Wang et al. 2012). These marked alterations were confirmed by the increase of IL-18 levels concomitant with the strong staining of both gastric TNF-α and NF-ҡB. By recruiting the inflammatory cells such as neutrophils which secrete interleukins like IL-18, TNF-α was speculated as an inflammatory mediator. Thus, our results confirmed that high TNF-α expression is associated with high IL-18 levels and was in agreement with Pittoni et al. 2002 findings. The gastro-protective effect of the extract (either plant or root, with different concentrations) which was clearly illustrated in the histopathological findings was strongly approved by the immune-staining; as both TNF-α and NF-ҡB were repressed alongside the enhancement of the gastric injury and congestions.

The current results, regarding omeprazole, were confirmed by earlier studies speculating that omeprazole negatively affected the activation process of NF-ҡB, and subsequently decreased the TNF-α values as well (El-Ashmawy et al. 2016a). Another study stated the potential anti-inflammatory activity of L. hebranicus related to the kaempferol fractions. Additionally, fermented L. hebranicus root showed an anti-ulcerogenic effect in rats via inhibiting NF-ҡB level (Yoo et al. 2020).

Conclusion and future perspective

In the current study, we proved that the true root culture is a cheap source that does not require the addition of external hormones or genetic transformation by A. rhizogenes. It gives large amounts of biomass of true root culture as raw materials in a short time, so it is an effective and important alternative to consuming natural resources. The extract of either L. hebranicus plant or true root was analyzed and proven to have an anti-ulcerogenic effect, mainly by managing the balance between oxidative stress and antioxidants along with halting the inflammatory mediators. Also, the extracts showed an empowering effect on the gastro-protective PGE2 as well as escalating the healing assistant mediators; Smad3 and TGF-β. Although the plant extract showed a fair protective role, still the root extract revealed a more significant effect.

Our future perspectives are scaling up a technology of true root cultures of L. hebranicus through bioreactors, application and improvement of the efficiency of true root cultures for phytoremediation research. In addition, we aim, in the near future to investigate, specifically and individually, each of the active compounds found in the extract to put hands on which one is responsible for the significant gastro-protective action.