Exploring the Multifaceted Healing Powers of Turnip Leaves from Saudi Arabia: Chemical Composition, Anti-Ulcer, Antibacterial, and Apoptosis Regulatory Properties

Introduction

Gastric and duodenal ulcers are prevalent global health issues, exerting substantial influence on the overall health of individuals and the functioning of healthcare systems. Gastro esophageal reflux disease (GERD) is a condition related to peptic ulcers that occurs when there is an imbalance between the stomach lining’s protective factors and harmful substances, especially acidic secretions. Various factors, such as viral infections, smoking, stress, certain drugs, and high alcohol consumption, can exacerbate this imbalance.^1-4 Bacterial infections are another health concern that is becoming more challenging due to the rise in bacterial strains that are resistant to a wide range of antibiotics. Emphasizing disease prevention is frequently regarded as more advantageous than depending on many therapies. The significance of diet in general health is paramount since it serves as the primary provider of both mental and physical energy. The functional characteristics of food play a crucial role in supplying needed nutrients and promoting and enhancing overall health.

The biennial herbaceous plant, called turnip, is formally classified as Brassica rapa L (BR). It is a member of the Cruciferae Brassica family. This adaptable plant often reaches maturity over two months and can be cultivated during various seasons, such as spring, late summer, and autumn. The flexibility of turnip to adjust to multiple growing seasons and its harvestable components can be used for both culinary and dietary purposes, making it a practical and readily available plant.⁵ Turnips offer substantial nutritional benefits, rich in carbohydrates, vitamins, and minerals, and have long been used in traditional medicine for treating various ailments.^5-9 Research has demonstrated that it possess several beneficial biological features, including anti-inflammatory and antioxidant characteristics and antibacterial and possibly anticancer benefits.^5,10  Numerous studies have also investigated the anti-diabetic and analgesic properties of turnips and their efficacy in reducing cholesterol levels and enhancing cardiovascular, renal, and hepatic well-being.¹¹ The potential of turnips in preventing osteoporosis and their efficacy in treating pulmonary edema and cerebral hypoxia highlight the wide-ranging and substantial health advantages linked to turnips.^5,12-14 The leaves , florets and various parts of medicinal plants play a crucial role in both traditional and modern medicine and offer a wealth of therapeutic benefits¹⁵. These leaves contain various bioactive compounds that are utilized for their healing properties.^15-17 Therefore, based on the traditional uses of turnip in disease treatment and on the pharmacological studies that revealed the high bioactivity of turnip, it has been decided to further investigate the therapeutic efficacy of turnip leaves from Saudi Arabia. Herein, we investigate the potential of turnip as an effective treatment for gastric ulcers and bacterial infection.

Materials and Methods

The ELISA kits employed in this study were procured from My BioSource and Abcam, USA whereas the remaining chemicals were obtained from Sigma Aldrich, USA

Plant collection, identification, and Cold maceration extraction

Samples of turnip leaves were obtained from a local market in Ha’il Saudi Arabia (27°29’18.8″N 41°41’09.6″E) and carefully rinsed with Millipore water to remove any impurities. Then, they were air-dried for 10days. The turnip plant was identified by Dr. Naila Alkefai (University of Hafr Al-Batin, Saudi Arabia) and a specimen of the stem and leaves were deposited at the Pharmacy College, University of Hail with the number UOHCOP014. The bioactive components of TLP were extracted by cold maceration with methanol by method followed by Syed, R. U et al 2022. ¹⁷

GC-MS analysis

TLP’s methanolic extract was analyzed using the Thermo Scientific GC–MS-AS 3000 autosampler with an IQS detector and equipped with a TR 5MS Capillary Standard non-polar column that has dimensions of 30 mm in length, 0.25 mm in internal diameter, and a 0.25 μm film coating. The identity of components was determined by utilizing the NIST and MAINLIB libraries, as well as by comparing their retention indices.¹⁷

In vivo experiments

Experimental animals

Male Wistar rats (150 g– 300 g) were used for the study. Each rat was housed individually in its specific pathogen free cage at temperatures between 22 and 28 °C and relative humidity between 50 and 60% at a controlled atmosphere with equal periods of light and darkness and  unrestricted access to clean water and food. The experiment protocol was approved by the Research Ethics Committee (REC) at the University of Ha’il, Saudi Arabia, with the reference number H-2024-102.

Gastric ulcer models

20 male Wistar rats took part in the study and were divided into 04 groups (G1to G4) of 05 rats each. The experimental strategy included creating ulcers using a slightly modified approach based on an already established procedure.^17-19 G 1: Normal control – These rats were not subjected to any intervention or therapy. G 2: Disease control (ulcer group) – ulcers were induced by administration orally of 95% ethanol at a dose of 5 mL/kg body weight. G3: Standard drug treatment – The rats in this group received oral administration of omeprazole (OMZ) (20 mg/kg body weight, diluted in distilled water (DW)) 2 hours before ethanol exposure. G 4: Treatment with the test drug. The rats in this group received 1 ml of turnip leaves powder (TLP) solution at a 400 mg/kg body weight, dissolved in DW, and administered orally 2 hours before exposure to ethanol. Before the rats were euthanized for the purpose of blood sampling, diethyl ether was administered as an anesthetic. Euthanasia was performed by surgically severing the jugular veins in the neck with a sterilized scalpel.

A dose of 400 mg/kg TLP was used based on previous studies that administered the same dose of the same plant species (Brassica rapa) to groups of mice and rats.^20,21

Determination of ulcer index and % inhibition of ulcer

The ulcer index (UI) was determined by a previously used method,²² through the following equation:

The ulcerated area was determined using a dissecting microscope, whereby 2 mm × 2 mm squares were counted throughout the ulcer’s length and breadth, and the sum of these squares represented the ulcerated region’s area. The following was used for calculating inhibition:

Macroscopic and biochemical gastric evaluations

The size of the ulcerated region was estimated according to a standard procedure. In addition, the calculation of percent inhibition was done by slightly modified method described in a previous study.^1,3,23 pH was measured and then neutralized with a sodium hydroxide solution.

Collection of Serum

The procedure began by withdrawing blood from the tail veins of the animals, without the use of anticoagulants. This blood was then collected. By tilting these tubes and then centrifuging. The supernatant was then centrifuged to obtain the serum, which was then stored in the refrigerator at temperatures between 2 and 8 °C. The next day, the serum was thoroughly analyzed using an enzymatic immunoassay (ELISA). This test specifically measured the levels of various pro-inflammatory cytokines.

Serum inflammatory cytokines

Cytokines were quantified using the sandwich ELISA technique.²³ Specific ELISA kits from MyBioSource and Abcam, USA, were used for this purpose. This method accurately measured the concentration of the individual cytokines in the serum. The ELx800 ELISA reader, also from the USA, was used to read the final measurements. The data collected was then plotted on a standard curve to accurately determine the concentrations of these pro-inflammatory cytokines in serum. The concentrations were then expressed in picograms per milliliter (pg/mL).

Serum apoptosis markers

The concentrations of apoptosis markers, in particular caspase-3 and caspase-9, were quantified in rat serum using a special ELISA kit from MyBioSource, USA. These markers were detected at an optical density of 450 nm.

Determination of antibacterial effect

Bacterial strains used

The study employed bacterial strains: Staphylococcus aureus ATCC 512477, Staphylococcus epidermidis ATCC 12228, Proteus mirabilis ATCC 299, Salmonella choleraesuis ATCC 10708, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922.

Standardization of bacterial culture

This was conducted following the protocol outlined by Syed et al.²⁴

Determination of antibacterial susceptibility

The test was conducted following the protocol outlined by Syed et al.²⁴

Statistical analysis

The statistical analysis was done via Prism 9 using one-way ANOVA, followed by Tukey’s test.

Results

Determination of bioactive constituents

Figure 1, the chromatogram depicting the analysis of TLP methanolic extract shows clear peaks, as evidenced by GC-MS analysis. GC-MS revealed the presence of over 100 compounds, mainly at trace levels, with 12 major compounds being particularly prominent (Table 1) (Figure 2). This study detected the fatty alcohols 1-hexadecanol and 2-hexadecanol with retenton times (RT) of 28.19 min and 38.54 min, respectively. Hexadecanoic acid methyl ester (methyl palmitate) was identified at an RT of 32.49 min, accounting for 6.90 % of the chromatogram. In addition, 7,10,13-hexadecatrienoic acid methyl ester, ageratriol and methyl tetradecanoate were also identified in TLP methanolic extract with characteristic RT values (see Table 1). Ageratriol, a sesquiterpene, was detected at a RT of 21.56 min. Methyl tetradecanoate, the methyl ester of tetradecanoic acid (myristic acid), was detected together with 2-myristynoylpantheline at an RT of 25.31 min, accounting for 2.04 % of the chromatogram. Furthermore, 9,12,15-octadecatrienoic acid methyl ester (methyl linoleate) or alpha-methyl linoleate, was identified within the extract at an RT of 40.43 min with a probability index of 12.44% and accounted for 10.23% of the chromatogram. Linalool, a naturally occurring terpene alcohol found in TLP extract, was determined at an RT of 12.90 min. In addition, 9-hexadecenoic acid (palmitoleic acid) was detected at an RT of 16.86 min, accounting for 1.27% of the chromatogram. Finally, ethyl iso-alcoholate and dasycarpidan-1-methanol acetate (esters) were identified in the extract, albeit to a lesser extent.

Figure 1: GC-MS chromatogram of CMETL ‎Click here to View Figure

Figure 2: Possible bioactive compounds and their chemical structures (1) 1-Hexadecanol, 2-methyl- (2) 2-Hexadecanol (3) Hexadecanoic acid, methyl esterClick here to View Figure

Table 1: Major chemical constituents of CMETL analyzed through GC-MS

Study on ulceration and treatment

Table 2 presents data on the effects of TLP on the healing of gastric ulcers in rats. In addition, Figure 3 shows the macroscopic view of rat stomachs, highlighting hemorrhagic lesions in the glandular region. The results show that the control group had an average size of 611 ± 32 mm². In contrast, rats treated with 400 mg/kg TLP showed a significant reduction in ulcer size to 124 ± 6.21 mm², a decrease of 79.7 ± 2.97%. Treatment with omeprazole resulted in an even smaller ulcer size of 90 ± 3.5 mm², corresponding to a healing rate of 85 ± 1.9 %.

Table 2: Effects of intra esophageal administration of Turnip leaves powder (TLP) and omeprazole on biochemical parameters of gastric juice recovered from rats.

TLP: Turnip leaves powder

Figure 3: Macroscopic inspection of hemorrhagic lesions in rat stomach. (I–IV) are representative photos from G 1, 2, 3, and 4, respectively. Click here to View Figure

Study on Biomarkers

In G 2, which represents the diseased animals, there was a significant increase in inflammatory cytokine levels compared to G 1 (the normal group). Conversely, inflammatory cytokine levels decreased significantly in G 3, treated with the standard OMZ, and in G 4, receiving the test drug TLP, with a significant decrease observed (Table 3).

Table 3: Cytokine network study on the effect of TLP treatment in ulcerated rats

^∗ Values are mean ± SD (n=5). ^$ Percentage decrease after treating with OMZ 20 mg/kg body weight; ^$$ Percentage decrease after treating with turnip leaves powder (TLP) at 400 mg/kg body weight.

Remarkably, after ulcer induction, TNF-α exhibited the greatest increase among the cytokines, with IL-1β and IL-6 also showing a notable increase. However, after administration of TLP for treatment, TNF-α showed the greatest decrease at 89.6%, closely followed by IL-2 and IL-1β, which decreased by approximately 89.4% and 87.2% with TLP treatment, respectively. The decrease in IL-6 and IFN-γ levels was much less pronounced than that of TNF-α and IL-1β, as shown in Table 3. Meanwhile, IL-4 and IL-10 were surprisingly increased upon induction of ulcer in the rats, however, TLP treatment resulted in a decrease in both cytokines by 54.3% and 74.7%, respectively (Table 3). It is crucial to note that omeprazole treated G 3 also decreased inflammatory cytokines, however, its effect was generally less than that resulting from TLP treatment. These results were summarized and illustrated in Table 3, Figure 4 and Figure 5.

Regarding apoptotic markers, caspases-3 and -9 in G 2 showed a significant increase after ulcer induction. However, both caspase-3 and caspase-9 levels decreased significantly after treatment with TLP (G 3) relative to disease G 2, as shown in Figure 6. It is noteworthy that the reduction in caspase-3 and caspase-9 levels was not significant in the TLP-treated animals (G 4) compared to the OMZ-treated group (G 3). The study highlights the immunomodulatory effect of TLP, which contributes to the treatment of ulcers.

Figure 4: Study on proinflammatory cytokines.Click here to View Figure

Figure 5: Study on anti-inflammatory cytokines. Click here to View Figure

Figure 6: Study on apoptosis markers. Click here to View Figure

Study on antibacterial effects

TLP exhibited its strongest antibacterial activity against Escherichia coli, while Staphylococcus aureus was the second most potent. It is noteworthy that the efficacy of the TLP extract was comparable for both Gram-positive and Gram-negative bacteria (Figure 7). As shown in Table 4, the efficacy of the methanolic TLP extract was lower than ciprofloxacin. Though TLP showed moderate activity in crude form, its performance can be increased by further purification of the compound.

Figure 7: Comparative antibacterial study of CMETL. All the results were not significant (ns) to each other at p > 0.05 level.Click here to View Figure

Table 4: Antibacterial activity of CMETL

CFU: Colony Forming Unit. # Values are mean ± SD (n=6).

Discussion

The comprehensive study presented sheds light on the multifaceted role of bioactive constituents in TLP in the context of gastric ulcer treatment, with a particular focus on their impact on inflammatory and apoptotic markers. The identification of over 50 compounds in TLP’s methanolic extract, with a focus on 12 major constituents, underscores the complexity and richness of natural extracts in offering a spectrum of bioactive molecules. Many bioactive compounds have been identified in the extracts which are displayed in Table 1. Research on turnips has revealed their potential in various health domains, including their anticancer, antimicrobial, anti-hypoxia, anti-diabetic, antioxidant, and nephroprotective properties. Consequently, turnips have become a focal point of significant interest in scientific investigations.⁹  9,12,15-Octadecatrienoic acid methyl ester, occurring naturally, has been detected in various plant species including Allamanda cathartica L. and Pithecellobium dulce.^25,26 It serves as a significant constituent in plants like Lepidium sativum, which display antibacterial properties.²⁷ Furthermore, it is a major component of several plants demonstrating antibacterial properties, such as Lepidium sativum and Plumeria alba.^27,28 Octadecadienoic acid (Z,Z)- (α-linoleic acid), may reduce the risk of heart disease.^3,23 n-Hexadecanoic acid (palmitic acid) has been identified as a significant bioactive compound. Recent research indicates that palmitic acid significantly inhibits prostate cancer.²⁹ It also showed anti-inflammatory properties and potent antibacterial effects against biofilm-forming bacteria.³⁰

The present study was meant to check the therapeutic efficacy of TLP in treating gastric ulcers by primarily investigating its effects on the cytokine network. The results showed that TLP has greater efficacy in modulating inflammatory cytokine levels than the usual administration of omeprazole. The importance of IL-1β, a vital cytokine, in regulating inflammation-related processes in the intestinal mucosa has been demonstrated in previous studies.^31,32 Interleukin-1β (IL-1β) maintains the integrity of the gastrointestinal mucosa. Its primary role is to inhibit the migration of neutrophils induced by chemotaxis. The present study observed a marked increase in IL-1β levels in G 2 ulcer-affected animals.

Interleukin-6 (IL-6) is another proinflammatory cytokine. According to Petrasek et al.,³³ the production of this cytokine occurs in response to tissue injury. It is a soluble mediator with multiple roles in inflammation and immunological response, particularly in peptic ulcers. TNF-α is a proinflammatory cytokine that is produced by macrophages and plays a central role in regulating inflammation and immunological responses, as well as in the modulation of immune cells and the body’s response to tissue damage or infection. It also combats in gastric ulcers, essentially gastric mucosa lesions. Promoting inflammation, stimulating cell apoptosis, and disrupting the protective mucosal barrier of the gastric mucosa can potentially exacerbate tissue damage. Studies have shown that gastric ulcers often have elevated TNF- α levels.^34,35 Neutrophils are the primary hosts of the IL-8 receptor (IL-8R), which significantly influences the inflammatory process. The receptor in question has a pronounced affinity for IL-8, a central component of the natural immune system that is synthesized by macrophages and an essential protein in the inflammatory response. In gastric ulcers, especially those due to Helicobacter pylori infection, the IL-8 gene shows the highest upregulation, highlighting its importance in the physiological response to these infections.^36-38 The effects of this phenomenon encompass all facets of the body’s immune system, emphasizing its importance in treating and responding to peptic ulcers.^39,40 IL-2 and IFN-γ are also pro-inflammatory cytokines that were investigated in this study. It can be clearly seen from the results that all these proinflammatory cytokines were upregulated in the ulcerated rats as expected, but treatment with TLP reduced their levels in a satisfactory extent.

The study also investigated the levels of IL-4 and IL-10 in ulcerated rats, and these are classified as anti-inflammatory cytokines.⁴¹ Surprisingly, these cytokines were actually significantly upregulated in ulcerated rats, however, significant upregulation of anti-inflammatory cytokines might result in a reverse effect and worsen inflammatory injury.⁴² Treatment with TLP also resulted in significantly decreasing IL-4 and IL-10 in the ulcerated rats, which further contributed towards the antiulcer effect of turnip. In general, the study underscored the immunoregulatory properties of TLP, attributed to its content of palmitic acid and alpha-linolenic acid, with the former associated with the promotion of pro-inflammatory responses and the latter with their suppression, as noted in previous studies.^43,44

The role of caspase-3 in the apoptotic pathway, essential for the breakdown of various cellular proteins and removing old, damaged, or dysfunctional cells, was also examined. Caspase-9, another critical enzyme in apoptosis, particularly regarding mitochondrial damage, was investigated. Research by Morandi et al.⁴⁵ demonstrated that an aqueous extract of olive leaves can mitigate apoptosis by lowering caspase-3 and -9 levels, an effect attributed to hydroxytyrosol and oleuropein. This aligns with findings from Yaguchi et al.⁴⁶, who reported that linoleic acid suppresses these caspases. These findings suggest a balance between pro-inflammatory and anti-inflammatory actions, which may contribute to the therapeutic potential of the extract in ulcer treatment. Additionally, TLP’s methanolic extract displayed a broad spectrum of antibacterial activities, supporting previous findings that turnip extract exhibits antibacterial properties.^46,47 The antibacterial properties of TLP could be due to hexadecanoic acid methyl ester, alpha-methyl linoleate, linalool and palmitoleic acid.^48-50

This comprehensive study on the effect of turnip leaves in treating gastric ulcers and bacterial infections illustrates the complexity of natural extracts and their potential therapeutic benefits. However, a limitation of the study is the inherent complexity of natural extracts, which contain various compounds that can interact in unpredictable ways, making it difficult to isolate the effects of individual constituents. Furthermore, while the study provides valuable insight into the anti-inflammatory activity of turnip and its effects on biomarkers associated with peptic ulcer, translating these findings to clinical practice requires additional research to fully understand the plant extract’s bioavailability, efficacy, and safety in human populations. The fact that the study relies on animal models and specific biomarkers also emphasizes the need for comprehensive clinical studies to validate these findings in humans and establish a clear therapeutic protocol for using turnip to treat peptic ulcers. It is also possible to continue this research in the future by conducting further isolation studies in order to develop a highly bioactive molecule instead of using the whole plant’s extract for treatment, and this is expected to increase efficacy and reduce side-effects.

Conclusion

This investigation firmly advocates for the therapeutic efficacy of TLP in addressing gastric ulcers, underscoring its significant potential. GC-MS analysis disclosed the presence of over 100 compounds, with 12 identified as necessary, demonstrating the natural extract’s rich and intricate nature in offering numerous bioactive molecules with profound health benefits. Particularly striking was the observed in reduction of ulcer sizes in rats treated with TLP, highlighting TLP’s substantial therapeutic promise, especially in its capacity to modulate cytokine networks crucial in the inflammatory processes linked to gastric ulcers. These findings align with existing research on these cytokines’ roles in inflammation and ulcer healing, reinforcing TLP’s therapeutic value. Moreover, the observed decrease in caspase-3 and -9 levels post-TLP treatment points to its potential to reduce apoptosis, which is vital for repairing and regenerating ulcerated tissues.

Additionally, TLP’s antibacterial properties against prevalent human bacterial pathogens add an extra dimension to its therapeutic profile, advocating for a comprehensive approach to ulcer treatment encompassing anti-inflammatory, immunomodulatory, and antibacterial actions. These outcomes not only bolster the accumulating evidence for the utility of botanicals in healthcare but also beckon further investigation into the precise mechanisms through which these bioactive components act and their broader medical applications. The study accentuates the promising role of TLP in developing new strategies for peptic ulcer therapy, offering insights that could lead to innovative, nature-based medical treatments.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethical Approval

Animal research commenced following approval from Research Ethical Committee at the University of Ha’il (Approval number: H-2024-102).

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