Anti-Heliobacter pylori and Anti-Inflammatory Potential of Salvia officinalis Metabolites: In Vitro and In Silico Studies

Due to its rising antibiotic resistance and associated inflammations, Helicobacter pylori poses a challenge in modern medicine. Salvia officinalis, a member of the Lamiaceae family, is a promising medicinal herb. In this regard, a phytochemical screening followed by GC-MS and LC-MS was done to evaluate the chemical profile of the total ethanolic extract (TES) and the essential oil, respectively. The anti-H. pylori and the anti-inflammatory activities were evaluated by a micro-well dilution technique and COX-2 inhibition assay. Potential anti-H. pylori inhibitors were determined by an in silico study. The results revealed that the main metabolites were flavonoids, sterols, volatile oil, saponins, and carbohydrates. The LC-MS negative ionization mode demonstrated 12 compounds, while GC-MS showed 21 compounds. Carnosic acid (37.66%), epirosmanol (20.65%), carnosol1 (3.3%), and 12-O-methyl carnosol (6.15%) were predominated, while eucalyptol (50.04%) and camphor (17.75%) were dominant in LC-MS and GC-MS, respectively. TES exhibited the strongest anti-H. pylori activity (3.9 µg/mL) asymptotic to clarithromycin (0.43 µg/mL), followed by the oil (15.63 µg/mL). Carnosic acid has the best-fitting energy to inhibit H. pylori (−46.6769 Kcal/mol). TES showed the highest reduction in Cox-2 expression approaching celecoxib with IC50 = 1.7 ± 0.27 µg/mL, followed by the oil with IC50 = 5.3 ± 0.62 µg/mL. Our findings suggest that S. officinalis metabolites with anti-inflammatory capabilities could be useful in H. pylori management. Further in vivo studies are required to evaluate and assess its promising activity.


Introduction
Since the beginning of time, people have embraced remedies from plants, animals, or marine organisms to combat and prevent disease. Fossil evidence demonstrates that humans have been using plants as remedies for at least 60,000 years [1]. Herbal medicine, often known as phytotherapy, is the practice of treating patients with herbal treatments [2].
In the last decade, resistance to conventional antibiotics has made the treatment of bacteria a tremendous challenge to all healthcare providers. There has been a renaissance in the investigation of plants as a potential source for new and robust antibiotics due to rising global demand for novel natural or synthetic forms of treatment [3]. Helicobacter pylori (H. pylori) is a Gram-negative bacteria that were identified as a group I carcinogen by the International Agency for Research and Cancer [4]. The World Health Organization (WHO) estimated in 1994 that around half of the world's population was infected with

Essential Oil Extraction
According to the procedure outlined in the European Pharmacopoeia [30] and as described by [3,31], the dried S. officinalis aerial parts (250 g) were hydro-distilled for 5 h using a Clevenger apparatus (plant to water ratio: 1:3 w/v). The volume (mL) of essential oil for every 100 g of the investigated plant was used to compute the yield percentage, and the oily phases were maintained after being separated and dried over anhydrous sodium sulfate.

GC-MS Analysis for Essential Oil
Recording of the mass spectra was done using Shimadzu GCMS-QP2010 (Kyoto, Japan) attached to Rtx-5MS fused bonded column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) (Restek, Las Vegas, NV, USA) with a split-split-less injector. The starting column temperature was kept at 45 • C for 2 min (isothermal) then programmed to 300 • C at a rate of 5 • C/min and kept constant at 300 • C for 5 min (isothermal). The injector temperature was 250 • C. The flow rate of the helium carrier gas was 1.41 mL/min. The recording of the mass spectra was done by applying the following condition: (system current) filament emission current, 60 mA; ionization voltage, 70 eV; and ion source, 200 • C. Diluted samples (1% v/v) were injected with split mode (split ratio 1:15).

In Vitro Evaluation of Anti-H. pylori Activity
The antibacterial activity of the S. officinalis essential oil and the ethanolic extract was rapidly screened to confirm their previously recorded activity [32,33]. Colorimetric broth micro-dilution method using XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2Htetrazolium-5-carboxanilide] reduction assay was adopted to determine the MIC against the reference strain of H. pylori ATCC 43504 according to [3,34,35]. The MIC was specified as the extract concentration that produced a 100% decrease in optical density compared with control growth results. Clarithromycin was used as a standard antibacterial agent. The volatile oils were serially diluted in 5% (DMSO) Dimethyl sulfoxide solution containing 0.1% Tween 80 (v/v) (10 mg/mL), and then, 50 µL of each dilution was added to wells in a microtiter plate containing 100 µL TSB. Fifty microliters of adjusted microbial inoculum (10 6 cells/mL) were added to each well, and then the microtiter plates were incubated in the dark at 37 • C for 24 h. After incubation, 100 µL of freshly prepared XTT were added and incubated again for 1 h at 37 • C [36]. Colorimetric variation in the XTT assay was measured using a microtiter plate reader at 492 nm.

In Silico Evaluation of Anti-H. pylori Activity
Molecular docking analysis (in silico) was done for the identified compounds in both the essential oil and the ethanolic extract regarding the active center of H. pylori glucose 6-phosphate dehydrogenase (HpG6PD), enzyme (PDB ID: 7SEH) was performed applying Discovery Studio 4.5 (Accelrys, Inc., San Diego, CA, USA). This enzyme is necessary for the metabolism of bacteria. The Protein Data Bank (http://www.pdb.org) accessed 5 December 2022, was used to obtain the protein (enzyme). The co-crystallized ligand nicotinamideadenine-dinucleotide phosphate (NADP) was used to determine the docking binding site. The free binding energies were computed for the most stable docking locations of the co-crystalized ligand, as well as for all identified molecules.

Anti-Inflammatory Assay
To examine the anti-inflammatory response by inhibiting the cyclooxygenase-2 (COX-2) enzyme, samples with concentrations between 125.0 and 0.98 g/mL were analyzed.
The COX (EC 1.14.99.1) activity was assessed as the result of the oxidation reaction between N, N, N, N-tetramethyl-p-phenylenediamine (TMPD) and arachidonic acid. With a few minor modifications, this experiment was carried out following the previously reported procedure [37,38]. Using a microplate reader, the inhibitory action was assessed by observing the rise in absorbance at 611 nm (BIOTEK, Santa Clara, CA, USA). Inhibitory activity (%) = (1 As/Ac) x − 100, where (As) is the absorbance in the presence of the test drug and (Ac) is the absorbance of the control, was used to compute the inhibitory percentages. The concentration eliciting the most inhibition of the COX-2 isoenzyme was used to measure the effectiveness of the extracts and the reference drug (celecoxib).

Phytochemical Screening
To assess a plant's potential therapeutic value, as well as to identify the active ingredients responsible for the known biological activity displayed by plants, a phytochemical screening is crucial. Additionally, it offers the base for more accurate identification of compounds and more accurate research. The first step in this work was the screening of bioactive molecules. The screening revealed the presence of flavonoids, carbohydrates, tannins, saponins, sterols, and volatile oil as presented in Table 1.

LC/MS of the Ethanolic Extract
The LC/MS analysis illustrates the chemical profile of the total ethanolic extract ( Figures 1 and 2 and Table 2). The appropriate spectrum parameters, including molecular ions, mass spectra, and retention time, were used for identification. pounds and more accurate research. The first step in this work was the screening of bioactive molecules. The screening revealed the presence of flavonoids, carbohydrates, tannins, saponins, sterols, and volatile oil as presented in Table 1.

LC/MS of the Ethanolic Extract
The LC/MS analysis illustrates the chemical profile of the total ethanolic extract (Figures 1 and 2 and Table 2). The appropriate spectrum parameters, including molecular ions, mass spectra, and retention time, were used for identification.

Extraction and GC/MS of the S. officinal Essential Oil
Hydro distillation of S. officinalis aerial parts results in the production of (0.7%) essential oil yield, which exhibited a light-yellow color with distinct strong aromatic fragrances. The chemical profile of S. officinalis oil resulted in the identification of 21 compounds. Several compounds were previously reported but with different percentages that may depend on the season, the geographic origin, extraction methods, and environmental factors [49][50][51][52]. Eucalyptol and Camphor were the major identified constituents (50.04%, 17.75%), respectively. Additionally, monoterpenes and sesquiterpenes were the most abundant classes (Figures 3 and 4 and Table 3).

Extraction and GC/MS of the S. officinal Essential Oil
Hydro distillation of S. officinalis aerial parts results in the production of (0.7%) essential oil yield, which exhibited a light-yellow color with distinct strong aromatic fragrances. The chemical profile of S. officinalis oil resulted in the identification of 21 compounds. Several compounds were previously reported but with different percentages that may depend on the season, the geographic origin, extraction methods, and environmental factors [49][50][51][52]. Eucalyptol and Camphor were the major identified constituents (50.04%, 17.75%), respectively. Additionally, monoterpenes and sesquiterpenes were the most abundant classes (Figures 3 and 4 and Table 3).

Extraction and GC/MS of the S. officinal Essential Oil
Hydro distillation of S. officinalis aerial parts results in the production of (0.7%) essential oil yield, which exhibited a light-yellow color with distinct strong aromatic fragrances. The chemical profile of S. officinalis oil resulted in the identification of 21 compounds. Several compounds were previously reported but with different percentages that may depend on the season, the geographic origin, extraction methods, and environmental factors [49][50][51][52]. Eucalyptol and Camphor were the major identified constituents (50.04%, 17.75%), respectively. Additionally, monoterpenes and sesquiterpenes were the most abundant classes (Figures 3 and 4 and Table 3).        Figure 5) compared to those of the standard used medication clarithromycin (MIC 0.48 g/mL). The highest activity was revealed by the TES (MIC 3.9 g/mL). However, oil displayed only modest action (15.63 MIC g/mL). The differences in chemical composition between TES and the oil could be the cause of this variation. This was further confirmed using the in silico study (Table 5 and Figure 6).

In Silico Evaluation of Anti-H. pylori Activity
Screening for enzyme inhibitors involved in pathogen metabolism and biosynthesis is one of the strategies performed nowadays to combat bacteria [62,63]. H. pylori was found to have enzymes from the pentose phosphate pathway (PPP), Glucose-6-phosphate isomerase, glucose 6-phosphate dehydrogenase (HpG6PD), and 6-phosphogluconolactonase were investigated by [64]. PPP may serve as a means of supplying NADPH for reductive processes and biosynthesis. Therefore, we focused on finding and testing compounds to inhibit the G6PD of H. pylori as a tactic for the rational design of drugs against this bacterium. We investigated 30 compounds that revealed HpG6PD's inhibition. All the compounds tested by molecular docking had the potential to bind with the active site

In Silico Evaluation of Anti-H. pylori Activity
Screening for enzyme inhibitors involved in pathogen metabolism and biosynthesis is one of the strategies performed nowadays to combat bacteria [62,63]. H. pylori was found to have enzymes from the pentose phosphate pathway (PPP), Glucose-6-phosphate isomerase, glucose 6-phosphate dehydrogenase (HpG6PD), and 6-phosphogluconolactonase were investigated by [64]. PPP may serve as a means of supplying NADPH for reductive processes and biosynthesis. Therefore, we focused on finding and testing compounds to inhibit the G6PD of H. pylori as a tactic for the rational design of drugs against this bacterium. We investigated 30 compounds that revealed HpG6PD's inhibition. All the compounds tested by molecular docking had the potential to bind with the active site of the enzyme. This may be explained by the studied compounds' potential to interact with the essential amino acids forming the enzyme through H-bonding and alkyl interactions ( Figure 6). Notably, most of the phenolic compounds and flavonoids identified from LC/MS have exceeded the fitting energy of co-crystallized ligand (NADP) nicotinamide-adeninedinucleotide phosphate (−29.6914 kcal/mol) suggesting possessing good inhibiting activity against the HpG6PD. This explains the potential of TES in anti-H. pylori activity, which could be related to its main components. On the other hand, the essential oil's main compounds had the same or even weaker fitting energy score compared to the co-crystallized ligand (NADP), which explains the moderate in vitro anti-H. pylori activity of the oil (Table 5).

COX-2 Inhibition Assay (Anti-Inflammatory Assay)
Given that H. pylori are frequently linked to a wide range of inflammations, the potential anti-inflammatory activity of both tested samples was assessed. Inflammation caused by the persistent infection can, in most cases, remain undiagnosed. Gastric inflammation, however, can develop into gastritis, gastric mucosa-associated lymphoid tissue MALT) lymphoma, peptic ulcer, and gastric cancer in some of the H. pylori-infected population [65]. The inhibition percentage using COX-2 assay was expressed as mean standard deviation, as seen in (Table 6 and Figure 7). The results revealed that, compared to the standard medication, celecoxib (positive control) had a IC 50 = 0.43 ± 0.12. The two tested samples inhibited COX-2 expression with various levels ( Figure 5), and TES revealed a strong anti-inflammatory activity approaching celecoxib with IC 50 = 1.78 ± 0.27. However, the oil had a weaker activity with IC 50 = 5.3 ± 0.62, confirming that the variation in the active metabolites resulted in a difference in the anti-inflammatory activity. Table 6. COX-2 inhibition assay of TES and the essential oil compared to the standard drug celecoxib.

Sample
Conc

Discussion
H. pylori infection is linked with numerous distinct chains of diseases in Egypt and other developing countries; however, this is not commonly seen in industrialized socie-

Discussion
H. pylori infection is linked with numerous distinct chains of diseases in Egypt and other developing countries; however, this is not commonly seen in industrialized societies. The prevalence of H. pylori infection among teenagers in the United States pales in comparison to infection rates of young children at 5 years of age in some regions of the developing world. While H. pylori are associated with gastritis, which can eventually lead to gastric carcinoma, and peptic ulcers, the infection appears to be linked with chronic diarrhea, malnutrition, and growth failure [66,67], as well as a predisposition to other enteric infections such as typhoid fever and cholera [68]. Treatment of H. pylori is seen as an additional challenge due to economic constraints such as the test-and-treat strategy, which lays a significant economic burden on many of these countries, as well as the prevalence of antibiotic resistance and poor patient compliance [69]. Proton pump inhibitor (PPI)-clarithromycin-amoxicillin or metronidazole treatment for two weeks is suggested as the first-line treatment for H. pylori infection [59,70,71]. Doctors usually prescribe anti-H. pylori therapy with an eradication rate of 90% at least for the treatment. However, multiple extensive clinical studies have revealed that the usual triple therapy eradication rate has generally decreased to unacceptable levels (i.e., less than 80%). Success rates in several countries are regrettably declining to reach as low as 25% [70]. The causes for this decline in efficacy over time are unknown, however, it may be connected to the rising prevalence of clarithromycin-resistant strains of H. pylori [70]. Thus, it was deemed necessary to search for novel therapeutic regimens that can be used as an extra constituent of antibiotic therapy or may replace current antibiotic treatments [72].
Plants have been a major source of novel pharmaceuticals and traditional treatments. They possess active metabolites that can be used to treat a variety of infectious diseases with little or no toxicity. The number of studies on medicinal plants and their potential as H. pylori agents has grown significantly in recent years [73][74][75]. Awareness of the use of medicinal plant metabolites as prophylaxis and therapeutics over synthetic drugs is now growing worldwide [74].
The Salvia genus is well known to possess significant pharmacological activity in the context of ethnopharmacological knowledge, especially in the treatment of bacterial infections [76]. The antimicrobial activity of S. officinalis was studied decades ago and was attributed to the presence of many active secondary metabolites [77].
In the current work, we focused on the chemical profile of the TES and essential oil, along with assessing the anti-H. pylori and anti-inflammatory effects. The results revealed a great variation in the chemical profile of both tested samples. The terpenoids (carnosic acid, β-sitosterol, rosmadial, 12-O-methyl carnosol, and carnosol); phenolic acids (rosmarinic acid); polyphenols (rosmanol and epirosmanol); and Flavonoids (dihydroxy kaempferol, Hispidulin, and cirsimaritin) were the dominant constituents in the TES.
However, monoterpenoids (eucalyptol, camphor, α-pinene, camphene, limonene, βmyrcene, α-terpineol, α-thujone, camphor, E-pinocamphone, endo borneol, myrtenol, and bornyl acetate) and sesquiterpenoids (caryophyllene, caryophyllene oxide, α-humulene, and viridiflorol) were dominant in the essential oil (Table 3). Due to the difference in the chemical profile, the two tested samples exhibited variation in biological activity. The TES displayed more potent anti-H. pylori activity is asymptotic to that of the standard antibiotic clarithromycin, in addition to a high percentage of inhibition of the COX-2 enzyme, indicating greater potential as an anti-inflammatory agent. This potent activity may be related to the active metabolites present in the TES.
According to the literature, the active metabolite carnosic acid and its derivatives are reported to have broad antimicrobial activity against Gram-positive and Gram-negative bacteria. It has been suggested that they exert their activity because of the lipophilic properties of these compounds allowing them to penetrate the bacterial membrane and interact with the membrane phosphorylated groups via their hydrogen bond-donor group (s) [78,79]. Moreover, carnosic acid and its derivatives may act as a modulator of the efflux pump through the dispersion of the membrane potential [78][79][80]. Despite showing moder-ate effect when used alone, synergisms were reported when added to antibiotics such as Erythromycin and tetracycline, improving their effectiveness by up to 8-and 16-fold against S. Aureus and, E. faecium, and E. faecalis, respectively [78]. On the other hand, carnosic acid demonstrates anti-inflammatory activity via stimulating the peroxisome proliferatoractivated gamma receptor (PPARγ) that modulates the genetic expression of inflammatory mediators. Furthermore, they act by inhibiting the formation of pro-inflammatory leukotrienes and the secretion of human leukocyte elastase, in addition to attenuating the formation of reactive oxygen species (ROS) [81][82][83]. Rosmarinic acid is another metabolite in the TES. Based on previous studies, it is considered one of the most active recorded anti-H. pylori phytochemicals with antioxidant and anti-inflammatory potential. It was proven to increase the activity of the antibiotic when combined with them [84]. Flavonoids found in the TES (dihydroxy kaempferol, Hispidulin, and Cirsimaritin) may contribute to the activity of the extract. Flavonoids were reported to be more active against Gramnegative bacteria [85]. They exhibit their anti-inflammatory effect via multiple pathways, including modulation of inflammatory signaling, reduction of inflammatory molecule production, decreased attraction and activation of inflammatory cells, regulation of cellular function, and antioxidative activity [86]. Despite the limited solubility of β-sitosterol, it was documented to possess wide antibacterial activity [87] and to inhibit the generation of inflammatory cytokines and partial inhibition of NF-κB in macrophages [88]. All those active metabolites in the TES may contribute to the pharmacological activity of the extract observed in the current study.
The oil demonstrated moderate antimicrobial activity as it contains volatile monoterpenes with eucalyptol and camphor being the major compounds. Both compounds are confirmed to possess antibacterial activity by [89]. Nonetheless, the oil demonstrated weaker anti-H. pylori activity, most of the compounds are well-established antioxidant agents such as limonene, pinene, and borneol [90,91]. Investigations in vivo and in vitro have revealed that compounds with high antioxidant activity, not only scavenge free radicals but also exhibit antibacterial activity against H. pylori [92].
To determine the potential of the identified compounds against H. pylori, an in silico investigation using a molecular docking technique was performed. Rosmarinic acid, βsitosterol, and carnosic acid had the highest energy score exceeding the co-crystallized ligand (NADP). This may assist in explaining the exceptional anti-H. pylori efficacy of TES. The oil constituents have a similar inhibitory activity to 7SEH or even less may explain the modest action of the essential oil compared to TES. However, more in vivo investigation is needed to confirm the activity.

Conclusions
The common inadequacy of conventional antibiotics to control H. pylori infections has generated interest in alternative curative agents. S. officinalis aerial parts are widely consumed traditionally for their therapeutic importance. In our study, the plant proved to be a potentially potent drug that can be used to eradicate the H. pylori infection or to alleviate the inflammation associated with it. The in silico study substantiates the activity of the identified constituents.
More studies are required to evaluate the Salvia metabolites' effects on bacteria, as well as how they impact antimicrobial resistance when combined with conventional antibiotics. Furthermore, in vivo research is required to develop a more simple, natural, cost-effective, and advantageous antibacterial and anti-inflammatory pharmaceutical product.