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Article

GC-MS Profiling, Anti-Helicobacter pylori, and Anti-Inflammatory Activities of Three Apiaceous Fruits’ Essential Oils

by
Hatun A. Alomar
1,
Noha Fathallah
2,
Marwa M. Abdel-Aziz
3,
Taghreed A. Ibrahim
4,5 and
Wafaa M. Elkady
2,*
1
Pharmacology and Toxicology Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
2
Department of Pharmacognosy and Medicinal Plants, Faculty of Pharmacy, Future University in Egypt, Cairo 11835, Egypt
3
Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo 11651, Egypt
4
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
5
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
*
Author to whom correspondence should be addressed.
Plants 2022, 11(19), 2617; https://doi.org/10.3390/plants11192617
Submission received: 10 September 2022 / Revised: 29 September 2022 / Accepted: 30 September 2022 / Published: 5 October 2022
(This article belongs to the Special Issue Plant Derivatives and Their Pharmaceutical Potential)
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Abstract

:
Eradication of Helicobacter pylori is a challenge due to rising antibiotic resistance and GIT-related disorders. Cuminum cyminum, Pimpinella anisum, and Carum carvi are fruits belonging to the Apiaceae family. Their essential oils were extracted, analyzed using GC-MS, tested for anti-H. pylori activity by a micro-well dilution technique, identified for potential anti-H. pylori inhibitors by an in-silico study, and investigated for anti-inflammatory activity using a COX-2 inhibition assay. Results showed that the main components of C. cyminum, P. anisum, and C. carvi were cumaldehyde (41.26%), anethole (92.41%), and carvone (51.38%), respectively. Essential oil of C. cyminum exhibited the greatest anti-H. pylori activity (3.9 µg/mL) followed by P. anisum (15.63 µg/mL), while C. carvi showed the lowest activity (62.5 µg/mL). The in-silico study showed that cumaldehyde in C. cyminum has the best fitting energy to inhibit H. pylori. C. cyminum essential oil showed the maximum ability to reduce the production of Cox-2 expression approaching celecoxib with IC50 = 1.8 ± 0.41 µg/mL, followed by the C. carvi oil IC50 = 7.3 ± 0.35 µg/mL and then oil of P. anisum IC50 = 10.7±0.63 µg/mL. The investigated phytochemicals in this study can be used as potential adjunct therapies with conventional antibiotics against H. pylori.

1. Introduction

Natural products are believed to have a great role in a wide range of therapeutic conditions. Complementary and alternative medicines are reported to be useful for of many health complaints [1]. Since ancient times, the active substances from plants have been used as remedies for various diseases in the form of hand-made preparations [2]. Several plants belonging to the Apiaceae family were used as spices, flavoring agents, and folk therapies as they have various biological activities [3]. This is because many species in this family contain significant amounts of essential oil as well as beneficial secondary metabolites [4]. In this study, three members are selected (Cuminum cyminum L., Pimpinella anisum L., and Carum carvi L.) that are endogenous to Egypt and the Middle East [5,6,7]. All of them are traditionally used extensively for culinary and medicinal purposes [8,9]. They are recommended for gastrointestinal and neurological complaints and have valuable antibacterial activity. They are also applied in different pharmaceutical, perfume, and cosmetic productions [10]. The three fruits possess essential oils as their main active constituents with recorded antimicrobial activity against various types of microorganisms [9,11,12]. The isolated essential oil of (C. cyminum L.) is distinguished by the presence of cuminaldehyde as the major ingredient. It’s commonly used to treat digestive and respiratory problems [13]. P. anisum L. is a traditional medicine for bronchitis, cough, colic, nausea, and sleeplessness; moreover, the gastroprotective potential of anise aqueous extract was previously proved [14]. Moreover, (C. carvi L.) is also useful for cleaning teeth and treating eczema and pneumonia [15].
Helicobacter pylori infection affects more than 50% of the population worldwide. This microorganism has been strongly linked to duodenal and peptic ulcers and different GIT inflammatory disorders, which the WHO categorized as group I carcinogens in 1994 [16]. In Europe and the United States, the common triple therapy of amoxicillin, clarithromycin, and a proton pump inhibitor (PPI) for 7–14 days is the first-line treatment for H. pylori infections [17]. However, the effectiveness of traditional triple therapy has declined in the past few years, with eradication rates of fewer than 80% due to the increase in clarithromycin resistance. This highlights the need for new first-line therapies. As a result, the discovery of alternative antimicrobial compounds derived from natural sources is required for improved medicinal uses [18,19,20,21].
The current work aims to extract the essential oils of the three chosen fruits and investigate the chemical profile of each oil separately using GC-MS, in addition to evaluating their anti-H. pylori and anti-inflammatory activities. Finally, this study also assesses the main constituents identified using in-silico analysis to identify the potential anti-H. pylori inhibitors. To our knowledge, this is the first comparison of these three fruits that are indigenous to Egypt against H. pylori.

2. Results

2.1. Essential Oil Extraction

Hydrodistillation of C. cyminum, P. anisum, and C. carvi fruits vary in their essential oil yields. C. cyminum exhibited the highest percentage yield (2.30%), followed by C. carvi (1.12%), whereas the P. anisum yield was (0.9%). All extracted essential oils had a golden yellowish color with distinct aromatic fragrances.

2.2. GC-MS Analysis of the Essential Oils

The phytochemical profile of the isolated essential oils was distinctly different despite belonging to the same family, Apiaceae. This proposes a potentially wide range of the potency of the biological activities. GC-MS resulted in the identification of 18 different compounds, mainly belonging to the monoterpenes and sesquiterpenes classes. Cuminaldehyde (41.26%), anethole (92.41%), and Carvone (51.38%) were the major constituents in C. cyminum, P. anisum, and C. carvi, respectively (Table 1, Figure 1 and Figure 2).

2.3. In Vitro Evaluation of Anti–H. pylori Activity

Results demonstrated that all examined essential oils have a promising potential against H. pylori activity (Table 2). C. cyminum showed the greatest activity with Minimum inhibitory concentration (MIC 3.9 µg/mL) comparable to the reference drug; clarithromycin. P. anisum showed moderate activity (MIC 15.63 µg/mL), while C. carvi showed the lowest activity (MIC 62.5 µg/mL). The variation in the activity could be related to the difference in the essential oil compositions.

2.4. Anti-Inflammatory Assay

It was deemed necessary to investigate the potential of the essential oils as anti-inflammatory agents because H. pylori infection is frequently linked to many types of gastric inflammations, ranging from gastritis to systemic and vascular inflammation [26,27]. The results were determined by the percentage of inhibition and expressed as mean ± standard deviation, as summarized in Figure 3. Overall, the results indicated that in comparison to the usual medication, celecoxib (positive control) IC50 = 0.43 ± 0.12, the three analyzed oils inhibited COX-2 expression but to various levels. As seen in Figure 3, it was noted that the oil of C. cyminum showed the strongest activity, approaching celecoxib with IC50 = 1.8 ± 0.41, followed by the C. carvi oil IC50 = 7.3 ± 0.35, and the weakest anti-inflammatory activity was observed in the oil of P. anisum IC5 0= 10.7 ± 0.63.

2.5. In Silico Evaluation of anti-Helicobacter pylori Activity

The strategy of drug discovery and development is searching for herbal medicinal metabolites as ailment inhibitors. Inhibiting quorum sensing, which is essential for bacterial cell proliferation, is an important target for studies aiming to create remedies for bacterial pathogens. It may prevent the formation of drug resistance bacterial strains; this is because preventing the synthesis of virulence factors may allow the human immune system to clear the infection. The 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzyme (3NM4) is vital in bacterial metabolism, especially in diseases where quorum sensing is important [28].
The in-silico study revealed that cumaldehyde, estragole, and p-cymene have high fitting scores compared to the co-crystallized ligand (TRIS) (−28.75, −28.72, −28.49 kcal/mol), respectively (Table 3), proposing that they should possess good inhibiting activity against the MTAN enzyme (3NM4). This may be explained by the studied compounds’ capacities to interact with the essential amino acids through H-bonding (Figure 4). Moreover, carvone and anethole have very similar fitting energy as TRIS (−28.22, −28.01, and −28.24, respectively). This could be related to the anti-H. pylori activity of the tested essential oil. C. cyminum essential oil has the greatest in vitro anti-H. pylori activity, which could be related to its main component, cumaldehyde (41.26%). In the same way, anethole, the major constituent in Pimpinella anisum (92.41%), could be the cause for its effect.

3. Discussion

H. pylori, is a Gram-negative microaerophilic bacterium which affects more than half of the human population worldwide [29]. H. pylori infection is the leading cause of gastric and duodenal ulceration and the main risk factor for gastric adenocarcinoma and lymphoma. This infection is usually acquired during early childhood, with higher rates in developing countries [30,31,32]. In Egypt, about 50% of children acquire the infection by 10 years of age [30].
Gastric inflammation, a physiological process that can be triggered by a bacterial infection or tissue injury, is an invariable finding in patients infected with H. pylori and appears as an immune response from the host to the organism [33,34]. LPS (lipopolysaccharides) are chemical signals originating from the host and play important roles in the host’s defense against this infection. They activate macrophages to generate high amounts of proinflammatory enzymes such as cyclooxygenase, inducible NO synthase (iNOS), and (COX-2) [27]. H. pylori hits various cellular proteins to alter the host inflammatory response, thus initiating multiple attacks on the gastric mucosa, resulting in gastritis and peptic ulceration [35].
The current conventional H. pylori treatment typically employs a triple-drug regimen with two antibiotics and a proton pump inhibitor (PPI) with a success rate of 80–90% [36]. However, antibiotic resistance is an issue that is provoking an alarming 90% prevalence of H. pylori recorded in some African countries [36]. Other major obstacles leading to low eradication rates are the lack of therapeutic compliance, the degradation of antibiotics at gastric pH, and their insufficient residence time in the stomach [32]. Therefore, there is a continuing need for new antimicrobial drugs and creative alternative treatment methods.
Phytochemicals, which are good for human health and may even help in preventing diseases, are mainly found in plants. Herbal drugs and their essential oils are known to possess a wide range of pharmacological activities [20,37,38]. For thousands of years, essential oils had been used as antibacterial and anti-inflammatory agents [39]. Modern in vitro studies have demonstrated that a variety of bacterial strains are susceptible to the bactericidal effects of essential oils [40,41].
Several essential oils were proven to have anti-H. pylori potential [37,42,43,44], while others were extensively analyzed for their anti-inflammatory effects and the mechanism of their action was recorded in the literature [45,46,47,48,49,50].
This study aimed to emphasize the chemical profile with the investigation of the anti-H. pylori and anti-inflammatory activities of the three tested essential oils extracted from C. cyminum, P. anisum, and C. carvi. They are all indigenous plants of Egypt’s Nile Delta. The chemical profile of each individual oil was obtained by using GC-MS technique. The chromatograms revealed different constituents among the three oils with the presence of numerous oxygenated monoterpenes, such as (limonene and estragole), sesquiterpenes such as (Zingiberene and β-Himachalene), and the monoterpenoid hydrocarbon (β-Pinene). They are known to have powerful antioxidant and antimicrobial activities [51,52,53,54]. Cuminaldehyde, anethole, and carvone were found as the major compounds in the oils of cumin, anise, and caraway, respectively. These compounds may contribute to the anti-H. pylori activities of the oils as they are reported to possess bactericidal activity against different types of microorganisms [7,22,55,56,57].
The in vitro findings against H. pylori noted cumin (C. cyminum) oil as the most potent, followed by anise (P. anisum), then caraway (C. carvi) with MIC values of 3.9 µg/mL, 15.63 µg/mL, and 62.5 µg/mL, respectively. The MIC values were evaluated against the standard drug, clarithromycin (MIC = 1.95 µg/mL). Additionally, the three oils revealed a potential for anti-inflammatory activity. However, they differed in their capacity to inhibit COX-2 expression. C. cyminum essential oil showed the highest efficacy (IC50 = 1.8 ± 0.41) and most closely resembles the common anti-inflammatory COX-2 inhibitor, celecoxib (IC50 = 0.43 ± 0.12). C. carvi and P. anisum had moderate and weak activity with IC50 = 7.3 ± 0.35 and IC50 = 10.7 ± 0.63, respectively. The main components of the isolated oils may play a main role in the activity variation. The main component of cumin oil, cuminaldehyde, was formerly considered to be a prominent anti-inflammatory agent, approaching NSAIDS as it develops a dual anti-inflammatory COX–LOX inhibition mechanism [58,59]. Limonene, found in anise oil, acts by regulating the iNOS, COX-2, PGE2, TGF-β, and ERK1/2 signaling pathways [60], and carvone in caraway oil works on LPS-initiated oxidative stress markers and pro-inflammatory cytokines [61].
These findings are very important, as scientific reports suggest that H. pylori infection represents an additional risk factor for peptic ulcer complications in aspirin/NSAID users, limiting their use for its eradication treatment protocol [62,63]. An in-silico analysis employing a molecular docking approach was carried out to clarify how these compounds affect H. pylori. A higher energy score compared to the co-crystallized ligand (TRIS) was recorded for cuminaldehdye, as shown in Table 3 and Figure 4. This may help to explain the cumin oil’s remarkable anti-H. pylori activity. The mild action of the caraway and anise essential oils in comparison to cumin may be explained by the fact that (-)-carvone and anethole demonstrated similar inhibitory activity to TRIS. However, further in vivo studies are required to confirm the activity.

4. Materials and Methods

4.1. Plant Material

The three fruits were purchased from Egypt’s local market in January 2022. They were verified throughout the herbariums of Cairo University’s Faculty of Science and National Research Center (NRC), Cairo, Egypt. Voucher specimens (CM-1, AN-1, and CR-1) were kept in the Herbarium of the Pharmacognosy department, Faculty of Pharmacy, Future University in Egypt.

4.2. Extraction of Essential Oils

Dried fruits of C. cyminum, P. anisum, and C. carvi were hydro-distilled for 5 h according to the method described in the European Pharmacopoeia [64] using a Clevenger apparatus (plant: water ratio 1:3, w/v). The % yield was calculated using the volume (mL) of essential oil for each 100 g of the studied plant. The oily phases were separated, dried over anhydrous sodium sulfate, and kept for further study.

4.3. GC-MS Analysis

The analysis was carried out using the approach proposed by Elkady et al. [65]. GC–MS analysis was performed on Shimadzu GCMS-QP 2010 (Tokyo, Japan) with Rtx-5MS capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) (Restek, USA). The capillary column was coupled to a quadrupole mass spectrometer (SSQ 7000; Thermo-Finnigan, Bremen, Germany). The oven temperature was kept at 45 °C for 2 min (isothermal), programmed to 300 °C at 5 °C/min, and kept constant at 300 °C for 5 min (isothermal); the injector temperature was 250 °C. Helium was utilized as a carrier gas with a constant flow rate set at 1.41 mL/min. Diluted samples (1% v/v) were injected with a split ratio of 15:1 and the injected volume was 1 μL. The MS parameters were as follows: interface temperature, 280 °C; ion source temperature, 200 °C; EI mode, 70 eV; and scan range, 35–500 amu. Components were identified using retention indices and mass spectra matching with the NIST-11 and Wiley library databases, as well as published data in the literature. The retention indices (RI) were computed in comparison to a homologous sequence of n-alkanes (C8-C28) administered under identical circumstances. The overall run duration was about 30 min.

4.4. In Vitro Evaluation of anti–H. pylori Activity

Rapid screening of the antibacterial activity of the fruit essential oils was done to confirm their previously reported activity by [66,67,68]. Colorimetric broth micro-dilution method using XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium- 5-carboxanilide] -reduction assay was adopted to determine the MIC against the reference strain of H. pylori ATCC 43504 according to [69,70]. 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 DMSO, and then 50 μL of each dilution were added to wells in a microtiter plate containing 100 μL TSB. 50 μL of adjusted microbial inoculum (106 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. Colorimetric variation in the XTT assay was measured using a microtiter plate reader at 492 nm.

4.5. Anti-Inflammatory Assay

The samples in the concentration range of 125–0.98 µg/mL were tested to investigate the anti-inflammatory response by inhibiting the COX-2 enzyme. The COX (EC 1.14.99.1) activity was monitored as the result of the N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) oxidation reaction with arachidonic acid. This assay was performed according to the previously described method [71,72] with slight modifications. The inhibitory activity was determined by monitoring the absorbance’s increase at 611 nm using a microplate reader (BIOTEK; USA). The inhibitory percentages were calculated according to the formula: Inhibitory activity (%) = (1 − As/Ac) ×100, whereas is the absorbance in the presence of the test substance and Ac is the absorbance of control. The efficacy of the extracts and the reference compound (celecoxib) to inhibit COX-2 isoenzyme was determined as the concentration causing 50% enzyme inhibition (IC50).

4.6. In Silico Evaluation of anti-H. pylori Activity

The in-silico molecular docking analysis for the discovered compounds in the extracted essential oils in the active center of H. pylori MTAN (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase) enzyme (PDB ID: 3NM4) was performed using Discovery Studio 4.5 (Accelrys, Inc., San Diego, CA, USA). This enzyme is essential for bacterial metabolism. The Protein Data Bank (http://www.pdb.org) accessed 5 May 2022, was used to obtain the protein (enzyme). The co-crystallized ligand tris[hydroxymethyl]aminomethane (TRIS) 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 [73].

5. Conclusions

Cumin, anise, and caraway essential oils are considered rich sources of bioactive compounds. The fruits are widely available and highly consumed due to their nutritional and medicinal value. The current work demonstrates the great anti-H. pylori activity of the cumin essential oil due to its major constituents. However, anise and caraway essential oils showed moderate activity. This could offer a natural solution in the pharmaceutical field to combat this bacterial infection. The in-silico study confirmed the activity of the identified constituents. Further in vitro and in vivo studies are required to investigate the synergistic or additive interactions of the oils with the antibiotics to obtain the best results.

Author Contributions

Conceptualization, W.M.E.; funding acquisition, H.A.A.; investigation, W.M.E.; methodology, N.F., M.M.A.-A. and W.M.E.; supervision, T.A.I.; writing—original draft, W.M.E.; writing—review and editing, H.A.A., N.F. and W.M.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no funding.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Süntar, I.; Akkol, E.K.; Tosun, A.; Keleş, H. Comparative pharmacological and phytochemical investigation on the wound-healing effects of the frequently used essential oils. J. Essent. Oil Res. 2014, 26, 41–49. [Google Scholar] [CrossRef]
  2. Bungãu, S.G.; Popa, V.-C. Between religion and science some aspects concerning illness and healing in antiquity. Transylv. Rev. 2015, 24, 3–18. [Google Scholar]
  3. Sun, W.; Shahrajabian, M.H.; Cheng, Q. Anise (Pimpinella anisum L.), a dominant spice and traditional medicinal herb for both food and medicinal purposes. Cogent Biol. 2019, 5, 1673688. [Google Scholar] [CrossRef]
  4. Daga, P.; Vaishnav, S.R.; Dalmia, A.; Tumaney, A.W. Extraction, fatty acid profile, phytochemical composition and antioxidant activities of fixed oils from spices belonging to Apiaceae and Lamiaceae family. J. Food Sci. Technol. 2022, 59, 518–531. [Google Scholar] [CrossRef]
  5. Akbar, S. Pimpinella anisum L. (Apiaceae/Umbelliferae). In Handbook of 200 Medicinal Plants; Springer: Berlin/Heidelberg, Germany, 2020; pp. 1419–1429. [Google Scholar]
  6. Laribi, B.; Kouki, K.; Bettaieb, T.; Mougou, A.; Marzouk, B. Essential oils and fatty acids composition of Tunisian, German and Egyptian caraway (Carum carvi L.) seed ecotypes: A comparative study. Ind. Crops Prod. 2013, 41, 312–318. [Google Scholar] [CrossRef]
  7. Wanner, J.; Bail, S.; Jirovetz, L.; Buchbauer, G.; Schmidt, E.; Gochev, V.; Girova, T.; Atanasova, T.; Stoyanova, A. Chemical composition and antimicrobial activity of cumin oil (Cuminum cyminum, Apiaceae). Nat. Prod. Commun. 2010, 5, 1355–1359. [Google Scholar] [CrossRef] [Green Version]
  8. Mnif, S.; Aifa, S. Cumin (Cuminum cyminum L.) from traditional uses to potential biomedical applications. Chem. Biodivers. 2015, 12, 733–742. [Google Scholar] [CrossRef]
  9. Akram, M.; Azhar, M.; Anjum, N.; Quddusi, N. Phytopharmacology of unani drug Zeerah Siyah (Carum Carvi Linn)—A review. J. Pharmacogn. Phytochem. 2019, 8, 2772–2782. [Google Scholar]
  10. Sayed-Ahmad, B.; Talou, T.; Saad, Z.; Hijazi, A.; Merah, O. The Apiaceae: Ethnomedicinal family as source for industrial uses. Ind. Crops Prod. 2017, 109, 661–671. [Google Scholar] [CrossRef] [Green Version]
  11. Kosalec, I.; Pepeljnjak, S.; Kuštrak, D. Antifungal activity of fluid extract and essential oil from anise fruits (Pimpinella anisum L., Apiaceae). Acta Pharm. 2005, 55, 377–385. [Google Scholar]
  12. Khanjari, A.; Bahonar, A.; Noori, N.; Siahkalmahaleh, M.R.; Rezaeigolestani, M.; Asgarian, Z.; Khanjari, J. In vitro antibacterial activity of Pimpinella anisum essential oil and its influence on microbial, chemical, and sensorial properties of minced beef during refrigerated storage. J. Food Saf. 2019, 39, e12626. [Google Scholar] [CrossRef]
  13. Sowbhagya, H. Chemistry, technology, and nutraceutical functions of cumin (Cuminum cyminum L.): An overview. Crit. Rev. Food Sci. Nutr. 2013, 53, 1–10. [Google Scholar] [CrossRef] [PubMed]
  14. Al Mofleh, I.A.; Alhaider, A.A.; Mossa, J.S.; Al-Soohaibani, M.O.; Rafatullah, S. Aqueous suspension of anise “Pimpinella anisum” protects rats against chemically induced gastric ulcers. World J. Gastroenterol. 2007, 13, 1112. [Google Scholar] [CrossRef]
  15. Ghanem, M.T.; Radwan, H.M.; Mahdy, E.-S.M.; Elkholy, Y.M.; Hassanein, H.D.; Shahat, A.A. Phenolic compounds from Foeniculum vulgare (Subsp. Piperitum) (Apiaceae) herb and evaluation of hepatoprotective antioxidant activity. Pharmacogn. Res. 2012, 4, 104. [Google Scholar] [CrossRef] [Green Version]
  16. Wang, Y.-C. Medicinal plant activity on Helicobacter pylori related diseases. World J. Gastroenterol. 2014, 20, 10368. [Google Scholar] [CrossRef]
  17. Dos Santos, A.A.; Carvalho, A.A. Pharmacological therapy used in the elimination of Helicobacter pylori infection: A review. World J. Gastroenterol. 2015, 21, 139. [Google Scholar] [CrossRef] [PubMed]
  18. Bille, B.E.; Nguepi, E. In vitro and in vivo anti-Helicobacter activities of Eryngium foetidum (Apiaceae), Bidens pilosa (Asteraceae), and Galinsoga ciliata (Asteraceae) against Helicobacter pylori. BioMed Res. Int. 2016, 2016, 2171032. [Google Scholar] [CrossRef] [Green Version]
  19. Ozen, F.; Ekinci, F.Y.; Korachi, M. The inhibition of Helicobacter pylori infected cells by Origanum minutiflorum. Ind. Crops Prod. 2014, 58, 329–334. [Google Scholar] [CrossRef]
  20. El-Sherbiny, G.M.; Elbestawy, M.K. A review–plant essential oils active against Helicobacter pylori. J. Essent. Oil Res. 2022, 34, 203–215. [Google Scholar] [CrossRef]
  21. Sayed, A.M.E.; Omar, F.A.; Emam, M.M.A.-A.; Farag, M.A. UPLC-MS/MS and GC-MS based metabolites profiling of Moringa oleifera seed with its anti-Helicobacter pylori and anti-inflammatory activities. Nat. Prod. Res. 2022, 1–6. [Google Scholar] [CrossRef]
  22. Pajohi, M.; Tajik, H.; Farshid, A.; Hadian, M. Synergistic antibacterial activity of the essential oil of Cuminum cyminum L. seed and nisin in a food model. J. Appl. Microbiol. 2011, 110, 943–951. [Google Scholar] [CrossRef]
  23. Iacobellis, N.S.; Lo Cantore, P.; Capasso, F.; Senatore, F. Antibacterial activity of Cuminum cyminum L. and Carum carvi L. essential oils. J. Agric. Food Chem. 2005, 53, 57–61. [Google Scholar] [CrossRef]
  24. Vieira, J.N.; Gonçalves, C.; Villarreal, J.; Gonçalves, V.; Lund, R.; Freitag, R.; Silva, A.; Nascente, P. Chemical composition of essential oils from the apiaceae family, cytotoxicity, and their antifungal activity in vitro against candida species from oral cavity. Braz. J. Biol. 2018, 79, 432–437. [Google Scholar] [CrossRef]
  25. Hassan, O.M.; Elhassan, I.A. Characterization of essential oils from fruits of Umbelliferous crop cultivated in Sudan I. Pimpinella anisum L. (Anise) and Anethum graveolens L. (Dill). J. Pharmacogn. Phytochem. 2017, 6, 109–112. [Google Scholar]
  26. Oshima, T.; Ozono, R.; Yano, Y.; Oishi, Y.; Teragawa, H.; Higashi, Y.; Yoshizumi, M.; Kambe, M. Association of Helicobacter pylori infection with systemic inflammation and endothelial dysfunction in healthy male subjects. J. Am. Coll. Cardiol. 2005, 45, 1219–1222. [Google Scholar] [CrossRef] [Green Version]
  27. Ruggiero, P. Helicobacter pylori and inflammation. Curr. Pharm. Des. 2010, 16, 4225–4236. [Google Scholar] [CrossRef]
  28. Ronning, D.R.; Iacopelli, N.M.; Mishra, V. Enzyme–ligand interactions that drive active site rearrangements in the Helicobacter pylori 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase. Protein Sci. 2010, 19, 2498–2510. [Google Scholar] [CrossRef] [Green Version]
  29. Algood, H.M.S.; Cover, T.L. Helicobacter pylori persistence: An overview of interactions between H. pylori and host immune defenses. Clin. Microbiol. Rev. 2006, 19, 597–613. [Google Scholar] [CrossRef] [Green Version]
  30. Galal, Y.S.; Ghobrial, C.M.; Labib, J.R.; Abou-Zekri, M.E. Helicobacter pylori among symptomatic Egyptian children: Prevalence, risk factors, and effect on growth. J. Egypt. Public Health Assoc. 2019, 94, 17. [Google Scholar] [CrossRef]
  31. Frenck, R.W., Jr.; Fathy, H.M.; Sherif, M.; Mohran, Z.; El Mohammedy, H.; Francis, W.; Rockabrand, D.; Mounir, B.I.; Rozmajzl, P.; Frierson, H.F. Sensitivity and specificity of various tests for the diagnosis of Helicobacter pylori in Egyptian children. Pediatrics 2006, 118, e1195–e1202. [Google Scholar] [CrossRef] [PubMed]
  32. Frenck, R.W., Jr.; Clemens, J. Helicobacter in the developing world. Microbes Infect. 2003, 5, 705–713. [Google Scholar] [CrossRef]
  33. Qadri, Q.; Rasool, R.; Gulzar, G.; Naqash, S.; Shah, Z.A. H. pylori infection, inflammation and gastric cancer. J. Gastrointest. Cancer 2014, 45, 126–132. [Google Scholar] [CrossRef]
  34. Bodger, K.; Crabtree, J.E. Helicobacter pylori and gastric inflammation. Br. Med. Bull. 1998, 54, 139–150. [Google Scholar] [CrossRef] [PubMed]
  35. Wang, F.; Meng, W.; Wang, B.; Qiao, L. Helicobacter pylori-induced gastric inflammation and gastric cancer. Cancer Lett. 2014, 345, 196–202. [Google Scholar] [CrossRef] [PubMed]
  36. Njume, C.; Afolayan, A.; Ndip, R. An overview of antimicrobial resistance and the future of medicinal plants in the treatment of Helicobacter pylori infections. Afr. J. Pharm. Pharmacol. 2009, 3, 685–699. [Google Scholar]
  37. Korona-Glowniak, I.; Glowniak-Lipa, A.; Ludwiczuk, A.; Baj, T.; Malm, A. The in vitro activity of essential oils against Helicobacter pylori growth and urease activity. Molecules 2020, 25, 586. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Kocić, B.D.; Đorđević, D.M.S.; Dimitrijević, M.V.; Marković, M.S.; Miladinović, D.L. Potentially Effective and Safe Anti-Helicobacter pylori Natural Products: Chemometric Study. Rev. Chim. 2020, 71, 267–273. [Google Scholar] [CrossRef]
  39. Burt, S. Essential oils: Their antibacterial properties and potential applications in foods—A review. Int. J. Food Microbiol. 2004, 94, 223–253. [Google Scholar] [CrossRef] [PubMed]
  40. Zaidi, S.F.H.; Yamada, K.; Kadowaki, M.; Usmanghani, K.; Sugiyama, T. Bactericidal activity of medicinal plants, employed for the treatment of gastrointestinal ailments, against Helicobacter pylori. J. Ethnopharmacol. 2009, 121, 286–291. [Google Scholar] [CrossRef]
  41. O’Mahony, R.; Al-Khtheeri, H.; Weerasekera, D.; Fernando, N.; Vaira, D.; Holton, J.; Basset, C. Bactericidal and anti-adhesive properties of culinary and medicinal plants against Helicobacter pylori. World J. Gastroenterol. 2005, 11, 7499. [Google Scholar] [CrossRef] [PubMed]
  42. Ohno, T.; Kita, M.; Yamaoka, Y.; Imamura, S.; Yamamoto, T.; Mitsufuji, S.; Kodama, T.; Kashima, K.; Imanishi, J. Antimicrobial activity of essential oils against Helicobacter pylori. Helicobacter 2003, 8, 207–215. [Google Scholar] [CrossRef]
  43. Esmaeili, D.; Mobarez, A.M.; Tohidpour, A. Anti-Helicobacter pylori activities of shoya powder and essential oils of Thymus vulgaris and Eucalyptus globulus. Open Microbiol. J. 2012, 6, 65. [Google Scholar] [CrossRef] [Green Version]
  44. Ruiz-Rico, M.; Moreno, Y.; Barat, J.M. In vitro antimicrobial activity of immobilised essential oil components against Helicobacter pylori. World J. Microbiol. Biotechnol. 2020, 36, 3. [Google Scholar] [CrossRef]
  45. Shen, C.-Y.; Zhang, T.-T.; Zhang, W.-L.; Jiang, J.-G. Anti-inflammatory activities of essential oil isolated from the calyx of Hibiscus sabdariffa L. Food Funct. 2016, 7, 4451–4459. [Google Scholar] [CrossRef] [PubMed]
  46. Chou, S.-T.; Lai, C.-P.; Lin, C.-C.; Shih, Y. Study of the chemical composition, antioxidant activity and anti-inflammatory activity of essential oil from Vetiveria zizanioides. Food Chem. 2012, 134, 262–268. [Google Scholar] [CrossRef]
  47. Miguel, M.G. Antioxidant and anti-inflammatory activities of essential oils: A short review. Molecules 2010, 15, 9252–9287. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Zhang, L.; Liang, X.; Wang, B.; Lin, Z.; Ye, M.; Ma, R.; Zheng, M.; Xiang, H.; Xu, P. Six herbs essential oils suppressing inflammatory responses via inhibiting COX-2/TNF-α/IL-6/NF-κB activation. Microchem. J. 2020, 156, 104769. [Google Scholar] [CrossRef]
  49. Sylvester, J.; Liacini, A.; Li, W.Q.; Dehnade, F.; Zafarullah, M. Tripterygium wilfordii Hook F extract suppresses proinflammatory cytokine-induced expression of matrix metalloproteinase genes in articular chondrocytes by inhibiting activating protein-1 and nuclear factor-κB activities. Mol. Pharmacol. 2001, 59, 1196–1205. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Abu-Darwish, M.; Cabral, C.; Ferreira, I.; Gonçalves, M.; Cavaleiro, C.; Cruz, M.; Al-Bdour, T.; Salgueiro, L. Essential oil of common sage (Salvia officinalis L.) from Jordan: Assessment of safety in mammalian cells and its antifungal and anti-inflammatory potential. BioMed Res. Int. 2013, 2013, 538940. [Google Scholar] [CrossRef] [Green Version]
  51. Quiroga, P.R.; Asensio, C.M.; Nepote, V. Antioxidant effects of the monoterpenes carvacrol, thymol and sabinene hydrate on chemical and sensory stability of roasted sunflower seeds. J. Sci. Food Agric. 2015, 95, 471–479. [Google Scholar] [CrossRef]
  52. Graßmann, J. Terpenoids as plant antioxidants. Vitam. Horm. 2005, 72, 505–535. [Google Scholar] [CrossRef] [PubMed]
  53. Kotan, R.; Kordali, S.; Cakir, A. Screening of antibacterial activities of twenty-one oxygenated monoterpenes. Z. Nat. C 2007, 62, 507–513. [Google Scholar] [CrossRef] [PubMed]
  54. Neerman, M.F. Sesquiterpene lactones: A diverse class of compounds found in essential oils possessing antibacterial and antifungal properties. Int. J. Aromather. 2003, 13, 114–120. [Google Scholar] [CrossRef]
  55. Abbaszadegan, A.; Gholami, A.; Ghahramani, Y.; Ghareghan, R.; Ghareghan, M.; Kazemi, A.; Iraji, A.; Ghasemi, Y. Antimicrobial and cytotoxic activity of Cuminum cyminum as an intracanal medicament compared to chlorhexidine gel. Iran. Endod. J. 2016, 11, 44. [Google Scholar] [PubMed]
  56. Park, J.S.; Baek, H.H.; Bai, D.H.; Oh, T.K.; Lee, C.H. Antibacterial activity of fennel (Foeniculum vulgare Mill.) seed essential oil against the growth of Streptococcus mutans. Food Sci. Biotechnol. 2004, 13, 581–585. [Google Scholar]
  57. Kwiatkowski, P.; Grygorcewicz, B.; Pruss, A.; Wojciuk, B.; Dołęgowska, B.; Giedrys-Kalemba, S.; Sienkiewicz, M.; Wojciechowska-Koszko, I. The effect of subinhibitory concentrations of trans-anethole on antibacterial and antibiofilm activity of mupirocin against mupirocin-resistant Staphylococcus aureus strains. Microb. Drug Resist. 2019, 25, 1424–1429. [Google Scholar] [CrossRef] [PubMed]
  58. Tomy, M.; Dileep, K.; Prasanth, S.; Preethidan, D.; Sabu, A.; Sadasivan, C.; Haridas, M. Cuminaldehyde as a lipoxygenase inhibitor: In vitro and in silico validation. Appl. Biochem. Biotechnol. 2014, 174, 388–397. [Google Scholar] [CrossRef]
  59. Ebada, M.E. Cuminaldehyde: A potential drug candidate. J. Pharmacol. Clin. Res. 2017, 2, 555585. [Google Scholar] [CrossRef]
  60. Yu, L.; Yan, J.; Sun, Z. D-limonene exhibits anti-inflammatory and antioxidant properties in an ulcerative colitis rat model via regulation of iNOS, COX-2, PGE2 and ERK signaling pathways. Mol. Med. Rep. 2017, 15, 2339–2346. [Google Scholar] [CrossRef] [Green Version]
  61. Rizwan, M.; Fatima, N.; Alvi, A. Epidemiology and pattern of antibiotic resistance in Helicobacter pylori: Scenario from Saudi Arabia. Saudi J. Gastroenterol. 2014, 20, 212. [Google Scholar] [CrossRef] [PubMed]
  62. Papatheodoridis, G.V.; Archimandritis, A.J. Role of Helicobacter pylori eradication in aspirin or non-steroidal anti-inflammatory drug users. World J. Gastroenterol. 2005, 11, 3811. [Google Scholar] [CrossRef] [PubMed]
  63. Tang, C.L.; Ye, F.; Liu, W.; Pan, X.L.; Qian, J.; Zhang, G.X. Eradication of Helicobacter pylori infection reduces the incidence of peptic ulcer disease in patients using nonsteroidal anti-inflammatory drugs: A meta-analysis. Helicobacter 2012, 17, 286–296. [Google Scholar] [CrossRef]
  64. Sintim, H.Y.; Burkhardt, A.; Gawde, A.; Cantrell, C.L.; Astatkie, T.; Obour, A.E.; Zheljazkov, V.D.; Schlegel, V. Hydrodistillation time affects dill seed essential oil yield, composition, and bioactivity. Ind. Crops Prod. 2015, 63, 190–196. [Google Scholar] [CrossRef]
  65. Elkady, W.M.; Gonaid, M.H.; Yousif, M.F.; El-Sayed, M.; Omar, H.A. Impact of Altitudinal Variation on the Phytochemical Profile, Anthelmintic and Antimicrobial Activity of Two Pinus Species. Molecules 2021, 26, 3170. [Google Scholar] [CrossRef]
  66. Al-Wendawi, S.A.; Gharb, L. Antioxidant, antibacterial and antibiofilm potentials of anise (Pimpinella anisum) seeds extracted essential oils. Iraqi J. Agric. Sci. 2021, 52, 348–358. [Google Scholar] [CrossRef]
  67. Simic, A.; Rančic, A.; Sokovic, M.; Ristic, M.; Grujic-Jovanovic, S.; Vukojevic, J.; Marin, P.D. Essential oil composition of Cymbopogon winterianus and Carum carvi and their antimicrobial activities. Pharm. Biol. 2008, 46, 437–441. [Google Scholar] [CrossRef]
  68. Kačániová, M.; Vukovič, N.; Horská, E.; Šalamon, I.; Bobková, A.; Hleba, L.; Mellen, M.; Vatľák, A.; Petrová, J.; Bobko, M. Antibacterial activity against Clostridium genus and antiradical activity of the essential oils from different origin. J. Environ. Sci. Health Part B 2014, 49, 505–512. [Google Scholar] [CrossRef]
  69. McLaren, A. In vitro susceptibility testing of H. pylori. In Helicobacter pylori Protocols; Springer: Berlin/Heidelberg, Germany, 1997; pp. 41–51. [Google Scholar]
  70. Fathallah, N.; Raafat, M.M.; Issa, M.Y.; Abdel-Aziz, M.M.; Bishr, M.; Abdelkawy, M.A.; Salama, O. Bio-guided fractionation of prenylated benzaldehyde derivatives as potent antimicrobial and antibiofilm from Ammi majus L. fruits-associated Aspergillus amstelodami. Molecules 2019, 24, 4118. [Google Scholar] [CrossRef] [Green Version]
  71. Petrovic, N.; Murray, M. Using N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD) to assay cyclooxygenase activity in vitro. In Advanced Protocols in Oxidative Stress II; Springer: Berlin/Heidelberg, Germany, 2010; pp. 129–140. [Google Scholar]
  72. Amessis-Ouchemoukh, N.; Madani, K.; Falé, P.L.; Serralheiro, M.L.; Araújo, M.E.M. Antioxidant capacity and phenolic contents of some Mediterranean medicinal plants and their potential role in the inhibition of cyclooxygenase-1 and acetylcholinesterase activities. Ind. Crops Prod. 2014, 53, 6–15. [Google Scholar] [CrossRef]
  73. Youssef, F.S.; Labib, R.M.; Gad, H.A.; Eid, S.; Ashour, M.L.; Eid, H.H. Pimenta dioica and Pimenta racemosa: GC-based metabolomics for the assessment of seasonal and organ variation in their volatile components, in silico and in vitro cytotoxic activity estimation. Food Funct. 2021, 12, 5247–5259. [Google Scholar] [CrossRef]
Plants 11 02617 g001 550
Figure 1. GC chromatograms of the essential oils of (A) C. cyminum, (B) P. anisum, and (C) C. carvi.
Figure 1. GC chromatograms of the essential oils of (A) C. cyminum, (B) P. anisum, and (C) C. carvi.
Plants 11 02617 g001
Plants 11 02617 g002 550
Figure 2. 2D structures of the main detected compounds of three oils L. by GC–MS analysis (Drawn by Chem-draw ultra-version 14).
Figure 2. 2D structures of the main detected compounds of three oils L. by GC–MS analysis (Drawn by Chem-draw ultra-version 14).
Plants 11 02617 g002
Plants 11 02617 g003 550
Figure 3. Anti-inflammatory activity cyclooxygenase COX-2 inhibitory % of CM (C. cyminum), AN (P. anisum), and CR (C. carvi) compared to standard drug, celecoxib.
Figure 3. Anti-inflammatory activity cyclooxygenase COX-2 inhibitory % of CM (C. cyminum), AN (P. anisum), and CR (C. carvi) compared to standard drug, celecoxib.
Plants 11 02617 g003
Plants 11 02617 g004a 550Plants 11 02617 g004b 550Plants 11 02617 g004c 550
Figure 4. 2D and 3D interaction diagram of the co-crystallized ligand and identified compounds docking pose interactions with the key amino acids in the 3NM4 binding site. The three-letter amino acid codes and positions of each residue are indicated. Between the receptor and the ligand, hydrogen-bonding interactions are illustrated by a green dashed line, whereas -alkyl interactions are represented by a purple dashed line.
Figure 4. 2D and 3D interaction diagram of the co-crystallized ligand and identified compounds docking pose interactions with the key amino acids in the 3NM4 binding site. The three-letter amino acid codes and positions of each residue are indicated. Between the receptor and the ligand, hydrogen-bonding interactions are illustrated by a green dashed line, whereas -alkyl interactions are represented by a purple dashed line.
Plants 11 02617 g004aPlants 11 02617 g004bPlants 11 02617 g004c
Table
Table 1. Chemical compositions of the isolated essential oils.
Table 1. Chemical compositions of the isolated essential oils.
CompoundRI expRI repC. cyminumP. anisumC. carviReferences
1- Monoterpenoids
Cumin aldehyde 1283128341.26n.dn.d[22]
Limonene102710270.771.5748.49[3,23]
Carvone 12441249n.dn.d51.38[23]
Anethole 918918n.d92.41n.d[3]
ɣ-Terpinene104910498.11n.dn.d[22]
1,4-p-Menthadien-7-al 1784178412.84n.dn.d[23]
Myrtenal 1237123719.26n.dn.d[23]
α-Pinene 9339300.43n.dn.d[22]
β-Pinene 96997310.27n.dn.d[22]
β-Myrcene 9809850.41n.d0.13[22]
p-Cymene 144114415.22n.dn.d[22]
Estragole 11971197n.d3.01n.d
trans-β-Ocimene 10341034n.d0.22n.d[3]
p-Menth-2-en-1-ol 113011291.26n.dn.d[22]
2- Sesquiterpenoids
β-Chamigrene14781478n.d0.20n.d[24]
Zingiberene14941494n.d0.26n.d[25]
β-Himachalene15051505n.d0.10n.d[25]
Longifolene 15321532n.d2.11n.d[25]
% of identification 99.8397.21100
All compounds were identified by Kováts index and mass spectral data by comparing values reported in the literature. RI exp Kovats index was determined experimentally relative to C8–C28 n-alkanes. RI rep reported Kovats retention indices.
Table
Table 2. Mean of H. pylori inhibitory % of three extracted essential oils against the standard drug clarithromycin.
Table 2. Mean of H. pylori inhibitory % of three extracted essential oils against the standard drug clarithromycin.
Sample conc. (µg/mL)Mean of H. pylori Inhibitory%
Clarithromycin *C. cyminum *P. anisum *C. carvi *
125100100.00100.00100.00
62.5100100.00100.00100.00
31.25100100.00100.0081.63 ± 1.3
15.63100100.00100.0070.64 ± 2.1
7.81100100.0079.21 ± 0.546.21 ± 0.59
3.9100100.0051.74 ± 1.321.74 ± 1.3
1.9510081.97 ± 0.9529.39 ± 0.68.29 ± 0.74
0.9892.45 ± 1.273.25 ± 1.317.92 ± 1.53.71 ± 0.91
0.4887.65 ± 0.559.31 ± 1.66.34 ± 0.740.00
0.2481.35 ± 1.527.28 ± 1.00.000.00
0.000.000.000.000.00
MIC1.953.915.6362.5
* All determinations were carried out in a triplicate manner and values are expressed as the mean ± SD.
Table
Table 3. Free binding energies (∆G) of the identified compounds within the 3NM4 active site calculated in kcal/mol using Discovery Studio 4.5, adopting both rule-based ionization techniques.
Table 3. Free binding energies (∆G) of the identified compounds within the 3NM4 active site calculated in kcal/mol using Discovery Studio 4.5, adopting both rule-based ionization techniques.
CompoundBinding Energy ∆G (Kcal/mol)
Rule-Based
Cumaldehyde−28.75
Estragole−28.72
p-Cymene−28.49
TRIS−28.24
(-)-Carvone−28.22
Anethole−28.01
ɣ-terpinene−25.32
Limonene−25.26
α Thujenal−22.32
β Pinene−21.88
Myrtenal−20.80
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Alomar, H.A.; Fathallah, N.; Abdel-Aziz, M.M.; Ibrahim, T.A.; Elkady, W.M. GC-MS Profiling, Anti-Helicobacter pylori, and Anti-Inflammatory Activities of Three Apiaceous Fruits’ Essential Oils. Plants 2022, 11, 2617. https://doi.org/10.3390/plants11192617

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Alomar HA, Fathallah N, Abdel-Aziz MM, Ibrahim TA, Elkady WM. GC-MS Profiling, Anti-Helicobacter pylori, and Anti-Inflammatory Activities of Three Apiaceous Fruits’ Essential Oils. Plants. 2022; 11(19):2617. https://doi.org/10.3390/plants11192617

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Alomar, Hatun A., Noha Fathallah, Marwa M. Abdel-Aziz, Taghreed A. Ibrahim, and Wafaa M. Elkady. 2022. "GC-MS Profiling, Anti-Helicobacter pylori, and Anti-Inflammatory Activities of Three Apiaceous Fruits’ Essential Oils" Plants 11, no. 19: 2617. https://doi.org/10.3390/plants11192617

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