Anti-Helicobacter Pylori Activity From Natural Products

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A hematoxylin and eosin stain of Helicobacter pylori in the lumen of a gastric foveola. Photo by Ed Uthman13

For many individuals, Helicobacter pylori, gram-negative bacteria, reside in the stomach that many times lead to duodenal and peptic ulcers. Even though most individuals infected with H. pylori never show symptoms, this bacterium causes around 85 percent of all ulcers. Antibiotics, clarithromycin and amoxicillin, and a proton pump inhibitor are typically used to inhibit H. pylori growth to cure the ulcer. This triple therapy for the elimination of H. pylori faces uprising resistance problems demanding new antibiotic candidates. Alternative therapies from natural plants and foods are gaining popularity in order to find products to help combat the drug-resistant strains. Many studies have examined the antimicrobial activity of Pakistanian, Indian, and Iranian medicinal plants can be used as potential bactericidal agents for curing ulcers. Additionally, studies have found that fractional isolates of honey and turmeric are responsible for antibacterial activity. These compounds provide new lead molecules that can help eradicate H. pylori.

Resistant Strains of H. pylori


Bactericidal Activity of Plant Extracts

Many different extracts from plants have been shown to have antimicrobial activity. GutGard™ is a flavonoid rich extract of Glycyrrhiza glabra Linn, which is commonly known as Licorice. The company sells this product to those who suffer from gut and digestion related issues. Many studies have been conducted to determine the validity of GutGard™. Furthermore, different acetone and methanol extracts were taken from traditional medicinal plants to determine the minimal inhibitory concentration on different strains of H. pylori.


Flavonoid Extract

The minimal inhibitory concentration of the components in GutGard and three different positive controls. Data from Asha et al.


Glycyrrhiza glabra is normally found in the Mediterranean and parts of Asia. Many older civilizations used the root of this plant for its medicinal properties to cure stomach ulcers, kidney stones, skin eruptions, and more. Due to the increasing drug resistant strains of H. pylori, studies are trying to demonstrate and verify the antimicrobial activity of this natural plant. Through a microbroth dilution method, the minimum inhibitory concentrations (MIC) against several variant strains of H. pylori were determined for GutGard™, glabrin, deglycyrrhizainated licorice (DGL), monoammonium glycyrrhizinate (MAG).3 Glabridin, a flavonoid rich extract of GutGard™, appeared to be the active ingredient causing anti-H. pylori activity.

GutGard’s™ mechanism of action is not quite confirmed, but through an in vitro study, GutGard™ demonstrated a protein synthesis inhibition via the reduction in (35)S methionine into H. pylori ATCC 700392 strain.3 Furthermore, this potential antibiotic exhibited an inhibitory effect on DNA gyrase and dihydrofolate reductase. Ciprofoxacin, levofoxacin, and fluoroquinolones, antibiotics that inhibit DNA gyrase, are being considered as possible drugs for the eradication of Helicobacter pylori.12 Thus, DNA gyrase may play an essential role in the growth and survival of H. pylori. Comparing the inhibition of DNA gyrase of GutGard™ to ciprofloxacin, GutGard™ had an MIC50 of 4.40 μg/ml while the positive control, ciprofloxacin, showed an MIC50 value of 12.31 ng/ml. If dihydrofolate reductase is blocked, the bacterial cells die because of its inhibition of DNA synthesis. When compared to the positive control methotrexate, GutGard™ was found to moderately inhibit H. pylori.

The chemical structures of different flavonoids. The flavonoids have very similar structures with only slight variations. The flavonoids compounds shown are a) naringenin, b) hesperidin, c) luteolin, d) quercetin, e) genkwanin, f) 7-O-butylnaringenin, g) kaempferol, h) apigenin, and i) hesperetin. Figure from Moon et al.



A double blind randomized study was conducted to evaluate the inhibitory effects of GutGard™. Participants were randomly placed into the placebo (n=52) and treatment (n=55) groups.11 Both groups orally received their respective drug once daily for 60 days. A 13C-urea breath test and Stool Antigen test were performed at days 0, 30, and 60 to measure the H. pylori levels in the stomach. Using the analysis of variance, chi-square, and Fisher’s exact probability test to compare the overall treatment conditions, the GutGard™ treatment group showed a statistically significant improvement in the management of H. pylori.

Even at a low stomach pH, flavonoids still seem to exhibit antibacterial activity. In general, flavonoids tend to have an antimicrobial effect against different H. pylori strains. The antibacterial effects of 7-O-Butylnaringenin, a novel flavonoid synthesized from citrus waste, and various natural flavonoids were studied.9 Hesperetin and naringenin exhibited the most antimicrobial effects for the natural flavonoids. However, 7-O-Butylnaringenin exhibited a higher inhibitory effect against urease activity of H. pylori than the natural flavonoids. The morphological changes of H. pylori show that 7-O-buthylnaringen and hesperetin damage the bacterial cell membrane at 200 μM. This study shows how different natural products can be slightly modified to yield a compound with greater inhibitory potential.


Acetone and Methanol Extracts

Many plants from the Middle Eastern, Mediterranean, and Indian area have traditionally been used to treat common ailments. Acetone and methanol extracts from these plants possess some antibacterial properties against different strains of H. pylori.2 Using the agar dilution method, the minimum inhibitory concentrations of acetone, methanol, and water extracts were determined from various plants in the Punjab province of Pakistan. The extracts’ ability to inhibit H. pylori urease was assessed by the phenol red method. The methanol and acetone extracts from Acacia nilotica and Calotropis procera had significant antibacterial activity and urease inhibition but were less powerful compared to amoxicillin and clarithromycin. The extracts from Acacia nilotica and Calotropis procera exhibited different mechanisms of inhibition: competitive and mixed, respectively.

Combretum molle from South Africa has been widely used to treat gastric ulcers. The agar diffusion method showed the MIC of five different solvent extracts: acetone, ethanol, water, crude extract, and ethyl acetate.10 All the solvent extracts demonstrated some anti-H. pylori activity; however, the acetone extract was highly bactericidal. At MIC90, the acetone extract exhibited inhibition at 2.5 to 5.0 mg/ml depending on the strain of H. pylori.



Bactericidal Activity of Natural Foods


Honey

Honey’s high acidity, high osmotic effect, concentration of hydrogen peroxide, and various photochemicals all help honey inhibit or kill different bacteria. Additionally, Krishna verified that the acidic medium of the stomach helps augment the antibacterial activity of honey.7 In the n-hexane extract of Goldcrest honey, TLC chromatography found four different column fractions – GCCL, GCF2, GCF3, and CGF4 – to exhibit anti-H. pylori activity. .7 Using the broth microdilution method, the column fractions had MIC50 that ranged from 5-10 mg/ml, which showed that there were probably different antimicrobial isolates in each of the fractions. GCF3 exhibited the best antimicrobial activity against various strains of H. pylori. The acetic acid, proponoic acid, and flavonoids components all help give honey its antimicrobial activity. In conclusion, the different actions of the volatile compounds present cause the antimicrobial activity.

Another study tested six different local South African honeys and their solvent extracts for anti-H. pylori activity. .8 The broth microdilution method determined the MIC50 of the two most active extracts from the six local honeys. All of the honeys had different inhibiting zones, but the Pure Honey and Champagne Royal Train had zone diameters that were not significantly different from amoxicillin, the positive control. The chloroform extract in pure honey had the most inhibiting effect from 42 to 72 hours. Similar to the 7-O- butylnaringenin, these honey extracts can serve as a start to make new molecules that are better antimicrobials.

CAPE binds to the PDF active site, blocking any substrate from entering. PDB 4E9A. Picture from PyMOL.4

Furthermore, other components from honey, propolis, has been found to exhibit anti-H. pylori activity. .4 An essential enzyme, H. pylori peptide deformylase (HpPDF), facilitates the removal of the formal group from the N-terminal of nascent polypeptide chains for newly synthesized proteins. Caffeic acid phenethyl ester (CAPE), an active component of propolis, competitively inhibits HpPDF by blocking substrate entrance. The MIC50 value of CAPE is 4.02 μM. The phenyl head of CAPE binds to the hydrophobic active site, while the CAPE tail protrudes to the entrance of the pocket. Hydrogen bonds are also formed with the CAPE and the surrounding amino acids, which exhibit pi-pi interaction. Most PDF inhibitors are pseudopeptides, but CAPE has a different structure. Compared to other PDF inhibitors, no chelation or disruption of metal-dependent catalysis occurs with the HpPDF-CAPE complex at the active site; this actually may lead to less harmful effects on the body.








Turmeric

The MIC of curcumin on the 65 different strains of H. pylori. Data from Kundu et al.5

Diferuloylmethane from turmeric, curcumin, has been shown to inhibit H. pylori growth. The antimicrobial activity was examined against 65 different clinical isolates of H. pylori. 5 Depending on the strain, the MIC of curcumin ranged from 5 μg/ml to 50 μg/ml. Looking at kinetic data, curcumin seems to act as a noncompetitive inhibitor in order to hinder the shikimate dehydrogenase (SHD) activity. SDH catalyzes the reduction of 3-dehydroshikimate to shikimate in the shikimate pathway.6 The difference in MIC values suggest that this enzyme may not always be essential for the bacteria’s survival.5 Furthermore, the effects of curcumin on a H. pylori infected mice was studied histologically. This test showed that curcumin was highly effective in treating H. pylori in the mice’s stomach and restoring the gastric damage. Curcumin serves potential as an alternative therapy because it has shown antimicrobial and mucoadhesive properties. The mucoadhesive microspheres of curcumin can serve as a drug to treat H. pylori in combination with other antimicrobials.1

Further Reading

[1]—Exploring alternative treatments for Helicobacter pylori infection

References

1. Ali, Sajid, Vinay Pandit, Mahendra Jain, and Kanhiya L. Dhar. "Mucoadhesive Microparticulate Drug Delivery System of Curcumin against Helicobacter Pylori Infection: Design, Development and Optimization." Journal of Advanced Pharmaceutical Technology and Research 5.1 (2014): 48-56. Print.
2. Amin, Muhammad, Farooq Anwar, Fauqia Naz, Tahir Mehmood, and Nazamid Saari. "Anti-Helicobacter Pylori and Urease Inhibition Activities of Some Traditional Medicinal Plants." Molecules 18.2 (2013): 2135-149. Print.
3. Asha, Mannanthendil Kumaran, Debnath Debraj, D'souza Prashanth, Jothie Richard Edwin, H.s. Srikanth, Nithyanantham Muruganantham, Shekhar Michael Dethe, Bhaskar Anirban, Balachandran Jaya, Mundkinajeddu Deepak, and Amit Agarwal. "In Vitro Anti-Helicobacter Pylori Activity of a Flavonoid Rich Extract of Glycyrrhiza Glabra and Its Probable Mechanisms of Action." Journal of Ethnopharmacology 145.2 (2013): 581-86. Print.
4. Cui, Kunqiang, Weiqiang Lu, Lili Zhu, Xu Shen, and Jin Huang. "Caffeic Acid Phenethyl Ester (CAPE), an Active Component of Propolis, Inhibits Helicobacter Pylori Peptide Deformylase Activity." Biochemical and Biophysical Research Communications 435.2 (2013): 289-94. Print.
5. De, R., P. Kundu, S. Swarnakar, T. Ramamurthy, A. Chowdhury, G. B. Nair, and A. K. Mukhopadhyay. "Antimicrobial Activity of Curcumin against Helicobacter Pylori Isolates from India and during Infections in Mice." Antimicrobial Agents and Chemotherapy 53.4 (2009): 1592-597. Print.
6. Han, Cong, Lirui Wang, Kunqian Yu, Lili Chen, Lihong Hu, Kaixian Chen, Hualiang Jiang, and Xu Shen. "Biochemical Characterization and Inhibitor Discovery of Shikimate Dehydrogenase from Helicobacter Pylori." FEBS Journal 273.20 (2006): 4682-692. Print.
7. Manyi-Loh, Christy E., Anna M. Clarke, and Roland N. Ndip. "Detection of Phytoconstituents in Column Fractions of N-Hexane Extract of Goldcrest Honey Exhibiting Anti-Helicobacter Pylori Activity." Archives of Medical Research 43.3 (2012): 197-204. Print.
8. Manyi-Loh, C. E., A. M. Clarke, and NDIP E. Green, RN. "Inhibitory and Bactericidal Activity of Selected South African Honeys and Their Solvent Extracts against Clinical Isolates of Helicobacter Pylori." Pak J Pharm Sci. 26.5 (2013): 897-906. Print.
9. Moon, Sun H., Jae H. Lee, Kee-Tae Kim, Yong-Sun Park, Seung-Yeol Nah, Dong Ahn, and Hyun-Dong Paik. "Antimicrobial Effect of 7-O-Butylnaringenin, a Novel Flavonoid, and Various Natural Flavonoids against Helicobacter Pylori Strains." International Journal of Environmental Research and Public Health 10 (2013): 5459-469. Print.
10. Njume, Collise, Anthony J. Afolayan, Amidou Samie, and Roland N. Ndip. "Inhibitory and Bactericidal Potential of Crude Acetone Extracts ofCombretum Molle (Combretaceae) on Drug-resistant Strains of Helicobacter Pylori." Journal of Health, Population and Nutrition 29.5 (2011): n. pag. Print.
11. Puram, Sreenivasulu, Hyung C. Suh, Seung Kim, Bharathi Bethapudi, Joshua Joseph, Amit Agarwal, and Venkateswarlu Kudiganti. "Effect of GutGard in the Management of Helicobacter Pylori: A Randomized Double Blind Placebo Controlled Study : Table 3." Hindawi (2013): 1-8. Hindawi, Feb. 2013. Web.
12. Rimbara, Emiko, Norihisa Noguchi, Takashi Kawai, and Masanori Sasatsu. "Fluoroquinolone Resistance in Helicobacter Pylori: Role of Mutations at Position 87 and 91 of GyrA on the Level of Resistance and Identification of a Resistance Conferring Mutation in GyrB."Helicobacter 17.1 (2012): 36-42. Print.
13. Uthman, Ed. Helicobacter pylori. 2007. Flickr. Web. <http://www.flickr.com/photos/78147607@N00/390307642/in/photolist-AuqS7-AuqS8-9hmf44-9pz3W4-fxzkmp>.

Edited by Amie Patel, a student of Nora Sullivan in BIOL168L (Microbiology) in The Keck Science Department of the Claremont Colleges Spring 2014.