Available online on 15.03.2023 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2023 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access   Full Text Article                                                                                                                                                 Research Article 

Determination of the Antibacterial Activity of Krameria pauciflora (Rose)

^a*Rodolfo Velasco-Lezama, ^bMa. Fernanda Aguilar-Carrillo, ^aRafaela Tapia-Aguilar, ^bMa. de Lourdes Velázquez-Vázquez, ^cReyna Cerón-Ramírez and ^cJorge Santana-Carrillo 

^aDepartamento de Ciencias de la Salud, División de Ciencias Biológicas y de la Salud-Iztapalapa, Universidad Autónoma Metropolitana, Mexico City, Mexico

^bLicenciatura de Biología Experimental, División de Ciencias Biológicas y de la Salud-Iztapalapa, Universidad Autónoma Metropolitana, Mexico City, Mexico

^cHerbario Metropolitano “Ramón Riba y Nava Esparza”, División de Ciencias Biológicas y de la Salud-Iztapalapa, Universidad Autónoma Metropolitana, Mexico City, Mexico

INTRODUCTON: 

According to the World Health Organization (W.H.O), in the year 2015, respiratory airways infections and the infections that accompany diarrhea caused 3.2 and 1.4 million deaths, respectively, occupying third and eighth places as causes of death at the worldwide level.¹ Diarrheal infections and gastroenteritis of infectious origin caused 5.1% of deaths in children less than 5 years of age at the worldwide level. These gave rise to the death of 525,000 children every year. Malnourished and immunocompromised children are those who present the greatest risk of potentially mortal diseases.² The report of the National Institute of Geography and Statistics (INEGI) in Mexico shows that diarrheal infections and gastroenteritis of infectious origin caused 5.1% of deaths in preschool children aged 1-4 years during 2015.³   

In Mexico, diarrheal disease has comprised an important public-health problem from the preHispanic era and the Viceroyalty. In the Cruz-Badiano Codices, the Florentine Codex, and Book of the Royal Protophysician Francisco Hernández, written in the XVI century, the existence is noted of “cocoliztli”, an infection considered mortal, an infection that caused abdominal pains and evacuations with loose or liquid feces occasionally with blood.⁴ The Mexican culture  utilized between 40 and 79 plants for the treatment of such an affectation ⁵, which was responsible for 50-90% of the deaths that occurred during the XVI century, being identified today with infections caused by enteric Salmonellas.^5,6 Infectious diseases are treated with antibiotics, and at present many of these have lost their effectiveness due to that some bacteria have developed mechanisms of defense,  expressing genes that confer on them resistance against antibiotics in clinical use, in addition to the secondary effects of these.⁷     

It is estimated that by the year  2050, bacterial diseases will be the primary cause of death, all of this due to the excessive and indiscriminate use of antibiotics.⁸ The latter has motivated the search for antimicrobials deriving from natural sources, such as medicinal plants and marine invertebrate organisms.⁹ In Mexico, plants of the genus  Krameria are employed to alleviate diverse illnesses, among these, infections of the respiratory airways and gastrointestinal diseases. This genus, belonging to the Krameriaceae family, has 18 species. Plants from this genus can grow principally as herbs or as the hemoparasitic bushes of perennial plants; the shrubs reach one meter in height, and the stem in some species is spiny. The leaves are simple or alternate, and the inflorescence is solitary with axillary or terminal flowers.¹⁰ 

These plants are native to the American continent, and they grow preferentially in tropical regions from 900-3,000 meters above sea level (masl).  In Ecuador, Peru, and Bolivia, these plants are utilized as mouthwashes or for gargling in cases of periodontis. Popularly, they are known with the name of ratania (rhatany), the name given to the root of the plant, which is one of those most frequently employed in popular medicine. Other medicinal employments include their use as astringents, as anti-inflammatories, to combat cancer of the intestine, stomach, and tongue. The plants are additionally utilized as suppositories, hemostatics, and in the treatment of hemorrhoids.¹¹  

In Mexico, the medical use of the plants of the genus Krameria is linked to indigenous groups from the North of the country (Sonora state), such as the Seri and the Yaqui. The former use Krameria grayl to regulate menstruation, as a tonic, and to the clean the kidneys. Meanwhile, the Yaquis utilize Krameria parvifolia to purify the blood, against venereal disease in males, and to increase the red globules.¹²  In the Mexican State of  Hidalgo, on the center of the country, the decoction is ingested of the rhizome or the leaves of Krameria pauciflora to combat gastrointestinal infections, some types of cancer, and anemia.    K. pauciflora is an herbaceous plant, a perennial, with a short main stem, with a woody base, with simple or trifoliate alternate leaves.¹³ Due to that this plant is employed popularly against gastrointestinal diseases, the objective of this study was to determine the capacity of the rhizome, the leaves, and a rhizome–leaves–stem  combination (the complete plant) to inhibit the growth of enterobacteria associated with gastrointestinal infections.    

MATERIAL AND METHODS

Plant material 

The aerial parts and the rhizome of Krameria pauciflora were collected in Tepeji del Río, Hidalgo state, Mexico, in July of 2017. The taxonomic identification of the samples was carried out by Professors Jorge Santana and  Reyna Cerón of the “Ramón Riba y Nava Esparza” Metropolitan Herbarium of the Metropolitan Autonomous University (UAM), where the samples were deposited for safekeeping. 

Preparation of the extracts The plant was left to dry at room temperature, including the rhizome, leaves, and the complete plant (leaves, stem, and rhizome). One hundred g of the pulverized material was macerated consecutively during 48 h in each of the following solvents: 1.2 L of hexane; dichloromethane; methanol (J.T. Baker, USA), and water during 48 h. The extracts were filtered, the organic solvents were eliminated at reduced pressure in a rotavapor (Buchii RII, Switzerland), and the water was eliminated by evaporation in a double boiler. Additionally, the decoction was prepared from the complete plant. With the solid extracts we prepared dissolution of dimethyl sulfoxide of 5-0.03 mg/mL 10% (DMSO)/H₂O (J.T. Baker, USA). A preliminary phytochemical study was carried out according that those reported by Alarcón and collaborators.¹⁴ 

Bacterial strains 

The bacteria employed were as follows: Salmonella typhimurium ATCC 13311; Shigella flexneri ATCC 29003; Salmonella typhi ATCC 6539; Escherichia coli SOS, and Proteus mirabilis. To determine the antibacterial activity and to know the Minimal Inhibitory Concentration (MCI), the protocol of Drummond and Waigh, modified by Satyajit, was followed, in which 96-multi-well plates were utilized, with resazurin as viabity indicator. This method is based on the capacity of reduction of the resazurin to resorufin by the oxidoreductase enzymes of the surviving bacteria. When the extract inhibits the bacterial growth, there is no activity of bacterial oxidoreductases. Thus, the resazurin remains blue in color, while when they survive,  the reduction to resorufin confers a pink color on the culture medium with the swerve of the indicator.¹⁵  

Antibacterial activity of the extracts 

The bacteria were seeded by crossed streak on plates with   Müeller–Hinton medium (Bioxón, México) and were incubated for 24 h at 37^oC. A colony was taken and was seeded in duplicate in 50 ml of Müeller–Hinton broth (Bioxón, México). The culture was incubated 24 h at 37^oC. After this, a 2.5-ml aliquot was taken; it was centrifuged (SOL-BAT, México) at 2,500 g during 5 min, the supernatant was eliminated, and the cell pellet was suspended in the same volume of sterile physiological saline solution (PSS.) The bacterial concentration was adjusted to 4 x 10⁶ CFU/mL, with the 0.5 McFarland-nephelometer    turbidity pattern, and the concentration was incubated for 24 h at 37°C. With the solid extracts, solutions were prepared separately with an initial concentration of 5 mg/mL in Dimethyl sulfoxide (DMSO, J.T. Baker, USA) at 1.0% and double dilutions were carried out. Fifty μL/well was deposited on 96 multi-well plates (Nunclon, Germany), 10 μL of the bacterial suspension (4 x 10⁶ CFU/mL) was added, in addition to 10 μL of Resazurin sodium (Sigma Chemical Co, St Louis MO, USA) 0.675% p/v in sterile distilled water and 30 μL of Müeller–Hinton 3X culture medium (320 mosm) (Bioxón, México). As negative control, DMSO 1.0% was utilized in sterile distilled water, and as positive control, we employed a Penicillin–Streptomycin solution 1 x 10⁴ IU/mL-1 x 10⁴ mg/mL (Sigma Chemical Co. St. Louis, MO, USA). The culture plates were Incubated at 37^oC during 22 h. (LabLine, USA) Each extract was proved in triplicate on at least three ocassions. Each extract was proved in triplicate on at least three occasions. 

RESULTS

 The preliminary phytochemical analysis of the hexanic and dichloromethanic extracts revealed the presence of terpenoid-type and fatty-acid compounds, while in the methanolic and aqueous extracts, soluble tannin compounds and flavonoids were detected. 

Antibacterial Activity of the Rhizome

In Table 1, it is shown that the methanolic and aqueous extracts of the rhizome presented better antibacterial activity than those of hexane and dichloromethane. The latter was that of least activity, solely inhibiting Shigella flexneri and Salmonella typhi, bacterial strains that were inhibited by all of the extracts. The MIC obtained was 0.156 mg/mL of the methanolic and aqueous extracts on S. typhi and S. flexneri, respectively (Table 1).

Table 1. Antibacterial activity of the extracts of the rhizome of Krameria pauciflora   

-- Signifies negative.

The extracts of the leaves presented similar activity to that of the rhizome. However, it can be observed that the activity of the leaf is lesser with respect to that of the rhizome. With the methanolic extract, an MIC was obtained of 0.312 on S. typhi and with the aqueous extract on   S. flexneri and S. typhi (Table 2).

Table 2. Antibacterial activity of the extracts of the leaf of  Krameria pauciflora    

---- Signifies negative.

Antibacterial Activity of the Extracts of the Complete Plant 

Different from the hexanic extracts of the rhizome and of the leaves, that obtained from the complete plant inhibited the growth of the strains employed. The hexanic, methanolic, and aqueous extracts of the complete plant inhibited all of the bacteria in concentrations lesser than those of the rhizome and the leaves, in particular on S. flexneri and S. typhi. However, it is important to highlight that the decoction presented the lowest MIC, that is, 0.078 mg/mL, of the study (Table 3).

Table 3. Antibacterial activity of the extracts and decoction of the complete plant of   Krameria pauciflora   

---- Signifies negative.

DISCUSSION

Neto and collaborators studied the effect of an ethanolic extract of Krameria trianda and its hexane, ethyl acetate, and methanol fractions on the growth of 18 bacterial strains, among these Escherichia coli. The raw extract inhibited such bacteria. However the hexane fraction did not inhibit any strain. In our study, the hexanic extract, particularly that of the leaf–stem–rhizome, inhibited all of the bacteria utilized, including E. coli. The difference in activity would be attributed to that, in the Neto study, the hexane fraction procedes from an ethanolic extract, a solvent that can extract some chemically non-polar compounds that are dissolved in hexane, while in our work, the hexanic extract was obtained directly with hexane as first extraction solvent, hence a greater number and variety of compounds.¹⁶   

Bussmann and collaborators ¹⁷ prepared ethanolic extracts and an aqueous decoction of 52 plants from the North of Peru    and evaluated their effect on the growth of Escherichia coli and  Staphylocccus aureus. These authors reported that 38 of the ethanolic extracts inhibited S. aureus and that only two extracts inhibited E. coli, one of these being K. lappaceae. The aqueous extract solely inhibited S. aureus, but not E. coli. These latter results are in contrast with ours, in that the aqueous extracts of the rhizome, leaf, and complete plant inhibited E. coli. This difference could be attributed to that our aqueous extract derives from another species. It is the residue of a material treated previously with three organic solvents chemically growing in polarity, thus less contaminated or more enriched with compounds that inhibit the proliferation of E. coli. 

With respect to K. lappacea, Genovese and collaborators¹⁸ reported the sensitivity of Methicillin-resistant S. aureus (MRSA) to the aqueous and ethanolic extracts of the root. Both extracts diminished the adhesive properties of the bacteria and their capacity to form biofilms. In results not included in this study, we evaluated the activity of the extracts of K. pauciflora on S. aureus ATTC 6538, observing susceptibility to the hexanic, dichoromethanic, methanolic, and aqueous extracts of the rhizome and to the decoction of the complete plant with an MIC of 0.156 mg/mL of the hexanic extract,¹⁹ which confirms the susceptibility of S. aureus to species of the genus Krameria. 

The antibacterial and antifungal activity of some species of the genus Krameria has been associated with the lignans and neolignans present in the plant’s root. Also reported have been catechin tannins, ratanitanic acid, and N-methyltyrosine, and cycloartanes, among other compounds.^20,21  

Lignans and neolignans are chemically non-polar compounds that could preferentially affect   the lipids of the external membrane of Gram-negative bacteria, achieving the disorganization of the membrane, which would explain the antibacterial activity of our extracts of hexane and dichloromethane.^22,23 The lignans of the root of Krameria lappacea have been linked with anti-inflammatory effects in vitro and in vivo, inhibiting acute inflation. Anti-inflammatory activity has also been reported in Krameria pauciflora, associated in this case with the cycloartanes isolated from the root of the plant.²⁴ 

Other compounds detected in the genus Krameria are the tannins. Our study showed the presence of tannins in the methanolic and aqueous extracts and in the decoction. Due to that tannin compounds are soluble in water and in organic polar compounds, it is possible that the antibacterial activity demonstrated by the methanolic and aqueous extracts and by the decoction could be related with the presence of tannins in these, because it is known that tannins precipitate proteins, including structural or enzymatic, impeding the microorganism from feeding and reproducing itself.²⁵ Although the majority of the phenolic compounds are hydrophobic, the hydroxyl group  (-OH) allows them to introduce aryl and alkaline groups into the proteins, modifying the three-dimensional (3D) structure of the latter. The OH- group can also affect the stability of the DNA, when such an interaction with amino and carbonyl groups of the pyrrhic and pyrimidine bases form new hydrogen bridges, rendering impossible the functionality of the microorganisms, in that they can disorganize the lipids of the bacterial membrane.²⁶  

In the literature, the antibacterial and antifungal activity is principally reported of the genus   Krameria in extracts of the root. However, in our study, we demonstrated that other structures also possess antibacterial activity. In popular Mexican medicine, the decoction is recommended of the rhizome of K. pauciflora for the treatment of gastrointestinal infections. It is observed that the antibacterial activity improves when the extract is prepared with the rhizome–leaves–stem of the plant (the complete plant). It is additionally observed that this activity is even greater if, instead of preparing the extract, a decoction is utilized of the complete plant (rhizome–leaves–stem). Our results are in agreed with the popular use of plant in the zone of its collection.      

CONCLUSIONS:

The minimal inhibitory concentration was obtained with the rhizome–stem–leaves decoction. 

The methanolic extract demonstrated better antibacterial activity than the remaining extracts. 

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

FINANCING

This work was developed with the budget assigned by the Universidad Autónoma Metropolitana (UAM), México. 

REFERENCES 

1. Organización Mundial de la Salud. Las diez principales causas de muerte en el mundo. OMS; 2017

2. Olaiz-Fernández GA, Gómez-Peña EG, Juárez-Flores A, Vicuña-de Anda FJ, Morales-Ríos JE and Carrasco OF. Historical overview of acute infectious diarrhea in Mexico and future preventive strategies. Salud Pública de México, 2020; 62(1):25-35. https://doi.org/10.21149/10002

3. Estadísticas a propósito del Día de Muertos: Instituto Nacional de Estadística y Geografía (INEGI). Base de datos México. 2017, Octubre 11.

4. Velasco LR, Tapia AR, Vega AE. Aspectos históricos para el estudio de las plantas medicinales. Contactos, 2004; 51: 1-20. Universidad Autónoma Metropolitana, México.

5. Estrada Erin. Memorias del Séptimo Diplomado Internacional de Plantas Medicinales de México. 1994. Universidad Autónoma de Chapingo.

6. Vágene AJ, Herbig A, Campana MG, Robles-García NM, Warinner C, Sabin S, et al. Salmonella enterica genomes from victims of a major sixteenth-century in Mexico suggests that Salmonella enterica paratyphi C (enteric fever) was responsible for a devastating epidemic that closely followed European presence in the region. Nature Ecology & Evolution, 2018; 2(3):520-528. https://doi.org/10.1038/s41559-017-0446-6

7. Martens E, Demian AL. The antibiotic resistance crisis, with a focus on the United States. Journal of Antibiotics, 2017; 70:520-526. https://doi.org/10.1038/ja.2017.30

8. Ventola CL. The antibiotic resistance crisis. Pharmacy and Therapeutics, 2015; 40(4):277-283.

9. Chandra H, Bishnoi P, Patni B, Mishra AP, and Nautiyal AR. Antimicrobial resistance and the alternative resources with special emphasis on plants-based antimicrobials. A review. Plants, 2017; 6 (16):1-11. https://doi.org/10.3390/plants6020016

10. Villarreal García Laura E. 2014. Potential antibacterial, actividad citotóxica y mutagénica de Krameria ramosissima, Larrea tridentata, Jatropha dioica y Leucophyllum frutescens. Universidad Autónoma de Nuevo León. pp. 68-71. http://eprints.uanl.mx/4000/1/1080253531

11. Chevallier A. The Encyclopedia of Medicinal plants. Atlas of the DK Publishing, Inc.; New York. 1996. p. 223.

12. Flora Medicinal Indígena de México I. Instituto Nacional Indigenista. México, 1994; p. 214, 284.

13. Argueta VA, Cano ALM y Rodarte ME. Atlas de las plantas de la medicina tradicional mexicana I. Instituto Nacional Indigenista. México, 1994; p. 466.

14. Alarcón AF, Vega AE, Almanza PJ, Velasco LR, Vázquez CL, and Román RR. Hypoglucemic Effect of Plantago major seeds in healthy and Alloxan-diabetic mice. Proceedings of the Western Pharmacology Society, 2006; 41:51-54.

15. Sarker DS, Nahar L, and Kumarasamy Y. Microtitre plate-based antibacterial assay incorporating Resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods, 2007; 42 (4):321-324. https://doi.org/10.1016/j.ymeth.2007.01.006

16. Neto CC, Owens CW, Langfield RD, Comeau AB, Onge J St, Vaisberg AJ, Hammond GB. Antibacterial activity of some Peruvian medicinal plants from the Callejón de Huaylas. Journal of Ethnophamacology, 2002; 79:133 - 138. https://doi.org/10.1016/S0378-8741(01)00398-1

17. Bussmann RW, Glenn A, Meyer K, Rothrock A, Townesmith A, et al. Antibacterial activity of medicinal plants of Northern Peru-Part II. Arnaldoa, 2009; 6 (1):93-103.

18. Genovese C, Di Àngeli F, Bellia F, Distefano A, Spampinato M, Attanasio F, Nicolosi D, Di Salvatore V, Tempera G, Lo Furno D, Mannino G, Milardo F, and Li Volti G. In vitro antibacterial, anti-Adhesive and anti-biofilm activities of Krameria lappacea (Dombery) Burdet & B.B Simpson root extract against Methicillin-resistant Staphylococcus aureus strains. Antibiotics, 2021; 10 (7):799. https://doi.org/10.3390/antibiotics10070799

19. Aguilar C, Ma F, Tapia AR, and Velasco LR. Evaluation of antibacterial activity of rhizome of Krameria prostrata Brandegee. XVII Reunión Internacional de Ciencias Médicas. 2017. León, Gto., México.

20. ESCOP Monographs. The Scientific Foundation for Herbal Medicinal Products. Ratanhiae radix. Rhatany Root, European Scientific Cooperative on Phytotherapy (ESCOP), 2017.

21. Achenback H, Utz W, Lozano B, Guajardo TEM, Moreno S. Lignans and neolignans from Krameria parvifolia. Phytochemistry, 1996: 43 (5):1093-1095. https://doi.org/10.1016/S0031-9422(96)00412-8

22. Kuklinski C. Farmacognosia. Ediciones Omega; Barcelona, España. 2003.

23. Baumgartner OL, Sosa S, Atanasov AG, Bodensieck A, Fakhrudin N, et al. Lignan derivates from Krameria lappacea roots inhibit acute inflammation in vivo and pro-inflammatory mediators in vitro. Journal of Natural Products, 2011; 74:1779-1786. https://doi.org/10.1021/np200343t

24. Ramírez-Cisneros, MA, Ríos-Gómez R, and Aguilar-Guadarrama AB. Cycloartanes from Krameria pauciflora and their in vitro PLA2, COX-1, and COX-2 enzyme inhibitory activities. Planta Medica, 2012; 78:942-1948. https://doi.org/10.1055/s-0032-1327882

25. Landrum JT. Carotenoids: physical, chemical and biological functions and properties. CRC Press, Taylor and Francis Group; Boca Raton, 2010. FL, USA.

26. Salih, NA, Arif ED, and Ali DJ. Antibacterial effect of nettle (Urtica dioica). AL-Qadisiya Journal of Veterinary Medical Science, 2014; 13(1):1-6. https://doi.org/10.29079/vol13iss1art270