Antimicrobial Potential of Naturally Occurring Bioactive Secondary Metabolites.

The use of traditional medicines of natural origin has been prevalent since ancient times globally as the plants produce a great diversity in their secondary metabolites. The naturally occurring bioactive constituents in food and other plant materials have shown widespread attention for their use as alternative medicine to prevent and cure microbial growth with the least toxic manifestations. The inclusion of these contents revealed their crucial role to improve the therapeutic efficacy of the classical drugs against various pathogenic microorganisms. Furthermore, several metabolites have also been explored in combination with antimicrobial agents to overcome the problems associated with drug resistance. This current review discusses the antimicrobial activities of secondary metabolites as well as their role in drug sensitivity against multiple-drug resistant pathogenic microbes. Copyright:

INTRODUCTION

The continuous expansion of multiple drug resistance in different microorganisms has made the strict barrier in combating infectious diseases.[12] The inappropriate use of antibiotics has been found as one of the factors for the development of drug resistance in pathogenic bacteria. However, the discovery of new antimicrobial drugs with novel mechanism could be vital, but the wide and constant increase of antimicrobial drug resistance, weaken such developments.[345] As evident from several reports, it has become a serious threat which needs global concern making the different strategies to overcome this problem. Moreover, the toxic manifestation of currently available antibiotics has sparked massive interest to investigate the antimicrobial potential of naturally occurring herbal products.[678] The use of herbal products and the development of effective combinations have opened several opportunities to drug designers.[9101112]

The plants and their products have been conventionally used in the treatment of several diseases since ancient times in various populations throughout human evolution and continued till the 20th century.[131415] However, the outcomes of allopathic medicines showed enormous consideration in the 20th century and the use of traditional medicines was replaced by it. As the allopathic antibiotics became popular, the misconceptions were also developed correspondingly in establishing it as miracle drugs in most of the developed countries. However, the repeated exposure of irrational and incomplete dose regimens of these antimicrobial drugs lost its potential. The studies suggested that the transformation of drug-resistant genes to other cells of microorganisms, play a crucial role in the development of resistance to the respective antibiotics. Subsequently, such transformations are occurred in the pathogens for other antibiotics make them multi-drug resistant (MDR) that are known as “superbugs.”[4161718] Nevertheless, the developing countries were continued to use these traditional therapeutic methods as the WHO reported that >80% population of these countries depend on naturally occurring herbal drugs due to its cost-effectiveness.[192021]

ANTIMICROBIAL POTENTIAL OF PHYTOCHEMICALS OR BIOACTIVE COMPOUNDS

Interestingly, there are several existing antibiotics, i.e., penicillin, cephalosporins, vancomycin, tetracycline, etc. also derived from natural sources.[2223] The development of advanced technologies such as high throughput screening methods, molecular docking has enabled the researchers to identify the molecules combating antibiotic resistance.[24] However, the approach of such molecules in clinical practice is limited as these molecules have not been shown the ability to penetrate the cell wall deviate mutational resistance and efflux pump.[2526] Therefore, widespread attention has been made among researchers to evaluate the antimicrobial effect of various products of plants’ origin. The high throughput screening of herbal products has replaced the traditional way as it utilized the current understandings of genomics, bioinformatics, and synthetic biology as well. The current tools related to drug discovery enable the drug designers in chemical modifications in the structure of certain bioactive secondary metabolites to make it more effective.[272829]

The herbal products show a broad range of biological activities depending on the occurrence of several secondary metabolites, i.e., phenolics, alkaloids, saponins, terpenoids, limonoids, polyacetylenes and secoiridoids, and so on. These secondary metabolites are the product of primary metabolism and key components of plants’ natural defense mechanisms. The drugs derived from these biologically active secondary metabolites are considered natural antibiotics as they are associated with the breakdown of the cell wall and cell membranes of microorganisms. As a result, cell death is occurred following the release of cellular contents, disruption of protein binding domain , and enzyme activation.[30313334]

POLYPHENOLS

Most of the plants contain different types of phenolic compounds, to protect them from microbial infections, ultraviolet radiations, and chemical stressors. The researchers have studied various polyphenols found fruits and vegetables from different origins against broad range of pathogens. The polyphenols are generally classified into flavonoids and nonflavonoids (phenolic acids).[3536] The extracts of Salvia officinalis have been shown as bactericidal and bacteriostatic effects due to the high content of phenolic acids and flavonoids.[37] Phytochemical analyses revealed that the extracts from Eugenia caryophyllata, Mentha piperita, Rosmarinus officinalis, and Prunus avium have been shown antimicrobial potential as they contain phenolic acids, flavonoids, and terpenoids as well. The presence of five phenolic compounds in the ethanolic extract of A. ampeloprasum as phenolic acid, cinnamic acid, p-coumaric acid, catechin, and sinapic acid showed great antimicrobial efficacy against Prunus aeruginosa and Micrococcus spp.[38] Pomegranate (Punica granatum) juice comprises several polyphenols as caffeic acid, gallic acid, and epigalactocatechin 3-gallate (EGCG) has been reported to inhibit the activity of different dental plaque microorganisms.[3940] The clove oil contains several phenolic compounds that has been shown to possess strong antifungal potential against opportunistic fungal pathogens such as Candida albicans, Cryptococcus neoformansi, and Aspergillus fumigatus.[41]

PHENOLIC ACIDS

The antimicrobial effects of phenolic acids have seen mainly against Gram-positive bacteria in comparison to Gram-negative. The hydrophobic structures of the outer membrane in Gram-negative bacteria guard them from various hydrophilic antibiotics including phenolic acids. However, the effects of phenolic acids have been observed in inhibiting the growth of some strains of Gram-negative bacteria.[424344] The methanolic extracts from the leaves Matricaria aureus, popularly known as golden chamomile has demonstrated significant growth inhibition against Bacillus subtilis, Streptococcus pyogenes, Staphylococcus aureus, and Klebsiella pneumoniae. The antifungal activity of golden chamomile extract was also noticed against several Colletotrichum gleosporoides, Aspergillus niger, and Aspergillus flavus.[45]

FLAVONOIDS

The flavonoids are occurred mainly in the fruits, vegetables, nuts, and seeds of edible plants. They are mainly subclassified into flavones, flavonols, flavanones, flavan-3-ol, isoflavone, and anthocyanidins. These flavonoids cover a wide range of secondary metabolites as luteolin, apigenin, diosmetin, chrysoeriol, tangeretin, sinensetin, gardenin, vitexin, baicalein, kaempferol, quercetin, galangin, datiscetin, morin, robinetin, isorhamnetin, tamarixetin, isoctytososide, quercetagetin, myricetin, hesperetin, taxifolin, eriodictyol, naringenin, catechin, epicatechin, genistein, daidzein, coumestrol, cyanidin, delphinidin, pelargonidin, peonidin, and so on.[4647] The antimicrobial activities of several flavonoids were investigated against some Gram-positive lactic acid bacteria of intestines, Gram-negative bacteria Escherichia coli CM 871 and Salmonella. The effect of myrecitin showed significant inhibition in the growth of Lactobacillus, while leutolin, pelargonidin, delphinidin as well as cyanidin-3-glucoside, were found to be effective against E. coli.[48] The bioactive constituent, isocytisoside isolated from the leaves of Aquilegia vulgaris has been shown to be effective against Gram-positive, Gram-negative and fungi as well.[49] The different fractions from the leave extract of Combretum erythrophyllum that contained apigenin, genkwanin, rhamnocitrin, kaempherol, quercetina, and rhamnazin were tested against Vibrio cholerae, Enterococcus faecalis, Micrococcus leuteu, and Shigella sonei. All the tested compounds showed significant growth inhibition against V. cholera and E. faecalis. However, rhamocitrin and quercetin also revealed their potential against M. luteus, S. sonei.[50] The plant Marrubium globosum has been used for the treatment of skin and urinary tract infections since ancient times. The methanolic extract of M. globosum contained flavonoids as rutin, naringenin demonstrated the MIC (8 mg/ml) against S. epidermidis and E. faecalis.[51] The high concentration of quercetin and kaempferol in the ethylene acetate extract of Argyreia speciosa showed strong potential against Micobacterium tuberculosis and Klebsiella pneumonie.[52] The kaempherol has been found effective against a broad range of pathogens such as Bacillus cereus, Listeria monocytogenesi and pseudomonas aeruginosa, M. luteus as well.[5354] The antimicrobial efficacy of flavone extracted from the fruits of Feijoa sellowiana has been reported against P. aeruginosa, P. mirabilis, Proteus vulgaris as well as Helicobacter pylori.[55] The study showed the antibacterial effect of morin and quercetin extracted from the fruit of psidium guajava commonly known as guava against Salmonella enteritidis, Bacillus cereus as well as numerous foodborne pathogenic bacteria.[5657] Thymoquinone is abundantly found in the seeds of Nigella sativa commonly known as black cumin has been shown potent antimicrobial agent against broad range of microrganisms.[5859]

NAPTHOQUINONES

Napthoquinones are found in plants, fungi comprise the one of largest groups of secondary metabolites that has been explored for their biological activities widely. Lapachol, and β-lapachone isolated from Tabebuia species are the naphthoquinones that have been reported to inhi bit the activity of C. albicans, Candida tropicalis and Cryptococcus neoformans.[60] The study showed that the broad-spectrum antimicrobial activity of two naphthoquinones, diospyrin, and isodiospyrin extracted from the root of Diospyros picatoria against several pathogenic bacteria.[61] Lapachol and anthraquinones-containing bark extracts of T. impetiginosa showed strong efficacy against H. pylori.[62] The antibacterial potential of 1,4-naphthoquinone sulfides has been shown as 7.6 and 31.3 mg/ml MIC recorded against S. aureus (Gram + ve) and E. coli (Gram–ve). The antifungal activity of 1,4-naphthoquinone sulfides showed decreased (23.4 mg/ml) in comparison to Amphotericin B (31.3 mg/ml) against C. albicans.[63] The efficacy of lawsone (2-hydroxy-1,4-naphthoquinone)-based compound was found more significant in comparison to conventional antibiotics against methicillin-resistant S. aureus (MRSA) in vitro as well as in vivo.[64]

ALKALOIDS

The studies suggested that several types of alkaloids, i.e., piperine, piperidine, quinoline, indole, pyrrolidine, quinazoline, isoquinoline, glyoxaline, lupinane, tropan, phenanthridine, imidazoline, alkaloidal amines, and terpenoid are naturally occurred in the plants.[6566] The indol type of alkaloid fractions extracted from Strychnos ptatorum showed growth-inhibiting potential against P. vulgaris, S. aureus, Salmonella typhimurium, V. cholera, M. tuberculosis A. niger and C. albicans as well.[676869] Noticeably, each class of alkaloids has been shown different modes of action against the pathogens. The indolizine class of alkaloids such as pergularinine and tylophorinidine, inhibit the nucleic acid synthesis through the inhibition of dihydrofolate reductase.[70] The effect of bisindole alkaloids such as deoxytopsentin and dibromodeoxytopsentin has been shown against MRSA due to its inherent targeting and inhibiting pyruvate kinase.[71] The study revealed that the natural occurring alkaloids, berberine, hydrastine, and candine extracted from Hydrastis candensis inhibited the bacteria through the Quorum quenching effect (QQE. The QQE is applied to control the disease by inhibiting the expression pathogenic genes instead of killing the bacteria.[72] The aqueous extract of Tinospora cordifoila contains several secondary metabolites including alkaloids have been shown to regenerate the weakened immune system and eradicate the systemic candidiasis in experimental animals.[73] The administration of AETC increased the survival and reduced bacterial load in the mice infected with S. typhimurium.[74]

ORGANOSULPHUR COMPOUNDS

The organosulphur thiosulfinates such as diallyl sufide, diallyl disulfide (DADS), diallyl trisulfide are main secondary metabolites occurred naturally in garlic, have been shown potent inhibitor of various pathogenic bacteria and yeast.[757677] DADS has been reported most active constituent, extracted from garlic in the inhibition of microbial growth. Other organosulfur compounds isolated from cabbage, i.e., Dimethyl trisulfide, methyl methanethiosulfinate, and methyl methanethiosulfonate have also showed significant growth inhibition against different bacterial and fungal species.[7879] The broad range of sulfur-containing metabolites is found in the large number of edible plants in the form of glucosinolates.[80] The brown mustard contains gluconapin, while white mustard comprises p-hydroxybenzyl glucosinolate. The high content of allyl, methylthiopropyl, and 2-hydroxy 3-butenyl was determined in horseradish, cabbage, and rapeseed, respectively.[8182] The antimicrobial activities of glucosinolates were noticed only in their hydrolytic products. As evident from several studies, isothiocyanates; sulforaphane, and benzyl isothiocyanate have been shown potent inhibitor of human pathogenic bacteria. 4-methyl sulfinyl butylisothiocyanate showed broad-spectrum antimicrobial potential.[8384]

SYNERGISTIC EFFECT OF SECONDARY METABOLITES

The synergistic interaction of plant extracts or their secondary metabolites with the existing antibiotics has become one of the effective tools for the management of MDR. Several studies showed that the coadministration of plant-derived extracts or bioactive secondary metabolites with the conventional antimicrobial drugs increased the potential of antibiotics.[85] Several studies suggested the different modes of mechanism in the synergism between the phytochemical and antibiotics against different pathogenic microbes including the MDR.[8687]

SYNERGISM AGAINST PATHOGENIC BACTERIA

The resistance to antibiotics has been observed in different types of bacteria by producing alternative target molecules as an enzyme or receptors to survive themselves. For example, the production of PB2a in MRSA, along with penicillin-binding proteins (PBPs), decreased the affinity of β-lactam, penicillin, and cephalosporin. Thereby, the cells continue to synthesize peptidoglycan and remain alive, suggested making the alternative strategy to inhibit such other active sites in the resistance.[8889] The coadministration of the crude extract of uva-ursi reduced the MIC of oxacillin or cefmitazole against MRCA. The corilagin was identified and isolated as a main bioactive secondary metabolite in uva-ursi, explored in combination with different β-lactams against MRSA that showed MIC up to thousands fold. However, it did not show such reduction in MIC against Methicillin-susceptible S. aureus strains. It showed that the extract of uva-ursi or corilagin targeted PBP2a only instead of PBPs.[9091] Similarly, several phenolic compounds, i.e., tiliroside, pinoresinol, magnatriol B, momorcharaside B, etc. have been shown for their synergistic effect with β-lactams by targeting other sites (PBP2a or PBP4) against MRSA.[92]

As β-lactamases catalyze the β-lactam antibiotics, they are recognized as one of the important factors for bacterial resistance. The determination of different secondary metabolites for their drug-metabolizing enzyme inhibiting activity can make a significant impact in the development of the effective combination of phytochemicals and antibiotics against drug resistance for both Gram-positive as well as Gram-negative bacterial species. The clavulanic acid, a potential β-lactamase inhibitor in combination with various β-lactam antibiotics has been used broadly to improve the therapeutic index of the drugs.[9394] The bioactive constituents isolated from green tea have been reported to inhibit β-lactamase and restored the drug antibiotic sensitivity in it.[95] The use of several flavonoids such as baicalin and silibinin also has also showed the synergism against these resistant bacterial strains reducing the MIC of the antibiotics.[9697]

The development of MDR in the bacterial strains prevents the entry of the antibiotic inside the cells due to lack of specific D2 porins, or the expression of efflux pumps, i.e., MexAB-OprM, MexCD-OprJ, etc.[98,[99100] Several studies suggested the role of different secondary metabolites as efflux pump inhibitors (EPI). The steroidal alkaloid, found in several plant species of family Apocynaceae, known as conessine, has been shown to inhibit the expression of MexAB-OprM and reversed the susceptibility to the respective drugs in P. aeruginosa.[101] It has been also noted the effect of secondary metabolites in the inhibition of efflux pump, only in combination with the respective antibiotics, but not alone. The use of monoterpene, especially (-)-a-Pinene, has been shown to increase the sensitivity of the drugs several folds against different strains of Campylobacter jejuni, but no effect was measured when it was examined alone.[102] The combination of active constituents (berberine and 5’-methoxyhydnocarpin) of the barberry plant blocked the efflux pump effect leading to increased sensitivity to the antibiotic.[103] The exposure of other phytochemicals such as kaempferol-3-O-α-L-(2,4-bis-E-p-coumaroyl) rhamnoside, indirubin, capsaicin, reserpine, carnosol, and carnosic acid have reported to inhibit the efflux transporter NorA of S. aureus.[104105106107108] Several studies also suggested the disintegration of the oily outer membrane of Gram-negative bacteria by and caffeic acid to increase permeability and allow the drug at high concentration inside the cells.[98]

SYNERGISM AGAINST FUNGAL INFECTIONS

Phytochemicals have been shown to play an important role against the resistance developed by the upregulation of efflux pump proteins and biofilm formation. The availability of a limited range of antifungal drugs encouraged the researchers to explore different natural bioactive constituents to overcome the resistance.[109110] The synergistic effect of garlic extracts was reported to increase the therapeutic efficacy against C. albicans.[111112] The MIC of fluconazole was found to be decreased by two-fold by adding the phenolic compound punicalagin against C. albicans.[113] The use of essential oil from Pelargonium graveolens has been shown to augment the potential of etoconazole against Trichophyton spp.[114] The synergistic effect of caffeic acid and its derivatives have also been evaluated to potentiate the effect of fluconazole and nystatin in various combinations against C. albicans.[115] The increased sensitivity of fluconazole was noticed against C. albicans in diabetic mice when the animals were exposed to thymoquinone along with antifungal agents.[116] As suggested from several studies berberine which is an active constituent of Berberis vulgaris, increase the sensitivity of fluconazole against drug-resistant C. albicans. Furthermore, it has been shown to inhibit the expression of fluphenazine activated drug resistance CDR1 gene.[117118119] Nyasol is one of the main constituents of Chinese herb, Anemarrhena asphodeloides has been shown for its antifungal as well as the synergistic effect with azoles.[120121] Curcumin is active constituent of turmeric that has been studied broadly against various diseases. It has been found to augment the generation of ROS and apoptosis against C. albicans when exposed in combination with azoles and polyenes.[122] Eugenol is the main constituent of essential oils from Syzygium aromaticum (clove) has been suggested from several studies to increase the sensitivity of fluconazole against drug-resistant fungal pathogens.[123]

CONCLUSIONS

Over the years, the researchers have identified hundreds of secondary metabolites and investigated their activities against different types of pathogens. The possible mode of microbial cell death or growth inhibition by several phytochemicals has been also established. Several studies revealed the potential of different secondary metabolites against microbial infections in vivo without affecting the beneficial bacteria in the gastrointestinal tracts. The phytochemicals were found effective in combination among them or with conventional antibiotics against broad range of microorganisms. Besides, it was also noticed that secondary metabolites can act synergistically and augment the effect of less efficient antibiotics against various pathogens including MDRs. All the possible consequences of secondary metabolites-drug interactions are suggested to be established as not so attention has been paid on this aspect. Finally, all the studies suggest the use of secondary metabolites as alternative medicine or in combination with conventional antimicrobial agents may play an important role in the development of future drugs of the 21st century.

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Conflicts of interest

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