Synergism between natural products and antibiotics against infectious diseases.

Abstract

Antibiotics have been effective in treating infectious diseases, but resistance to these drugs has led to the emergence of new and the reemergence of old infectious diseases, One strategy employed to overcome these resistance mechanisms is the use of combination of drugs, such as [beta]-lactams together with [beta]-lactamase inhibitors. Several plant extracts have exhibited synergistic activity against microorganisms. This review describes in detail, the observed synergy and mechanism of action between natural products including flavonoids and essential oils and synthetic drugs in effectively combating bacterial, fungal and mycobacterial infections. The mode of action of combination differs significantly than that of the same drugs acting individually; hence isolating a single component may lose its importance thereby simplifying the task of pharma industries.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Infectious diseases; Antibiotic resistance; Natural products; Resistance modifying agents; Synergy

Introduction

Infectious diseases are caused by bacteria, viruses, parasites and fungi, and it is due to a complex interaction between the pathogen, host and the environment. The discovery of antibiotics had eradicated the infections that once ravaged the humankind. But their indiscriminate use has led to the development of multidrug-resistant pathogens. Around 90-95% of Staphylococcus aureus strains worldwide are resistant to penicillin (Casal et al., 2005) and in most of the Asian countries 70-80% of the same strains are methicillin resistant (Chambers, 2001). There are considerable reports on the progress of resistance to the last line of antibiotic defense, which has led to the search for reliable methods to control vancomycin-resistant Enter-ococci (VRE) and S. aureus (VRSA), and methicillin-resistant S. aureus (MRSA). In addition, the synergy between tuberculosis and the AIDS epidemic, along with the surge of multidrug-resistant isolates of Mycobacterium tuberculosis, has reaffirmed it as a primary health threat. Multidrug-resistant TB (MDRTB) is associated with high death rates (50-80%), spanning within a relatively short period of time (4-16 weeks) from diagnosis to death (WHO, 2004). In developing countries, MDRTB has increased in incidence and it interferes with TB control programs.

Plant-derived antibacterials are always a source of novel therapeutics. A quick look at the way nature, especially plants, are tackling the issue of infection will provide a deeper understanding of the methodology, which needs to be adopted for the design and development of novel highly effective antiinfectious agents in general, and antimycobacterials in particular. The scarcity of infective diseases in wild plants is in itself an indication of the successful defense mechanisms developed by them. Plants are known to produce an enormous variety of small-molecule (MW < 500) antibiotics--generally classified as 'phytoalexins'. Their structural space is diverse having terpenoids, glycosteroids, flavonoids and polyphenols. Be that as it may, it is interesting to note that most of these small molecules have weak antibiotic activity--several orders of magnitudes less than that of common antibiotics produced by bacteria and fungi. In spite of the fact that plant-derived antibacterials are less potent, plants fight infections successfully. Hence, it becomes apparent that plants adopt a different paradigm--"synergy"--to combat infections. A case in study to reiterate this view is the observation on the combined action of berberine and 5'-methoxyhydnocarpin, both of which are produced by berberry plants. Berberine, a hydrophobic alkaloid that intercalates into DNA, is ineffective as an antibacterial because it is readily extruded by pathogen--encoded multidrug resistance pumps (MDRs). Hence, the plant produces 5'-methoxyhydnocarpin that blocks the MDR pump (Stermitz et al., 2000). This combination is a potent antibacterial agent (Lewis and Ausubel, 2006). Using this cue, Ball et al. (2006) reported that covalently linking berberine to IN[F.sub.55], an inhibitor of MDR, results in a highly effective antibiotic that readily accumulates in bacteria.

This paper introduces and provides examples of synergistic interactions of the secondary metabolites of plants with antibiotics in the treatment of infectious diseases. The understanding of the molecular mechanisms of synergy would pave a new strategy for the treatment of infectious diseases, overcome drug-resistant pathogens, and decrease the use of antibiotics and hence the side effects created by them.

Synergy towards bacterial infection

The development of antibiotic resistance can be natural (intrinsic) or acquired and this can be transmitted within same or different species of bacteria. Natural resistance is achieved by spontaneous gene mutation and the acquired resistance is through the transfer of DNA fragments like transposons from one bacterium to another. Bacteria gains antibiotic resistance due to three reasons namely: (i) modification of active site of the target resulting in reduction in the efficiency of binding of the drug, (ii) direct destruction or modification of the antibiotic by enzymes produced by the organism or, (iii) efflux of antibiotic from the cell (Sheldon, 2005). One strategy employed to overcome these resistance mechanisms is the use of combination of drugs. Inhibitors of [beta]-lactamases have been long known and they are administered with antibiotics as co-drugs. The most successful strategy that has been adopted to overcome the resistance to penicillinase is by administering clavulinic acid, with the drugs sulbactam and tazobactam (Lee et al., 2003b). But the frequent use of clavulanate has led to the emergence of resistant bacterial strains (Blasquez et al., 1993; Enright et al., 2002). The appearance of extended spectrum [beta]-lactamase and resistance against IMP-1 (a new [beta]-lactam), cephalosporins and carbapenems have further necessitated the need for developing new [beta]-lactamase inhibitors (Chaibi et al., 1999).

[FIGURE 1 OMITTED]

The secondary metabolites from plant are good sources for combination therapy. As shown in Fig. 1, there are a wide range of phytochemicals which act as multidrug resistance modifiers depicted and their mechanism of action is discussed in the following sections.

Receptor or active site modification

For selective antimicrobial action the target site plays a vital role. Introduction of mutations in the target site alters it, leading to a reduction in the activity of the drug towards the microbe. Two examples of receptor (target) modification are (a) mutations in RNA polymerase and DNA gyrase, rendering rifamycins and quinolones inactive (Heep et al., 2000; Willmott and Maxwell, 1993), and (b) modification in the structural confirmation of penicillin-binding proteins (PBPs) resulting in the development of penicillin resistance. The most important example of a target change is the production of PBP2a, an altered transpeptidase.

[beta]-Lactam antibiotics (BLA) are highly specific inhibitors of the metabolism of peptidoglycan and they target the membrane bound D,D-peptidase domain of the PBPs (Ghysen, 1994). These peptidases cross link the bacterial peptidoglycan cell wall which maintain the integrity of the latter (Berger-Bachi and Rohrer, 2002). S. aureus acquires resistance to all penicillins and cephalosporins with the acquisition of the gene mecA (Enright et al., 2002), which is carried on a large genetic element called the staphylococcal cassette chromosome mec (SCC mec). This is acquired by a parasexual horizontal transfer from a coagulase-negative Staphylococcus sp. (Ito et al., 2003). The house keeping PBPs are BLA sensitive whereas the MecA (PBP 2a) have reduced affinity to BLA (Lu et al., 1999). A number of BLAs, including modified cephalosporins (Vouillamoz et al., 2004), carbapenems (Kurazono et al., 2004) and trinem (Ferrari et al., 2003) have been designed with enhanced activity against PBP2a.

Another approach to overcome resistance is to include inhibitors of the PBP2a in the treatment strategy. A number of reports are available listing the synergistic interactions of BLA with natural compounds to overcome resistant microorganisms. The later includes catechins (Camellia sinesis) (Takahashi et al., 1995), EGCg (Epigallocatechin gallate) from green tea (Suresh et al., 1997), tellimagrandin I and rugosin B from rose red (Rosa canina) (Shiota et al., 2000), baicalin from Scutellaria amoena (Liu et al., 2000) and corilagin from Arctostaphylos uva-ursi (Shimizu et al., 2001). Corilagin, a polyphenol from Arctostaphylos uva-ursi is found to markedly reduce the Minimum Inhibitory Concentration (MIC) of [beta]-lactams in MRSA. Shimizu et al. (2001) suggest that there are two possibilities regarding the mechanism of action of corilagin, namely inhibition of PBP2a activity or inhibition of its production. They later reported that the PBP2a of MRSA cells grown in the presence of corilagin or tellimagrandin I lost its ability to bind to BOCILLIN FL, a fluorescent-labeled benzylpenicillin (Shiota et al., 2004).

Studies through reverse transcription-PCR and a semiquantitative PBP2a latex agglutination assays indicted that, EGCg did not suppress either the mRNA expression of PBP2a or its production. But the synergy between EGCg and BLA was achieved since both directly or indirectly attacked the same target site namely, peptidoglycan present on the cell wall (Yam et al., 1998; Zhao et al., 2001). EGCg was found to synergistically enhance the activity of carbapenems against MRSA but the mechanism of action has not been studied (Hu et al., 2002). Nicolson et al. (1999) have shown that diterpene derivative 416 potentiated the activity of methicillin by significantly reducing the expression of PBP2a.

There is a wide list of phytochemicals which act as inhibitors and a few of them are glycosylated flavones suppressing topoisomerase IV activity (Bernard et al., 1997), myricetin inhibiting DnaB helicase (Griep et al., 2007), allicin inhibiting RNA synthesis (Feldberg et al., 1988) and compounds from the plant Polygonum cuspidatum inhibiting bacterial DNA primase (Hegde et al., 2004). These phytochemicals when used in combination with other classes of antibiotics have the potential to either inhibit the modified targets or exhibit a synergy by blocking one or more of the other targets in the metabolic pathway. Table 1 lists the synergy observed between natural products and commercial antibiotics against bacteria.

Table 1. Synergism between natural products and antibiotics
against bacterial infection

Natural product        Antibiotics               Microorganisms

Carnosic acid      Tetracycline                 Tet (K) possessing
                                                strains

Carnosol           Erythromycin                 Msr (A) MSSA [beta]-
Epigallocatechin-  Ampicillin/sulbactam         lactamase producing
gallate (EGCg)                                  S. aureus

EGCg               Penicillin                   Penicillinase producing
                                                S. aureus

                   Ampicillin

EGCg               Carbapenems                  MRSA

EGCg               [beta]-Lactam                MSSA, MRSA

EGCg               Tetracycline                 S. aureus with Tet (K)
                                                MDR pump

Tea catechin       Oxacillin                    MRSA

Totatrol           Methicillin                  MSSA, MRSA

Berberine          5'-Mehoxyhydnocarpin         Nor (A) mutant
(Berberis
plant)

Green tea extract  Levofloxacin                 Escherichia coli 0157
                                                in gnotobiotic mouse
                                                model

Craneberry juice   -                            Helicobacter pylori
extact
Blueberry,
Grape seed and
oregano extract

Oregano and        Lactic acid                  Vibrio

cranberry extract                               parahemolyticus

Isoflavone         Mupirocin                    MRSA
Bidwillon B from
Erythrina
variegata

[alpha]-Mangostin  Vancomycin                   MRSA and Vancomycin
                                                enterococci

                   Gentamycin

Aqueous crude      Tetracycline                 Streptococcus sanguis,
khat extracts                                   Fusobacterium nucleatum

Corilagin from     [beta]-Lactams such as       MRSA

Arctostaphylos     oxacillin. cefmetazole
uva-ursi

Baicalin           [beta]-Lactam antibiotics    MRSA

Tellimagrandin I   [beta]-Lactams               MRSA
from rose red
tree,

Rugosin B from     [beta]-Lactams               MRSA
rose red tree

Diterpenes from    Tetracycline                 S. aureus possessing
Lycopus europaeus                               Tet (K), Msr (A) MDR
                                                pumps

                   Erythromycin

A penta-           Erythromycin                 S. aureus possessing Tet
substituted                                     (K),

pyridine from                                   Nor (A) MDR pumps
Jatropha
elliptica
                   Ciprofloxacin

Pomegranate        chloramphenicol,             MRSA
extract            gentamicin, ampicillin,
                   tetrcycline, and oxacillin

                                                MSSA

Myricetin          Amoxicillin/clavulanate,     Extended-spectrum
                   amplicillin/sulbactam and    -lactmases
                   cefoxitin                    (ESBL) producing

Isopimaric acid    Reserpine                    MRSA
from Pinus nigra

Totarol,           Isonicotinic acid hydrazide  Mycobacterium
ferulenol          (INH)                        intracellulare,
(from Ferula                                    M. smegmatis,
communis) and                                   M. xenopei and
plumbagin (from                                 M. chelonei
Plumbago
zeylanica)

Erybraedin A or    Vancomycin                   Vacomycin-resistant
eryzerin C                                      enterococci (VRE) and
isolated                                        MRSA S. mutans, non-
from the roots of                               susceptible E. coli
Erythrina                                       and C. albicans
zeyheri,
Butylated
hydroxyanisole,
(BHA) green tea

Sopheoraflavanone  Vancomycin                   MRSA
G                  hydrochloride, fosfomycin,
                   methicillin, cefozonam,
                   gentamicin, minocycline
                   and levofloxacin

Essential oil-1,   Antibiotics                  Bacterial species
8
cineol, linalool,
alpha-terpineol
and terpinen-4-o1
from Melaeuca
leucodendron and
oil from Ocimum
gratissimum

Novoimanin from    Ampicillin, kanamycin,       Staphlococcus
Hypericum          fusidic acie and rifocin     aureus 209
perforatum L.


Natural product       Mechanism of action        References

Carnosic acid        Inhibit the MDR pumps,       Oluwatuyi et
                     Tet (K) and Msr (A)          al. (2004)

Carnosol

Epigallocatechin-    Inhibits [beta]-lactamase    Hu et al. (2001)

gallate (EGCg)

EGCg                 Inhibits penicillinase       Zhao et al. (2001)

EGCg                 -                            Hu et al. (2002)

EGCg                 EGCg directly binds t o the  Yoshida et al. (1990)
                     peptidoglycan and inhibits
                     cell wall

EGCg                 Blocks MDR efflux pumps      Roccaro et al. (2004)

Tea catechin         -                            Takahashi et al.
                                                  (1995)

Totatrol             PBP 2a production            Pao et al. (1998)

Berberine (Berberis  Inhibits Nor (A) MDR         Smith et al. (2005)

plant)               pump

Green tea extract    -                            Isogai et al. (2001)

Craneberry juice     -                            Vattem et al.
extract Blueberry,                                (2005)
Grape seed and
oregano extract

Oregano and          -                            Lin et al. (2005)
cranberry extract

Isoflavone           Bidwillon B and mupirocin    Sato et al. (2004)
Bidwillon B from     inhibited the
Erythrina variegata  incorporation
                     of thymidine, uridine,
                     glucose and isoleucine

[alpha]-Mangostin    -                            Sankagami et al.
                                                  (2005)

Aqueous crude        -                            Al-hebshi et al.
khat extracts                                     (2006)

Corilagin from       Inhibits PBP2a production    Shimizu et al. (2001)
Arctostaphylos       or activity
uva-ursi

Baicalin             Inhibits [beta]-lactamase    Liu et al. (2000)

Tellimagrandin I     -                            Shiota et al. (2000)
from rose red tree,

Rugosin B from       -                            Shiota et al. (2000)
rose red tree

Diterpenes from      Blocks MDR pumps             Gibbons et al. (2003)
Lycopus europaeus

A penta-             Blocks MDR pumps             Marquez et al' (2005)
substituted
pyridine from
Jatropha elliptica

Pomegranate          Blocks Nor (A) pump          Braga et al. (2005)
extract

Myricetin            -                            Lin et al. (2005)

Isopimaric acid      Blocks Nor (A) pump          Simonetti et al.
from Pinus nigra                                  (2004)

Totarol, ferulenol   -                            Mossa et al. (2004)
(from Ferula
communis) and
plumbagin (from
Plumbago
zeylanica)

Erybraedin A or      -                            Sato et al. (2004)
eryzerin C isolated
from the roots of
Erythrina zeyheri,

Butylated            -                            Shiota et al. (2004)
hydroxyanisole,
(BHA) green tea

Sopheoraflavanone    -                            Sakagami et al. (1998)
G

Essential oil-1, 8   -                            Jedlickova et al.
cineol, linalool,                                 (1992)
alpha-terpineol
and terpinen-4-o1
from Melaeuca
leucodendron and
oil from Ocimum
gratissimum

Novoimanin from      -                            Avenirova et al.
Hypericum                                         (1975)
perforatum L.

Enzymatic degradation and modification of the drug

Bacterial cells spend a considerable amount of energy to resist antibiotics. One way the cells achieve active drug resistance is by the synthesis of enzymes that selectively target and destroy or modify the antibiotics. The various enzymatic strategies that lead to antibiotic inactivation are through hydrolysis, group transfer or redox mechanisms (Wright, 2005). Hydrolytically susceptible chemical bonds (such as ester or amide bonds) are cleaved by enzymes that are expressed by the resistant organisms. The modification of the active group in the drug through acylation, phosphorylation, glycosylation, nucleotidylation or ribosylation by the organism could make the former innocuous. Redox mechanism involves the oxidation-reduction of the antibiotics leading to the information of inactive compound (Wright, 2005).

[beta]-Lactamases are one such family of enzymes that cleave the [beta]-lactam ring of cephalosporins and penicillins. They act through the serine residue in the active site of the enzyme or through the activation of the Z[n.sup.2]+center (Bush, 1998, 2002). inhibitors of [beta]-lactamases have long been known. The combination of ampicillin and sulbactam inhibits [beta]-lactamase and increases the spectrum of activity of the former. Zhao et al. (2002) have confirmed that EGCg inhibits the penicillinase produced by S. aureus thereby restoring the activity of penicillin. It acts in a dose-dependent manner, with 50% inhibition at a concentration of 10[micro]g/ml. The combination of ampicillin and sulbactam is effective but is not powerful enough against MRSA and strains producing [beta]-lactamases. When they are further combined with EGCg, the MI[C.sub.90] of this combination is reduced to 4mg/ml from an initial value of 16mg/ml (Hu et al., 2001). The potent synergy between these concoctions could possibly have clinical use.

Reduced accumulation of the antibiotic within the bacterial cell

Reduced accumulation of the antibiotic inside the microorganism could be because of two reasons namely decreased permeability of the drug through the outer membrane of the cell or, the efflux of the accumulated drug out of the cell.

Decreased outer membrane permeability

Cells of Gram-negative bacteria are surrounded by an additional membrane (outer membrane, OM), which provide them with a hydrophilic surface and functions as a permeability barrier for many external hydrophobic agents including detergents, hydrophobic dyes and antibiotics (Helander et al., 1997a; Vaara, 1992, 1999; Nikaido and Vaara, 1985). This barrier is due to the presence of lipopolysaccharide (LPS) molecules in the outer leaflet (Nikaido, 2003; Nikaido and Vaara, 1985), which makes up to 75% of the total membrane surface and forms specific contacts with integral outer membrane proteins (Omp), such as porins (Alexander and Rietschel, 2001; Bos and Tommassen, 2004). Bacterial lipoproteins anchor the OM to the periplasmic peptidoglycan layer (Brade et al., 1999). Divalent cations are tightly associated with the anionic membrane-proximal regions of the LPS molecules, strengthening the structure (Vaara, 1992). Some Gram-negative bacteria are known to contain glycosphingolipids instead of LPS in their OM (Kawahara et al., 1991).

Bivalent cations contribute to the stability of the OM by creating electrostatic interactions between the proteins and LPS (Leive, 1965; Vaara, 1981, 1999). EDTA is a chelator which sequesters these ions. Treatment with EDTA releases a large proportion of LPS from the OM, exposing the phosholipids and creating a hydrophobic pathway (Leive, 1965). EDTA has been reported to potentiate the activity of cell wall degrading agents including lysozyme, nisin and biocides (Leive, 1965; Vaara, 1981; Walsh et al., 2003a, b). In addition, there are a wide range of permeabilizers such as polycationic polymyxin B nonapeptide, which interact with and disorganize the anionic LPS thereby sensitizing the bacteria to hydrophobic antibiotics (Vaara and Vaara, 1983a, b). Essential oils such as thymol and carvacrol as membrane permeabilizers (Fig. 1) have been studied by Helander and co-workers (Helander et al., 1998). Magnesium chloride disrupts the activity of EDTA and polyethylenimine, but it has no effect on the activity of carvacrol or thymol. This indicates that essential oils neither chelate nor intercalate with LPS by replacing the divalent cations which stabilize the OM (Helander et al., 1997a; Vaara, 1992).

Active efflux

The development of multidrug resistance pumps (MDRs) is one of the defense mechanisms employed by bacteria against the accumulation of antimicrobial drugs inside the cell. These efflux pumps either use ATP hydrolysis or ion gradient to expel the antibiotics. They are grouped into five major classes namely, the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily (Veen and Konings, 1998; Veen et al., 1996), the major facilitator superfamily (MFS) (Pao et al., 1998), the small multidrug resistance family (SMR) (Paulsen et al., 1996), the resistance-nodualtion-cell division RND superfamily (Saier et al., 1994) and the multidrug and toxic compound extrusion (MATE) family (Brown et al., 1999). Of these the RND, SMR and MATE classes are unique to prokaryotes (Lynch, 2006).

The efflux pumps of S. aureus Qac A (MFS family), Smr (SMR family) and Nor A (MFS family) have been well characterized. The Nor A efflux pump is responsible for fluoroquinolone resistance (Yoshida et al., 1990); Qac A is responsible for acriflavine and ethidium bromide resistance (Littlejohn et al., 1992) and Tet (K) and Msr (A) transporters are specific to tetracycline and macrolide efflux (Renau et al., 1999). Renau et al. (1999) have developed the first broad-spectrum RND pump-inhibitor, M[C.sub.-207,110] (phenylalanyl-arginyl-[beta]-naphthy-lamide), which potentiates the activity of levofloxacin, particularly the RND pumps against wild-type P. aeruginosa. Although these compounds are not effective antimicrobial agents by themselves, they reverse the resistance by blocking the efflux pumps.

Secondary metabolites of plants have shown to possess considerable activity against Gram-positive bacteria but not against Gram-negative species or yeast. In Gram-negative species, the outer membrane is a fairly effective barrier for amphiphatic compounds (Lewis and Lomovskaya, 2001). A set of multidrug resistance pumps (MDRs) extrude amphiphatic toxins across the outer membrane (Lewis, 2001; Nikaido, 1999). Tegos et al. (2002) have shown that MDR inhibitors M[C.sub.207,110] and IN[F.sub.271] dramatically increase the effectiveness of a set of 11 plant antimicrobials (e.g. Rhein, resveratrol, gossypol, berberine) against Gram-negative bacteria. By contrast, certain plant-derived natural products can modulate MDR. For example carnosic acid (from Rosmarinus officinalis) (Oluwatuyi et al., 2004), and a penta substituted pyridine (from Jatropha elliptica) (Marquez et al., 2005), act as inhibitor of the Nor A efflux pump and restore the level of intracellular drug concentration.

Two isopirmarane diterpenes from the Lycopus europaeus enhance the activities of tetracycline and erythromycin against two strains of S. aureus. Otherwise these strains are highly resistant to these antibiotics due to the presence of multidrug efflux pumps, Tet (K) and Msr (A) (Gibbons et al., 2003). EGCg increases the accumulation of tetracycline in S. aureus strains by inhibiting the Tet (K) and Tet (B) efflux pumps (Roccaro et al., 2004). EGCg also enhances the activity of norfloxacin against a Nor A harboring S. aureus strain (Gibbons et al., 2004). Isoflavones isolated from Lupinus argenteus act in synergy with norfloxacin against a mutant of S. aureus by inhibiting the MDR pump. Reserpine, a plant alkaloid potentiates the activity of fluoroquinolones (Schmitz et al., 1998) and tetracycline against multidrug-resistant S. aureus strain (Gibbons and Udo, 2000). Reserpine has been shown to inhibit LmrA, the MDR ABC efflux system of L. lactis (Marquez et al., 2005), but unfortunately bacterial resistance to this natural product has been observed (Ahmed et al., 1993). The calcium channel antagonist verapamil, another known inhibitor of P-gp, also inhibits several bacterial ABC efflux pumps, including LmrA (Lee et al., 2003a; Pasca et al., 2004; Choudhuri et al., 2002).The efflux pump inhibitors from natural sources discussed so far can be co-administered with the antibiotic to decrease the degree of resistance of the bacteria to the drugs, reverse the acquired resistance of the microorganism or reduce the emergence of resistant bacterial strains (Marquez et al. 2005).

Synergy and MDRTB therapy

Tuberculosis has established itself as a primary health threat. Few new agents are in development today for treating TB, and none has been designed specifically to shorten the treatment regimen and provide the break-through in therapy that is sorely needed if the epidemic is to be brought under control. Drug design targeting the latency stage and synergistic interaction between the various drug candidates might prove to be good alternatives.

Antimycobacterial treatment has always been a combination therapy. Today's TB treatment, which dates back to 1970s, is long and burdensome, requiring at least 6 months of multidrug chemotherapy. Novel targets are being identified alongside developing better drugs for known targets. Synergistic interaction between these drug like molecules is also gaining sufficient attention from the researchers (Chen et al., 2006; Vinogradova et al., 1999). Combination studies with natural products from plants and synthetic drugs are limited to few reports. Totarol ferulenol and plumbagin were observed to increase the potency of isonicotinic acid hydrazide by fourfold against Mycobacterium sp. (Mossa et al., 2004). A napthoquinone 7-methyljuglone, isolated from the roots of Euclea natalensis in combination with isoniazid or rifampicin resulted in a four-to sixfold reduction in the MIC of the synthetic drugs (Bapela et al., 2006). An aqueous extract from Cuminum cyminum seeds produced a 35% enhancement of rifampicin levels in rat plasma. This activity was due to a flavonoid glycoside, 3', 5-dihydroxyflavone-7-0-[beta]-D-galacturonide 4'-0-[beta]-D-glucopyranoside, found in the natural product. The altered bioavailability profile of rifampicin could be attributed to the permeation enhancing effect of this glycoside (Sachin et al., 2007).

Antifungal agents and synergism

Fungi have higher number of chromosomes and complex nuclear membrane, cell organelles and cell wall composition. Since the last three decades, the rate of death every year due to fungal infections has risen significantly. With the increased use of antifungal agents there is an increase in the number and variety of fungal strains resistant to these drugs. Also the present antifungal therapeutics is often toxic. Alternative therapy needs to be developed to suppress the emergence of antifungal resistance. This can be achieved by the use of combinations of existing agents or the development of new, safer and effective agents primarily from plant sources which can exhibit synergy with drugs. Table 2 lists the reported synergy observed between natural products and drugs towards fungal species.

Table 2. Combination of natural products and synthetic
drugs to combat fungal infection

Nautral products            Synthetic     Fungal species     References
                             drugs

Allium sativum             Ketaconazole  T. rubrum,           Pyun and
                                         T. Erinacei and      Shin
                                         T. soudanense        (2006)

Essential oil fraction of  Ketaconazole  Aspergillus          Shiota et
P. graveolens and its                    niger and            al. (2000)
main components,                         A. flavus
geraniol and citronellol

Essential oil from         Ketaconazole  T. erinacei,         Shin and
Agastache                                T. mentagrophytes,   Kang
rugosa and its main                      T. rubrum,           (2003)
component,                               T. schoenleinii
estragole                                and
                                         T. soudanense

Euphorbia characias        Ketoconazole  Candida              Giordani
latex                                    albicans             et al.
                                                              (2001)

Scopoletin, vanillin,                    Fusarium             Carpinella
4-hydroxy-                               verticillioides      et al.
3-methoxycinnamaldehyde,                                      (2005)
and + Pinoresinol
isolated from
Melia azedarch L. fruits

Santolina oil              Clorimazole   Candida              Suresh et
                                         albicans             al.
                                                              (1997)

Anethole                   Miconazole    Candida              Lee and
                                         albicans             Kin
                                                              (1999)
                           Amphotericin
                           B

Essential oil from         Amphotericin  Candida              Giordani
Thymus                     B             albicans             et al.
vulgaris thymol                                               (2004)
chemotype

Scopoletin, a hydroxycoumarin isolated from the fruits of Melia azedarach L. enhances the effect of two synthetic drugs namely, mancozeb and carboxin against Fusarium verticillioides (Carpinella et al., 2005). Synergistic interaction between EGCg and antimycotics such as amphotericin B and fluconazole has been reported against C. albicans. EGCg possibly attacks the cell membrane and causes cell lysis (Toyoshima et al., 1993). Amphotericin B below the minimum fungicidal concentration (MFC) is known to enhance the permeability of catechin through the fungal membrane, thereby increaseing its uptake into the cell (Hirasawa and Takada, 2004). A few herbal essential oils (Shin and Lim, 2004) particularly estragole, an oil from Agastache rugresa (Shin and Kang, 2003), Tea tree (Melaleuca alternifolia) oil (Hammer et al., 2000) and volatile oils from Allium plants and Euphorbia characigs (Giordani et al., 2004) have demonstrated significant synergism with ketacona-zole against certain fungal species.

In a recent study by Han (2007), a synergistic effect of grape seed extract (GSE) with amphotericin B was observed in both in vitro and in murine model of disseminated candidiasis due to Candida albicans. Mice treated with combination of amphotericin B and GSE or amphotericin B alone survived 62.4 and 38.4 days, respectively. The combination therapy reduced more than 75% of amphotericin B required to achieve the same level of inhibition.

In vitro evaluation of synergy

The accurate prediction of synergy between commercial drugs or between a drug and a natural product based upon the results of in vitro testing is very crucial. A number of methods are used to detect synergy. However, the checkerboard and time-kill curve methods are the two most widely used techniques and the former is a relatively easy test to perform (White et al., 1996). The checkerboard is prepared in microtiter plate for multiple combinations of two antimicrobial agents in concentrations equal to, above, and below their minimal inhibitory concentrations for the microorganism that is being tested. Each row (x axis) in the plate will contain the same diluted concentration of the first antimicrobial compound; while the concentration in each subsequent row will be half this value. Similarly each column (y axis) in the plate will contain the same diluted concentration in each subsequent column will be half this value. The drugs combination in which the growth is completely inhibited is taken as effective MIC for the combination.

The time-kill method assesses the bactericidal activity of the individual as well as different concentration of the combination of drugs as a function of time. It is a labor intensive and time-consuming process (White et al., 1996). Tubes containing individual compounds and combination of compounds with concentrations ranging from one-quarter to twice the MIC for the bacterial strain of interest (NCCLS, 1987) are prepared. The tubes are inoculated with about 5 X [10 sup.5] colony forming units/ml of the strain, and they are incubated overnight. Aliquots of the samples from Oh of incubation (reflecting the initial inoculum) and 24 h of incubation (reflecting exposure of bacteria to the compound) are plated onto agar plates. Synergy is defined as a 100-fold or greater decrease in colony count at 24 h by the combination of agents with reference to the starting inoculum and also when compared to the most active single agent (Saiman, 2007).

E test is another method of recent origin. It consists of two plastic strips coated with a continuous gradient of each of the compound on one side. For evaluation of synergy, one compound strip is placed onto an agar plate for 1 h and then removed, and the second compound strip is placed on top of the gradient left behind by the first. The MIC of the combination is taken as the value at which the two inhibition zones intersect. If the use of the E strip could be standardized for testing the synergy of drugs and the results obtained could be demonstrated to be similar to those determined by established methods, this new test method would represent an attractive alternative to the labor-intensive procedures. Further, this method could be performed on a routine basis in a clinical microbiology laboratory (White et al., 1996). The standardization of these techniques for routine routine laboratory testing is the need because of the common use of combination therapies against the growing numbers of multiple drug-resistant strains.

Analysis of the synergy data

In all the above methods the interaction between the two antimicrobial agents is estimated by calculating the fractional inhibitory concentration of the combination (FIC) index. The FIC of each drug is calculated by dividing the concentration of the compound present in that well in combination where complete inhibition of growth of the microorganism is observed by the MIC of that compound alone to inhibit the microorganism. The FIC of the combination is then the sum of these two individual FIC values. When the FIC index of the combination is equal to or less than 0.5, the combination is termed as synergistic; when FIC index falls between 0.5 and 4.0, it indicates 'no interaction' between the agents, and a value above four indicates antagonism between the two compounds (Odds, 2003).

A convenient graphical way of representing the results of combination studies is by the use of an 'isobologram', introduced by Loewe and Muischnek (1926). It is independent of the mechanism of action, makes no assumption about the behavior of each compound. So it is applicable to multiple component mixtures. Combination of drugs X and Y that shows inhibition of the growth of the organism are represented in a graph using rectangular coordinates as (x,y) for the respective doses. In this format, the dose of drug X alone as (a) and drug Y alone as (b) are represented along the axes as (a,0) and (0,b). The straight line connecting these points is called the 'line of additivity'. This line provides a convenient means for visually discriminating additive from non-additive interactions on the basis of whether or not the coordinate of the combination falls on (additive), below (superadditive) or above (subadditive) this line. The determination requires statistical evaluation (Tallarida et al., 1989) because the technique obtains the individual or combination of doses as random variables from the dose response data and there is always an error involved in the estimation (Tallarida and Raffa, 1996). If synergy is occurring, the dose of the combination needed to produce the same effect will be less than the sum of the individual components and then the curve will be concave. In antagonism, the dose of combination will be greater than expected and the curve will be convex. The 'isobole' method has been well explained with several examples by Williamson (2001).

Synergistic interactions in other therapies

The successful use of combinations of plant extracts is not only observed in antiinfective therapy, but also seen in the treatment of several disorders including cancer, HIV, inflammatory, stress-induced insomnia, osteoarthritis and hypertension (Williamson, 2001). Conventional medicine applies the "silver bullet" method, where single target therapy is employed. The recent trend has been the "herbal shotgun" method like Ayurveda, where multitargeted approach of the herbals and drugs is used. Today illness such as cancer, AIDS, hypertension, etc., are successfully treated with combination of 3-5 synthetic drugs. Cannabis extract is found to act in synergy as antispastic agent in mice than tetrahydrocannabinol at an equivalent dose (Baker et al., 2000; cit. at Wagner, 2006). Ginkgolide A and B has been seen to act in synergy in the inhibition of PAF- induced thrombocyte aggregation (Wagner, 2001, 2006) In the case of cancer chemotherapy, the molecular targets for the phytochemicals is diverse hence it necessitates the need to understand the degree of its interaction with synthetic drugs. Multitargeted therapy approach involving the application of phytochemicals or phytoextracts and synthetic drugs as anticancer agents has been detailed in a review (Hemaiswarya and Doble, 2006).

Conclusions

Before prescribing an antibiotic treatment the guidelines usually suggest that a specimen containing the suspected organism is sent for culture and sensitivity. Microbiology departments, for their part, use in vitro sensitivity of isolates taken from patients with bacterial infections to recommend which antibiotic(s) to prescribe or to use as an empirical guide for treatment in other situations. There are a number of reports available on the different antibiotic combinations tested in vitro and applied to clinical scenario. But there are no reports on the use of natural products and synthetic drug combinations used in the clinical settings. As discussed in the review, there is plenty of hope for the purified natural products to be used in combination with antibiotics as antiinfective drugs. EGCg, demonstrates a synergistic behavior with antibiotics by destroying the [beta]-lactamase activity as well as by acting on the peptidoglycan of the cell wall. The safe consumption of tea for thousands of years indicates its low toxicity. EGCg, the principal constituent of tea, is absorbed through the digestive tract and distributed to many organs in animals and humans. This indicates the high bioavailability of EGCg which could enhance the activity of antibiotic under in vivo conditions. Thus the undesirable side effects of antibiotics on human and animal health could be possibly reduced by replacing at least in part the synthetic substances by negligibly toxic, highly specific antimicrobial compounds.

Several reports are available that describe the action of secondary metabolites from plants as antimicrobial agents. While the screening of natural compounds for antimicrobial activity is by itself a research area of major significance, the development of compounds with resistance modifying action is of interest since currently there are no known agents presently in use in clinics. In order to select a compound that could act in synergism with a drug it is necessary to understand the complete molecular mechanism of the drug action in the presence and absence of the natural compound. The problems that still need to be addressed are stability, selectivity and bioavailability of these natural products, and any adverse herb-drug interaction. To overcome multidrug resistance in the antimicrobial therapy a combination of drugs has to be used. The maximum benefit can be achieved when the pharmacokinetics of natural product and the antibiotic combination match. This does not mean that pharmacokinetic profiles for both agents should be identical. The optimal ratio and dosing regimens should be explored for higher efficacy and decreased toxicological profiles. Animal models with engineered strains lacking the particular resistant genotype can be used to very precisely define the pharmacokinetic and pharmacodynamic targets followed by regulated clinical trials. Even in vitro screening procedures for drug combination are time-consuming process which should be speeded up to achieve quick breakthroughs in combination therapy. Techniques such as isobologram can be used successfully to demonstrate regions of synergy between drug combinations from other regimes.

The recent developments in genomics, proteomics and metabolomics have created a new platform to distinguish the synergistic efficacy of phytoextracts and for the determination of their mode of action. By the application of the "-omic" technologies it should be possible to detect the mechanism of action as the gene/ protein expression profiles of the combination of drugs can be entirely different from the ones induced by the single drugs. This may lead to new phyto-based paradigms towards the use of complex plant mixtures in medicine (Ulrich-Merzenich et al., 2007).

As seen from this review, the number of natural compounds acting in synergy with synthetic drugs towards fungal and Mycobacterium species are minimal. This could be due to limited understanding of the mechanism of action of drugs against these organisms or insufficient screening of natural compounds. So research should be focused towards this direction to identify more natural compounds which exhibit synergistic behavior.

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Shanmugam Hemaiswarya (a), Anil Kumar Kruthiventi (b), Mukesh Doble (a).*

(a) Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India

(b) Division of Medicinal Chemistry, Institute of Life sciences, Hyderabad 500 046, India

* Corresponding author. Tel.: + 914422574107;

fax: [phone].

E-mail address: [e-mail] (M. Doble).