WO1998036750A1

WO1998036750A1 - Anti-microbial product

Info

Publication number: WO1998036750A1
Application number: PCT/GB1998/000512
Authority: WO
Inventors: Robert Michael Edward Richards; David Garnet Durham; Iain Xiaojun Liu
Original assignee: Btg International Limited
Priority date: 1997-02-20
Filing date: 1998-02-18
Publication date: 1998-08-27
Also published as: GB9726401D0; BR9807444A; AU6108198A; CA2281524A1; EP0973523A1; JP2001512473A; AU726471B2

Abstract

A product comprises, for simultaneous, separate or sequential administration, a flavonoid of formula (I), or a pharmaceutically acceptable salt thereof; and an antibiotic. The flavonoid may be present in such an amount so as to potentiate the action of the antibiotic. When the antibiotic is a β-lactam antibiotic, the resulting medicament may be used for treating or preventing bacterial infections which are at least partially resistant to treatment by the β-lactam antibiotic alone.

Description

ANTI-MICROBIAL PRODUCT

The present invention relates to flavonoids and their use in medicaments for combating microbial infections.

The discovery of penicillin was a major breakthrough in antimicrobial therapy and β-lactam antibiotics remain of great importance in providing effective and safe treatment for a wide range of infections. The efficacy of such antibiotics has. however, gradually been eroded due to the appearance and gradual increase of β-lactam-resistant staphylococci.

Microbial resistance to β-lactam antibiotics is usually achieved by the microbes producing the enzyme β-lactamase, which cleaves the β-lactam ring of the antibiotic destroying its ability to prevent microbial growth.

Today over 90% of S. aureus strains are β-lactamase positive (O'Brien, T. F. and the International Survey of Antibiotic Resistance Group 1981 ). Additionally, strains of methicillin-resistant Staphylococcus aureus (MRSA) are usually multiply-resistant to many antibiotics (A-Massandi, S. B. et al. 1991 ), and now pose a serious problem to hospitalised patients and their care givers (Mulligan, M. E. et al. 1994). Although several types of antibiotics are available for the treatment of MRSA infection, their use is frequently associated with unwanted side-effects and the risk of resistant mutants appearing (Brumfitt, W. and Hamilton-Miller. J. 1989).

The search for new anti-microbial agents therefore remains of great importance. A few agents, for example clavulanic acid and sulbactam. which inhibit the microbial β-lactamase enzyme and promote the efficiency of β-lactam antibiotics are known, but there remains a real need for further agents.

WO 95/23607-A1 (Royal Free Hospital School of Medicine) discloses an antibacterial agent containing tea extract or active fraction thereof and a β-lactam antibiotic. The combination is said to act synergistically in the treatment of MRSA infections. The tea is extracted using water and contains polyphenols such as catechins of the following formula:

in which Ri is a hydrogen atom or a hydroxy group and R? is a hydrogen atom or a 3.4.5-trihydroxybenzoyl group. The particular examples of compounds given are mainly catechins, in which R₂ is a hydrogen atom.

Liu and Matsuzaki in an article entitled "Antibacterial activity of Flavonoids against Methicillin-resistant Staphylococcus aureus (MRSA)", Dokkyo Journal of Medical Sciences 22 (1995) 253-261 , showed that various flavonoids may have activity against MRSA. Six different types of compound may have activity: (a) flavones, (b) flavonols, (c) catechins, (d) isoflavones (e) flavanones and (f) flavanonols. Particularly interesting compounds are baicalein, myricetin, datiscetin, quercetagetin, (-)-epigallocatechin and (-)-epigallocatechin gallate.

It has now been found that certain flavonoids work with antibiotics to give improved antimicrobial compositions. These compounds can be used to promote the antimicrobial activity of such antibiotics.

The present invention therefore provides a product comprising, for simultaneous, separate or sequential administration: (a) a flavonoid of formula:

in which: each group R³, R⁵, R⁶, R⁷, R⁸, R^a, R^b, R^c, R^d and R^e represents independently hydrogen or a group OZ;

• Z is hydrogen, lower alkyl, a glycosyl group or a leaving group which in vivo is transformed into a hydrogen group; and

• X and Y are each hydrogen or X and Y together represent a double bond; or a pharmaceutically acceptable salt thereof; and

(b) an antibiotic.

The invention includes within its scope: products in which the flavonoid is a flavone of formula:

products in which the flavonoid is a flavonol of formula:

products in which the flavonoid is a flavanonol of formula:

and products in which the flavonoid is a flavanone of formula:

in which R⁵, R⁶, R⁷, R⁸. R^a, R^b, R^c, R^d and R^e are as defined as above, and pharmaceutically acceptable salts thereof.

Particularly good activity is obtained for compounds in which X and Y together represent a double bond. When X and Y are hydrogen, the most active compounds have been found generally to be those in which then R³ represents a group OZ. The best compounds are those in which at least three of R³. R⁵, R°, R⁷. R⁸, R^a, R^b, R^c, R^d and R^e represent a group OZ.

When there are at least four of these groups representing a group OZ, there is a wide degree of flexibility as to where the groups may be. However, we have generally found the compounds in which R³ and R⁷ represent a group OZ to be particularly effective, especially those compounds in which only three of the above groups represent a group OZ. In the case where R^J represents a group OZ. it appears especially desirable to have R³ and R⁷ represent a group OZ.

When Z represents a glycosyl group, a wide variety of such groups may be employed, either those formed from simple sugars or from derivatives thereof such as uronic acids.

Note that all glycosyl groups, i.e. groups formed by removal of the anomeric hydroxyl group in a sugar or a derivative thereof, are easily metabolised to the corresponding hydroxy compound. Thus the exact nature of the sugar moiety is of secondary importance. Commonly occurring glycosides that fall within the scope of the present invention include the monosaccharide derivative apigetrin/cossmetin (apigenin 7-glucoside), the disaccharide derivatives apiin (apigenin 7-(apiosylglucoside)) and acaciin (apigenin 7-(rhamnosylglucoside)), and the uronic acid derivative baicalin (baicalein 7-glucuronoside).

A preferred glycosyl group is that of formula:

wherein X represents CH₂OH or COOH.

We especially prefer the product to use a flavonoid in which each group R³, R\ R⁶, R⁸. R^a, R^b, R^c, R^d and R^e represents independently hydrogen or an OH group and R⁷ represents a group OZ in which Z is hydrogen or a group of formula:

As will be appreciated, the above two groups are glucosyl and glucuronosyl, respectively.

As indicated above, compounds such as baicalin may be used in the present invention:

Baicalin is the glucuronide of baicalein and may be isolated from the Chinese herb Xi-nan Huangqin. However, especially useful are the compounds galangin, luteolin and apigenin of formulae:

galangin luteolin

apigenin

These are all commercially available, as are the 7-glucoside of apigenin and further disaccharide derivatives of apigenin.

The compounds are mostly polyphenols and are weakly acidic. Pharmaceutically acceptable salts include phenolic salts with bases. However, they are particularly suitable for administration as salts when there is a C0₂H group present such as in the glucuronic acid residue mentioned above. This can be converted to CO₂M. where M is a metal, especially an alkali metal such as sodium or potassium. The glucuronic acid residue also helps solubility of the compound.

The flavonoid would normally be present in such an amount so as to potentiate the action of the antibiotic, so that the combination is synergistic in the treatment of microbial infections. Suitable compositions include those in which the ratio of flavonoid to antibiotic is in the range of from 2:1 to 1 :25 by weight (this might also be expressed as 'from 1 :0.5 to 1:25 by weight'), preferably from 1 :1 to 1 :25 by weight. The flavonoid may be present in such an amount so as to give plasma concentrations in the range of 0.1-100 μg mL^"1, preferably 1-10 μg mL^"1. It will be appreciated that the ranges may also be expressed as '0.1-100 mg L^"1, preferably 1-10 mg L^"1'. The antibiotic may be effective against fungi, protozoa or viruses, but preferably the antibiotic is an antibacterial agent. The term 'antibiotic', as used throughout this specification, is intended to include synthetic agents such as 4-quinolones, a family of antibacterial agents including ciprofloxacin. Other suitable antibiotics may comprise aminoglycosides and tetracyclines, but a β-lactam antibiotic is especially preferred. Suitable β-lactam antibiotics include penicillins (including penicillin G, penicillin V and others), methicillin, ampicillin, cefotaxime, amoxicillin, cloxacillin. cefoperazone and piperacillin.

The compositions of the present invention may comprise other ingredients. Mention may be made of inter alia antimicrobial agents, viscosity adjusting agents, osmolarity adjusting agents, buffers. pH adjusting agents, flavourings, stabilisers, colourings, preservatives, solubilisers and the like. Delayed release or controlled release formulations are also included in the present invention.

The compositions of the present invention may be formulated for administration by any suitable means. Particular mention may be made of oral, enteral, parenteral, subcutaneous and nasal routes of administration. The compositions may be prepared as a spray, solution, suspension, colloid, concentrate, powder, granules, tablets, pressed tablets, capsules (included coated and uncoated tablets and capsules), suppositories and the like. Delayed release or controlled release formulations are also included.

Advantageously the compositions will be sterile and suitable for medical use.

In another aspect the present invention provides the use of a flavonoid of formula:

in which: each group R³. R⁵, R⁶, R⁷, R⁸, R^a, R^b, R^c. R^d and R^e represents independently hydrogen or a group OZ; Z is hydrogen, lower alkyl, a glycosyl group or a leaving group which in vivo is transformed into a hydrogen group; and • X and Y are each hydrogen or X and Y together represent a double bond; or a pharmaceutically acceptable salt thereof; in the manufacture of a medicament for combating microbial infection, said medicament comprising an antibiotic.

The flavonoid would normally be used in such an amount so as to potentiate the action of the antibiotic, so that the combination is synergistic in the treatment of microbial infections. Suitable compositions include those in which the ratio of flavonoid to antibiotic is in the range of from 2:1 to 1 :25 by weight. The flavonoid may be present in such an amount so as to give plasma concentrations in the range of 0.1-100 μg mL^"1, preferably 1-10 μg mL^"1. It will be appreciated that the ranges may also be expressed as '0.1-100 mg L^"1, preferably 1-10 mg L^"1'. The antibiotic may be effective against fungi, protozoa or viruses, but preferably the antibiotic is an antibacterial agent. As indicated above, the term 'antibiotic' is intended to include synthetic agents such as 4-quinolones. a family of antibacterial agents including ciprofloxacin. Other suitable antibiotics may comprise aminoglycosides and tetracyclines, but a β-lactam antibiotic is especially preferred. Suitable β-lactam antibiotics penicillins (including penicillin G, penicillin V and others), methicillin, ampicillin. cefotaxime, amoxicillin, cloxacillin. cefoperazone and piperacillin.

It is not essential, although it may be convenient, for the flavonoid compound and antibiotic to be mixed together and administered in a single formulation. Simultaneous or sequential administration of two separate formulations is also suitable and falls within the scope of the invention. Optionally, where two separate formulations are to be administered each formulation may be administered by a different route. The composition of the present invention is particularly useful in treating or preventing bacterial infections or infections by other micro-organisms which are at least partially resistant to treatment by a β-lactam antibiotic alone. However, the composition of the present invention may also be used to treat any microbial infection and may be used to prevent further spread of resistance to β-lactam antibiotics.

The present invention is particularly useful against β-lactam resistant bacteria such as Staphylococcus spp., in particular Staphylococcus aureus.

In antimicrobial tests, the results obtained showed that baicalin, baicalein, apigenin, galangin and others significantly enhanced the activity of β-lactams against MRSA. With baicalein as the example, the following results were obtained. MICs were reduced from 125 to 4 μg mL^"1 for methicillin, 125 to 4 μg mL^"1 for cefotaxime, 250 to 8 μg mL^"1 for ampicillin and 250 to 16 μg mL^"1 for penicillin G. The compound also significantly enhanced the activity of penicillin G and ampicillin against penicillin- resistant S. aureus 9968, with a reduction of MIC of 125 to 4 μg mL^"1 for penicillin and 250 to 2 μg mL^"1 for ampicillin. Against β-lactamase-producing S. aureus 11561 similar synergistic activity was recorded. The correlation curves based on the MICs of β-lactams versus the concentration of baicalin not only showed significant potentiation of β-lactams by baicalin but also demonstrated a directly proportional activity-dose relationship in the combination (Fig. 1). The curves indicated that the MICs of β-lactams decreased with the increase of the concentration of baicalin. For example, 16 μg mL^"1 of baicalin reduced the MIC of ampicillin to 8 μg mL^"1. while 16 μg mL^{" 1} of baicalin reduced the MIC of methicillin to 4 μg mL^"1 against MRS A respectively.

The viable counting test results were equally impressive (see Fig. 2 to Fig. 8). In combination with baicalin. methicillin at 12.5 μg mL^"1 was bactericidal and killed approximately 99% of MRSA cells in 6 hours. In addition, activity of cefotaxime was enhanced to kill approximately 99.9% of MRSA cells in 6 hours at the concentration of 12.5 μg mL^"1 in combination with baicalin. Ampicillin also showed moderate bactericidal activity in combination with baicalin against MRSA although it was not as strong as that of methicillin and cefotaxime. On the other hand, this combination enhanced the activity of 10 μg mL^"1 of penicillin G and ampicillin to kill 99.9% of S. aureus 9968 cells in 4 h (Figs. 6 and 7). Furthermore, ampicillin at 12.5 μg mL^"1 was active enough to kill 99.9% of S. aureus 1 1561 cells in 4 hours in combination with baicalin. Penicillin G in combination with baicalin also showed similar bactericidal activity to ampicillin against S. aureus 1 1561 even though it was slightly less at the concentration of 12.5 μg mL^*1 but greater at 50 μg mL^"1 than the corresponding concentrations of ampicillin.

Results of viable count determinations were in good agreement with those of the checkerboard tests. Both sets of results suggested that baicalin combined with β-lactams had significant synergistic activity. The role played by baicalin as an enhancer of β-lactams was found to be strong enough to inhibit and to kill MRSA and/or penicillin- resistant Staphylococcus aureus at a relatively low ranges of concentrations (2 to 16 μg mL^"1) in vitro. This concentration was regarded as being a breakpoint for clinical susceptibility (Livermore, D. M., 1993).

Nevertheless, one of the factors which must be evaluated is the toxicity of baicalin baicalein, apigenin, galangin and others. As indicated above baicalin is a main constituent of the Chinese herb Huangqin with a content as high as 4% (Chinese Pharmacopoeia Committee 1985). This herb has been in use for more than one thousand years in China and Japan (Huang. H-C, el al. 1994) with no toxicity recorded in the literature (Chinese Pharmacopoeia Committee 1985). Literature reports indicate that oral administration of aqueous extract of the herb 4 or 5 g kg^"1 to dogs thrice daily for 8 weeks did not produce any significant abnormalities in the routine blood tests and histology of internal organs. Loose bowel movement occurred only in the high dosage group but it disappeared upon discontinuation of the administration of the drug (Wang, Y. S. 1983). This suggested a low toxicity for the compound. Pharmacological studies showed that baicalin has a wide range of bioactivity such as aldose-reductase inhibitory, anti-inflammatory, antiallergic (Williamson, E. M. and Evans, F. J. 1988) and anti- anaphylactic activity (Abe, K-I. Inoue, O.. and Yumioka, E., 1990). The anti-thrombotic activity (Harborne. J. and Baxer. H. 1993) of the compound, which prolongs the clotting time of fibrinogen by thrombin in high concentration (0.1-1 mM), may be a possible side-effect of baicalin. However, this concentration is much higher than the 25 μg mL^"1 or less used in the present study to enhance the activity of β-lactams against MRSA and penicillin-resistant S. aureus. In summary, the traditional use of its parent herb Huangqin, and recent studies of the compound suggested that baicalin has very low toxicity.

The present invention also provides the use of a medicament containing a flavonoid of the above formula, or a pharmaceutically acceptable salt thereof, and an antibiotic to combat a microbial infection. The invention further provides a method of treatment of a microbial infection by administering a medicament containing a flavonoid of the above formula, or a pharmaceutically acceptable salt thereof, and an antibiotic to a patient afflicted with such an infection.

The present invention will now be further illustrated with reference to the following, non-limiting examples and drawings in which —

Fig. 1 shows the potentiation of β-lactams selected from penicillin G, ampicillin, cefotaxime and methicillin by baicalin against methicillin-resistant Staphylococcus aureus NCTC 11940 (Fig. la), penicillin-resistant NCTC 11561 (Fig. 1 b) and penicillin-resistant NCTC 9968 (Fig. 1 c);

Fig. 2 shows the effect of methicillin combined with baicalin on the viable counts of methicillin-resistant Staphylococcus aureus (NCTC 11940), the values plotted being the means of 4 observations, and the vertical bars indicating the standard errors of the means;

Fig. 3 shows the effect of cefotaxime combined with baicalin on the viable counts of methicillin-resistant Staphylococcus aureus (NCTC 11940), the values plotted being the means of 4 observations, and the vertical bars indicating the standard errors of the means; Fig. 4 shows the effect of ampicillin combined with baicalin on the viable counts of methicillin-resistant Staphylococcus aureus (NCTC 11940), the values plotted being the means of 4 observations, and the vertical bars indicate the standard errors of the means;

Fig. 5 shows the effect of penicillin G combined with baicalin on the viable counts of penicillin-resistant Staphylococcus aureus (NCTC 9968), the values plotted being the means of 4 observations, and the vertical bars indicating the standard errors of the means;

Fig. 6 shows the effect of ampicillin combined with baicalin on the viable counts of penicillin-resistant Staphylococcus aureus (NCTC 9968), the values plotted being the means of 4 observations, and the vertical bars indicating the standard errors of the means;

Fig. 7 shows the effect of ampicillin combined with baicalin on the viable counts of penicillin-resistant Staphylococcus aureus (NCTC 1 1561), the values plotted being the means of 4 observations, and the vertical bars indicating the standard errors of the means; and

Fig. 8 shows the effect of penicillin G combined with baicalin on the viable counts of penicillin-resistant Staphylococcus aureus (NCTC 11561), the values plotted being the means of 4 observations, and the vertical bars indicate the standard errors of the means.

Example 1. Enhanced activity of β-lactams by baicalin against β-lactam-resistant Staphylococcus aureus

Materials and Methods

Chemistry

Scutellaria amoena C. H. Wright was collected in Xichang. Sichuan, China. Dried and powdered root (1 kg) was extracted with hot water followed by chromatography isolation and purification for active constituents. A yellow crystalline powder was isolated and identified as baicalin by chemical and spectroscopic methods compared with a reference compound from the Central Drug Control Institute, State Public Health Administration. Beijing, China. The structure was further confirmed by X-ray crystallography analysis with methyl ester derived from the isolate.

Microbiology

Materials

Staphylococcus aureus NCTC 6751, NCTC 9968. NCTC 1 1940 and NCTC 11561 were obtained from The National Collection of Type Cultures. Colindale, London. Strain NCTC 9968 and NCTC 1 1561 are penicillin-resistant and the latter is also a β-lactamase producer. Strain NCTC 1 1940 is methicillin-resistant. β-Lactam antibiotics were obtained from Sigma, Poole. England. Iso-sensitest™ broth and agar, nutrient broth were obtained from Oxoid. Basingstoke. England. Ammonia solution (NH₄OH) and lecithin were obtained from BDH, Poole, UK. Tween™ 80 was obtained from ICI, Leatherhead. UK. Microtiter plates were obtained from Bibby Sterilin Ltd., Stone. UK.

Methods

Preparation of chemicals and bacterial inoculum β-lactams were dissolved in sterile water. Baicalin was dissolved in ammonia solution and diluted with sterile water to the required test concentration. The concentration of solvent in the test solution was 1%. Nutrient broth containing 0.125% lecithin and 3% Tween™ 80 was used as a combined inactivation-culture medium for viable counting. Iso-sensitest™ broth was used as the culture medium for all micro-organisms. Overdried Iso-sensitest™ agar plates were used for determining the number of colony- forming units (CFU). The subcultures of the organisms to be tested were incubated in 20 mL of Iso-sensitest™ broth for 18 hours at 37 °C. The cell cultures were centrifuged and the cell pellets were washed with saline, recentrifuged, and resuspended in saline. The cell concentrations were adjusted with saline using a spectrophotometer at 500 nm to contain 10 CFU mL^" . This cell suspension was then diluted with double strength broth to 10⁶ CFU mL^"1. Calibration curves of absorbance readings versus colony counts of inoculum suspensions for each of the organisms were determined to confirm the validity of the counting method according to the method already described (Richards, R. M. E. and Xing, D. K. L. 1993).

MIC Determinations MIC determinations were carried out using a modified microtiter method according to that described in the literature (American National Standards Institute, 1991). Test solution, 100 μL, was added to the first row of wells then two-fold serial dilutions were performed by transferring 50 μL with a microtiter pipette to the wells of the next row containing 50 μL of sterile water and so on until the seventh row. A 50 μL inoculum (10⁶ cells mL^"1 in double strength medium) was inoculated into each of the wells of the plates. The final concentration of the inoculum in all the wells was 5 x lO³ cells mL^"1. To prevent drying, the plates were covered with a plastic cover and then incubated at 37 °C for 18 hours. The microdilution plates were examined from below with a reflective viewer or read visually from the top. The MIC was taken as the lowest concentration of chemical at which the micro-organism tested did not show visible growth. Controls were performed using the corresponding concentrations of solvents.

Checkerboard tests were performed as previously described (Lorian, V. 1991) with modification. Solutions of β-lactam (A) and solutions of baicalin (B) were prepared containing 4 times the desired concentrations (MIC) respectively. Solution A of 100 μL was added to each well of the far right-hand column of a microtiter plate (A) and solution B of 100 μL to each well of the first row of a microtiter plate (B). Two-fold serial dilutions were performed along the x-axis by transferring 50 μL dilution beginning with the far right-hand column with a microtiter pipette to the wells of the next column containing 50 μL of sterile water up to the far left-hand column for solution A. Dilution for solution B was carried out by the same method but beginning with the first row along the y-axis and continuing until the last row. Above dilutions of A and B in the microtiter plates were combined by transferring the dilution in each well of plate (B) to the well at the same column and row of plate (A) respectively to make a checkerboard, and duplicate plates were made. The inoculum (10⁶ cells mL^"1 in double strength medium) of 50 μL was inoculated into each of the wells. The final concentration of the inoculum in all the wells was 5 x 10³ cells mL^"1. To prevent drying, the plates were covered with a plastic cover and then incubated at 37 °C for 18 hours. The microtiter plates were examined and MICs noted following the procedure described in MIC determinations.

Viable Cell Counting

Viable cell counting was performed as previously described (Richards. R. M. E. and Xing. D. K. L. 1993) with modification. Compounds in test solutions were diluted alone or in combination with sterile water to the concentrations to be tested respectively. Cell suspensions of organisms obtained as described earlier were diluted with double strength medium to 10⁶ cells mL^"1. A solution of 100 μL of each chemical singly or in combination was mixed with 100 μL of cell suspension in the first row of a microtiter plate to yield final concentrations of 5 x 10³ cells mL^"1. At contact time of 0, 0.5. 1, 2, 4, 6 and 24 hours after incubation at 37 °C, 10 μL of each incubated mixture was inactivated and diluted for counting. The wells in rows 2-10 of the microtiter plate were primed with 90 μL of an antibacterial inactivation broth, and 10 μL samples from row 1 were added to row 2 and diluted through ten serial 10-fold dilutions by adding 10 μL samples from row 2 to row 3. from row 3 to row 4 and so on to row 10. Each dilution was plated on overdried Iso-sensitest™ agar plates in quadruplicate (x 4) and incubated at 37 °C for 18 hours before counting, the numbers of colony forming units (CFU) per 10 μL were determined for each sample. The detectable limit of the counting method was 10^J CFU mL^"1. Cells suspensions mixed with corresponding solvent were used as controls. Results

MIC determinations The minimum inhibitory concentration of baicalin and β-lactams for 4 strains of Staphylococcus aureus are presented in Table 1.

Table 1. Minimum inhibitory concentrations of baicalin and β-lactams used alone against Staphylococcus aureus ( μg mL^"1)

*penicillin-resistant, ** methicillin-resistant. n not selected

Baicalin was found active against all the strains of S. aureus with MICs of 64 μg mL^" Overall, the MIC of baicalin did not differ among the Staphylococcus test strains whether they were susceptible or resistant to the β-lactams. Two penicillin-resistant strains, NCTC 9968 and 1 1561, were resistant to penicillin and ampicillin with MICs of 125 to 500 μg mL^"1, but susceptible to methicillin with an MIC < 8 μg mL^'1. However, the methicillin-resistant strain of Staphylococcus aureus (MRSA) was resistant to all the β-lactams with MICs of 125-250 μg mL^'1 respectively.

Checkerboard results

The results of the test combination of baicalin and β-lactams against MRSA and penicillin-resistant S. aureus are shown in Table 2. Table 2. Minimum inhibitory concentrations of baicalin and β-lactams used in combination against penicillin and methicillin-resistant Staphylococcus aureus (μg mL^'1)

* penicillin-resistant, **methicillin-resistant, n not tested

Baicalin at 16 μg mL^"1 (one quarter of the MIC) enhanced the activity of methicillin against MRSA (NCTC 11940), reducing the MIC of methicillin from 125 to 4 μg mL^"1. Baicalin at the same concentration also enhanced the activity of other β-lactams against MRSA, reducing the MIC of cefotaxime from 125 to 4 μg mL^"1, of ampicillin from 250 to 8 μg m^"1 of baicalin with penicillin reduced the MIC of penicillin from 125 to 4 μg mL^"1 against S. aureus 9968 and from 250 to 16 μg mL^"1 against S. aureus 1 1561. The MIC of ampicillin was lowered by the same concentration of baicalin from 250 to 2 μg mL^{" 1} against 11561 respectively. Fig. 1 shows the synergistic activity for all the concentrations of the combinations of baicalin and β-lactams tested. A decrease of MICs for β-lactams is clearly indicated with the increase in the concentration of baicalin in the combination.

Viable Counting

The effects of baicalin combined with β-lactams on the viable counts of MRSA and penicillin-resistant S. aureus over 24 hours are presented in Figs 3 to 9.

Baicalin at 25 μg mL^"1 combined with methicillin at either 50 or 12.5 μg mL^" caused a reduction of colony forming units (CFU mL^"1) of viable counts of MRS A from approximately 5 x 10³ to 5 x 10^J in 6 hours and to below 10³ CFU mL^"1 (lowest detectable limit) in 24 hours (Fig. 2).

Viable counts of MRSA were not only reduced from 5 x 10³ to below 10³ CFU mL^"1 in 6 hours but also maintained under the detectable limit of 10³ CFU mL^"1 over 24 hours by 25 μg mL^" of baicalin combined with 12.5 or 50 μg mL^'1 of cefotaxime (Fig. 3).

Baicalin at 25 μg mL^"1 in combination with ampicillin, 50 μg mL^"1; reduced more than 2.5 log cycles of the viable counts of MRSA over 6 hours and maintained the reduced counts below 10 CFU mL^"1 at 24 hours. The same concentration of baicalin combined with ampicillin at 12.5 μg mL^"1 reduced the viable counts of MRSA from 5 x 10³ to 5 x 1 100^JJ CCFFUU mmLL^""11 oovveerr 66 hhoouurrss.. HHoowweevveerr,, tthhee counts recovered and increased to approximately 10 CFU mL^"1 at 24 hours (Fig. 4).

Against penicillin-resistant S. aureus 9968, both penicillin G and ampicillin at either 50 or 10 μg mL^"1 in combination with baicalin at 25 μg mL^"1 reduced the CFU mL^"1 by 3 log cycles over 4 hours. The reduced counts did not recover in 24 hours (Fig. 5 and Fig. 6).

For the penicillin-resistant strain S. aureus 1 1561 (β-lactamase-producer), ampicillin at 50 or 12.5 μg mL^"1 in combination with 25 μg mL^"1 of baicalin reduced the viable counts of the bacteria from 5 x 10³ to 10^J CFU mL^"1 in 4 hours and continued to keep the counts under the detectable limit at 24 hours (Fig. 7).

In combination with 25 μg mL^"1 of baicalin. a fairly low concentration of penicillin G (50 μg mL^"1, 10% MIC) in combination with baicalin reduced the CFU by 2 log cycles in 4 hours, but the counts began to increase after 6 hours and reached to approximately 10⁶ CFU mL^"1 in 24 hours (Fig. 8). Example 2. Enhanced activity of β-lactams by other flavonoids against β-lactam- resistant Staphylococcus aureus

Materials and Methods

Materials

Bacterial strains, culture medium, antibiotics and microtiter plates were obtained from the same sources as those detailed in the Example 1. Clinical isolates of staphylococci (β-lactamase producing, coagulase negative, 426805 and 426895) were obtained from the Microbiology Department, Aberdeen Royal Infirmary, Aberdeen, UK. Cloxacillin- resistant MRSA was induced by the following method. Methicillin-resistant Staphylococcus aureus 1 1940 was tested for MIC of cloxacillin and then subcultured with Iso-sensitest™ broth containing one half of the MIC of cloxacillin at 32 °C. The grown inoculum was cultured in the fresh media containing various concentrations of cloxacillin. The bacteria grown in culture containing the highest concentration of cloxacillin were subcultured in the higher concentrations of cloxacillin respectively. This process was repeated for about ten generations till the MIC of cloxacillin against the strain reached 1000 μg/mL. The flavonoids were purchased from Aldrich, Lancaster Synthesis or Sigma. Ammonia solution (NH OH) was obtained from Koch-Light Laboratory Ltd., Colinbrook. Berks., England. Dimethyiformamide (DMF) was obtained from Hopkin & Williams. Chadwell Heath, Essex. England.

Preparation of chemicals and bacteria inoculum β-lactams were dissolved in sterile water. Flavonoids were dissolved in ammonia solution or DMF and diluted with sterile water to application concentration. The concentration of solvent in application solution was 1% for ammonia and 50% for DMF. Iso-sensitest™ broth was used as culture medium for all micro-organisms. The subcultures of the organisms to be tested were incubated in 20 mL of Iso-sensitest™ broth for 24 h at 32 °C for MRSA (Hewitt, J. H. et al. 1969) and 37 °C for the rest of the bacteria. The cell cultures were centrifuged and the cell pellets were washed with saline, recentrifuged, and resuspended in saline. The cell concentrations were adjusted with saline under a spectrophotometer at 500 nm to a concentration of 10 CFU mL • This cell suspension was then diluted with double strength broth to 5 x 10³ CFU mL^"'. Calibration curves of absorbency readings versus colony counts of inoculum suspensions for each of the organisms had previously been plotted according to the method described in the literature (Richards, R. M. E. and Xing, D. K. L. 1993) to ensure the final CFU values were valid.

MIC Determination

MIC determination was carried out with a microtiter method according to a modification of that described in the literature (American National Standards Institute, 1991). Antibiotic application solution of 100 μL was added to the first row of wells then two-fold serial dilutions were performed by transferring 50 μL sequential dilutions with a microtiter pipette to the wells of the next row containing 50 μL of sterile water till the eleventh row. The inoculum (5 x 10^" cells mL in double strength medium containing 50 μg mL^" flavonoid) of 50 μL was inoculated to each of the wells on the plates. The final concentration of the inoculum in all the wells was 2.5 x 10^" mL and the final concentration of flavonoid was 25 μg mL^"1 or as otherwise specified. To prevent drying, the plates were covered with a plastic cover and then incubated at 32 °C for 24 hours for MRSA (Hewitt, J. H. et al. 1969) and 37 °C for the rest of the strains.

The microdilution plates were examined from below with a reflective viewer or read visually from the top. The endpoint of MIC was taken as the lowest concentration of chemical at which the micro-organism tested did not show visible growth. Controls were set using corresponding solvents and water.

Results

The structures of the flavonoids tested are given in Table 3. Table 3. The structures of the flavonoids tested

Flavone derivatives

1 R⁵ = R⁶ = R⁷ = R⁸ = R^b = R^c = H

2 R⁵ = OH, R⁶ = R⁷ = R⁸ = R^b = H

3 R⁵ = OH, R⁷ = OMe, R⁶ = R⁸ = R^b = R^c = H

4 R⁵ = R⁷ = OH, R⁶ = R⁸ = R^b = R^c = H

5 R⁵ = R⁶ = OH, R⁷ = OMe, R⁸ = R = R^c = H

6 R⁵ = R⁶ = R⁷ = OH, R⁸ = R^b = R^c = H

7 R⁵ = R⁶ = OH, R⁷ = O-glucuronyl, R⁸ = R° = R^c = H

8 R⁵ = R⁶ = R⁷ = OMe, R⁸ = R^b = R^c = H

9 R⁵ = R⁷ = R = OH, R⁶ = R⁸ = R^b = H

10 R⁵ = R⁷ = R^b = R^c = OH, R⁶ = R⁸ = H

11 R⁷ = R⁸ = R^b = R^c = OH, R⁵ = R⁶ = H

Flavonol derivatives

12 R³ = OH. R³ R⁷ = R^a = R R^c = R = H 13 R³ = R³ = R⁷ — O ^r H^tJ, R ^{ϋ aa} = R^D = R r> ^cc = R^d = H 14 R³ = R⁵ = R⁷ = R^c = OH. R^a = R^b = R^d = H 15 R³ = R³ = R⁷ = R^b = R^c = OH, R^a = R^d = H 16 R³ = R^3" = R⁷ = R^a = R^c = OH. R^b = R^d = H 17 R³ = R⁵ = R⁷ = R^b = R^c = R^d = OH, R^a = H

Flavanone derivatives

18 R⁵ = R⁷ = R^c = OH, R³ = R^b= H 19 R ) 5³ _ = r R> 7' = R^D= OH, R^c = OMe, R^J = H 20 R³ = R⁵ = R⁷ = R^b = R^c = OH

In the evaluation of twenty flavonoids tested in combination with β-lactams (Table 4 to 10), galangin (13), luteolin (10), 3',4',7,8-tetrahydroxyflavone (11), baicalein (6), apigenin (9) and baicalin (7) showed significant enhancement of β-lactam activities against methicillin-resistant Staphylococcus aureus (MRSA). Galangin (13) and luteolin (10) brought down the MICs of the antibiotics from 149-420 μg mL^"1 to 0.076- 0.15 μg/mL. This indicated a more-than-one-thousand times enhancement of the activity of the antibiotics when used with those compounds. On the other hand, clavulanic acid when tested against MRSA reduced the MICs from 149-354 μg mL^"1 to 106 μg mL^"1 for cefotaxime and methicillin, and to 11 and 23 μg mL^"1 for amoxicillin and ampicillin. This evidence suggested that the most active flavonoids tested, such as galangin (13) and luteolin (10), had several hundred times stronger activity than clavulanic acid in enhancing the activity of β-lactams against MRSA. The order of potency for the flavonoids was galangin (13) > luteolin (10) > apigenin (9) > 3',4',7,8-tetrahydroxyflavone (11) > baicalein (6) > baicalin (7). Other flavonoids, such as kaempferol (14), quercetin (15), morin (16), myricetin (17) and taxifolin (20) showed some activity. Table 4. Minimum inhibitory concentration (MIC* μg mL^"1) of β-lactams used alone and in combination with clavulanic acid (25 μg mL^"1) and flavonoids (25 μg mL^"1) against methicillin-resistant Staphylococcus aureus NCTC 11940

methiamoxi- ampicefocillin cillin cillin taxime

Control (antibiotic alone) 210 250 354 149

(63) (0) (144) (63)

NH₄OH (0.05%) 177 297 250 149

(72) (125) (157) (63)

DMF (2.5%) 297 420 297 210

(125) (125) (125) (63) clavulanic acid 106 1 1 23 106

(31 ) (5) (9) (31) flavones 1 flavone 210 250 297 177

(63) (0) (125) (72)

2 5-hydroxyflavone 210 354 354 149

(63) (144) (144) (63)

3 5-hydroxy-7-methoxy- 250 250 250 149 flavone (0) (0) (0) (63)

4 5 , 7-dihydroxyfiavone 297 297 297 177

(chrysin) (125) (125) (125) (72)

5 5,6-dihydroxy-7- 177 297 250 126 methoxyflavone (72) (125) (0) (78)

6 5,6,7-trihydroxyflavone 5.7 2.8 2.8 2.4

(baicalein) (2.3) (1.2) (1.2) (1)

7 baicalein 7-0- 23 45 38 9.5

D-glucuronide (baicalin) (9) (19) (16) (4)

8 5.6.7-trimethoxyflavone 250 297 250 106

(0) (125) (0) (31 )

9 4',5.7-trihydroxyflavone 0.15 45 38 0.1 1

(apigenin) (0.063) (19) (16) (0.031)

10 3',4',5,7-tetrahydroxy- 0.11 0.15 0.15 0.11 flavone (luteolin) (0.031) (0.063) (0.063) (0.031)

11 3'.4',7,8-tetrahydroxy- 4 2.4 2.4 2.4 flavone (2.5) (1 ) (1) (1) flavonols 12 3 -hydroxy flavone 250 297 354 125

(0) (125) (144) (0)

13 3 , 5 , 7-trihy droxy flavone 0.125 0.089 0.076 0.15

(galangin) (0) (0.035) (0.031) (0.063)

14 4',3,5,7-tetrahydroxy- 14 45 45 1 1 flavone (kaempferol) (4) (19) (19) (5) methiamoxi- ampi- cefo- cillin cillin cillin taxime

15 3',4',3,5,7-penta- 38 54 38 54 hydroxyflavone (16) (16) (16) (16)

(quercetin)

16 2',4',3,5,7-penta- 76 149 149 38 hydroxyflavone (morin) (31) (63) (63) (16)

17 3',4'5',3,5,7-hexa- 11 32 38 19 hydroxyflavone (5) (0) (16) (8)

(myricetin) flavanones 18 4',5,7-trihydroxy- 125 149 106 89 flavanone (naringenin) (0) (63) (31) (35)

19 3',5,7-trihydroxy-4'- 149 177 76 106 methoxyflavanone (63) (72) (31) (31)

(hesperetin)

20 3',4',3,5,7-penta- 38 54 76 38 hydroxyflavanone (16) (16) (31) (16)

(taxifolin)

* MIC presented as Geomean (standard deviation in brackets underneath) of 3-5 observations

Table 5. Minimum inhibitory concentration (MIC) of flavonoids and clavulanic acid used alone against methicillin-resistant Staphylococcus aureus NCTC 1 1940

Compound MIC (μg mL^"')

methicillin 250 NH₄OH (0.05%) >250 DMF (2.5%) >250 clavulanic acid 250 flavones 1 flavone >250 2 5 -hydroxyflavone >250 3 5-hydroxy-7-methoxyflavone >250 4 5,7-dihydroxyflavone (chrysin) >250 5 5,6-dihydroxy-7-methoxyflavone >250 6 5,6,7-trihydroxyflavone (baicalein) 32 7 baicalein 7-O-D-glucuronide (baicalin) 64 8 5,6,7-trimethoxyflavone >250 9 4',5,7-trihydroxyflavone (apigenin) 250

10 3',4',5,7-tetrahydroxyflavone (luteolin) 32 11 3',4',7,8-tetrahydroxyflavone 64 flavonols 12 3 -hydroxyflavone >250 13 3,5,7-trihydroxyflavone (galangin) 32 14 4',3,5,7-tetrahydroxyflavone (kaempferol) 125 Compound MIC (μg mL^"')

15 3',4',3,5,7-pentahydroxyflavone (quercetin) 125

16 2',4',3,5,7-pentahydroxyflavone (morin) 250

17 3',4'5',3,5,7-hexahydroxyflavone 125 (myricetin) flavanones 18 4',5,7-tπhydroxyflavanone (naringenin) >250

19 3',5,7-trihydroxy-4'-methoxyflavanone >250 (hesperetin)

20 3',4',3,5,7-pentahydroxyflavanone 125 (taxifolin)

Table 6. Minimum inhibitory concentration (MIC) of β-lactams in combination with flavonoids against clinical isolates of staphylococcus (48605, β-lactamase producing strain)

Compound methicillin amoxicillin ampicillin cefotaxime antibiotic alone with NH OH 2 16 8 1

(0.05%)

5.6,7-trihydroxyflavone 1 0.5 0.5 1

(baicalein) baicalein 7-O-D-glucuronide 2 2 4 1

(baicalin)

4'.5,7-trihydroxyflavone 1 <0.25 <0.25 <0.25

(apigenin)

3'.4',5,7-tetrahydroxyflavone 2 1 1 1

(luteolin)

3.5.7-trihydroxyflavone 0.5 <0.25 <0.25 0.25

(galangin)

Table 7. Minimum inhibitory concentration (MIC) of β-lactams in combination with flavonoids (25 μg mL^"1) against clinical isolates of staphylococcus (48695. β-lactamase producing strain)

Compound methicillin amoxicillin ampi- cefotaxime cillin

antibiotic alone with NH₄OH 8 8 8 2

(0.05%)

5.6.7-trihydroxyflavone 4 1 2 2

(baicalein) Compound methicillin amoxicillin ampi- cefotaxime cillin baicalein 7- -D-glucuronide 4 4 2 2

(baicalin)

4',5,7-trihydroxyflavone <0.25 <0.25 <0.25 <0.25

(apigenin)

3',4',5,7-tetrahydroxyflavone 2 0.5 0.5 0.5

(luteolin)

3,5,7-trihydroxyflavone 0.25 <0.25 <0.25 0.25

(galangin)

Table 8. Minimum inhibitory concentration (MIC) of flavonoids used alone against clinical isolates of staphylococcus (β-lactamase producing)

Compound MIC (μg mJA) strain 428605 strain 428695 methicillin 2 8

5,6.7-trihydroxyflavone (baicalein) 125 125 baicalein 7-<3-D-glucuronide (baicalin) 125 125 4',5,7-trihydroxyflavone (apigenin) >250 >250 3',4',5,7-tetrahydroxyflavone (luteolin) 125 125 3,5,7-trihydroxyflavone (galangin) 125 250

Against penicillin-resistant Staphylococcus aureus, the activity of the flavonoids was equally impressive. Galangin and apigenin. as well as clavulanic acid, reduced the MICs of penicillin G. amoxicillin and ampicillin from 250-500 μg mL^"1 to below 0.25 μg mL^"1 against penicillin-resistant Staphylococcus aureus NCTC 1 1561 and 9968 (Table 9 and 10).

Table 9 Minimum inhibitory concentration (MIC) of β-lactams in combination with clavulanic acid (25 μg m nLL^""11)) aanndd flflaavvoonnooiiddss ((2255 μμgg mmLL^""11)) aagg;ainst penicillin-resistant Staphylococcus aureus NCTC 9968

Compound penicillin G amoxicillin ampicillin cefotaxime

Antibiotic alone with NH₄OH 250 500 250 1

(0.05%)

Clavulanic acid <0.25 <0.25 <0.25 <0.25 Compound penicillin G amoxicillin ampicillin cefotaxime

5,6,7-trihydroxyflavone 2 0.5 0.5 <0.25

(baicalein)

4',5.7-trihydroxyflavone <0.25 <0.25 <0.25 <0.25

(apigenin)

3',4'.5,7-tetrahydroxyflavone 4 1 1 0.5

(luteolin)

3,5.7-trihydroxyflavone <0.25 <0.25 <0.25 <0.25

(galangin)

Table 10. Minimum inhibitory concentration (MIC) of β-lactams i n combination with clavulanic acid (25 μg mL^"1) and flavonoids (25 μg mL^"1) against penicillin-resistant

Staphylococcus aureus NCT 1 1561

Compound penicillin G amoxicillin ampicillin cefotaxime

Antibiotic alone with NH OH 500 500 250 2

(0.05%)

Clavulanic acid <0.25 <0.25 <0.25 <0.25

5,6,7-trihydroxyflavone 8 4 4 2

(baicalein)

4',5,7-trihydroxyflavone <0.25 <0.25 <0.25 <0.25

(apigenin)

3',4',5,7-tetrahydroxyflavone 4 1 2 1

(luteolin)

3.5.7-trihydroxyflavone <0.25 <0.25 <0.25 <0.25

(galangin)

Galangin, apigenin and baicalein also enhanced the activity of cloxacillin and penicillin V against cloxacillin-resistant MRSA 1 1940 by bringing down the MICs up to more than a thousand times (Table 11).

Table 1 1 Minimum inhibitory concentrations (MICs) of β-lactams in combination with flavonoids against cloxacillin-resistant MRSA (induced from NCTC 11940)

Compound cloxacillin penicillin V antibiotic alone with NH₄OH (0.05%) Ϊ000 500 5,6.7-trihydroxyflavone (baicalein. 25 μg mL^"') 1 8 Compound cloxacillin penicillin V

4',5,7-trihydroxyflavone (apigenin, 25 μg mL^"') Ϊ25

3, 5, 7-trihydroxyflavone (galangin, 12.5 μg mL^"1) 2 8

When used alone, some of the flavonoids such as galangin, luteolin, baicalin, baicalein. apigenin and 3',4',7,8-tetrahydroxyflavone showed weak activity against MRSA NCTC 1 1940 with MICs in the range 32-250 μg mL^"1 (Table 5). These results are in agreement with those previously reported (Liu, M. and Matsuzaki, S. 1995).

References

Abe, K. I., Inoue. O., and Yumioka,. E. (1990) Biliary Excretion of Metabolites of Baicalin and Baicalein in Rats, Chem. Pharm. Bull. 38:208-211.

A-Massandi, S. B., Day, M. J. and Russel. A. S. D. (1991) Antimicrobial resistant and genetransfer in Staphylococcus aureus. Journal of Applied Bacteriology 70:279-290.

American National Standards Institute (1991) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 2nd Edition, NCCLS Document M7-A2, Vol. 10, No. 8, pp. 12-21.

Brumfitt, W. and Hamilton-Miller. J. (1989) Methicillin-resistant Staphylococcus aureus. N. Engl. J. Med. 320:1188-1196.

Chinese Pharmacopoeia Committee (1985) Chinese Pharmacopoeia I. People's Health Publishing House and Chemical Industry Press. Beijing, pp. 271-272.

Harborne. J. and Baxer, H. (1993) Phytochemical Dictionary, A Handbook ofBioactive Compounds from Plants, Taylor & Francis, London, Washington DC, p. 392.

Hewitt, J. H., Coe, A. W. and Parker, M. T. (1969) The detection of methicillin resistance in Staphylococcus aureus, Journal of Medical Microbiology 2:443. Huang, H-C, Wang, H-R and Hsieh, L.-M. (1994) Antiproliferative effect of baicalein. a flavonoid from a Chinese herb, on vascular smooth muscle cell, European Journal of Pharmacology, 251:91-93.

Liu, Q., Markham, H. R., Pare. P. W., Dixon, R. A. and Mabry, T. J. (1993) Flavonoids from elicitor-treated cell suspension cultures of Cephalocereus senilis, Phytochemistry, 32:925-928.

Liu, M. and Matsuzaki, S. (1995) Dokkyo Journal oj Medical Sciences, 22:253-61.

Livermore, D. M., (1993) Activity of inhibitor combinations. J. Antimicrob. Chemother. 1993. 31 (suppl.): 9-17.

Lorian, V. ( 1991 ) Antibiotics in Laboratory Medicine, 3rd Edition. Williams & Wilkins, Baltimore, USA, pp. 436-441.

Markham, K. R. (1982) Techniques of flavonoid identification, Academic Press, London, pp. 37-39.

Mulligan, M. E.. Murray-Leisure, K. A., Ribner. B. S.. Standiford. H. C, John, J. F.. Korvick, J. A.. Kauffman. C. A. and Yu. V. L. (1993) Methicillin-resistant Staphylococcus aureus: a consensus review of the microbiology, pathogenesis. and epidemiology with implications for prevention and management. American Journal of Medicine 94:313-328.

O'Brien. T. F. and The International Survey of Antibiotic Resistance Group, Resistance to antibiotics at medical centre in different parts of the world (1986) J. Antimicrob. Chemother. 1 (suppl. C):243-253.

Richards, R. M. E. and Xing, D. K. L. (1993) fn vitro Evaluation of the Antimicrobial Activities of selected Lozenges, Journal of Pharmaceutical Sciences 82:218-1220. Wang, Y. S. et al. (1983) Pharmacology and Applications of Chinese Herbs, People's Health Publishing House Beijing, pp. 1022-1027.

Williamson, E. M. and Evans, F. J. (1988) Potter's New Cyclopaedia of Botanical Drugs and Preparations, Revised Ed., The C. W. Daniel Co. Ltd., Saffron Walden, Essex, UK, p. 362.

Claims

A product comprising, for simultaneous, separate or sequential administration: (a) a flavonoid of formula:

in which: each group R³, R⁵, R⁶, R⁷, R⁸, R^a, R^b, R^c. R^d and R^e represents independently hydrogen or a group OZ;

Z is hydrogen, lower alkyl, a glycosyl group or a leaving group which in vivo is transformed into a hydrogen group; and

X and Y are each hydrogen or X and Y together represent a double bond; or a pharmaceutically acceptable salt thereof; and (b) an antibiotic.

? A product as claimed in claim 1. in which the flavonoid is of formula:

in which R⁵, R . R⁷, R⁸, R^a, R^b, R^c, R^d and R^e are as defined in claim 1 ; or a pharmaceutically acceptable salt thereof.

3. A product as claimed in claim 1 , in which the flavonoid is of formula:

in which R⁵, R⁶, R⁶, R⁷. R⁸, R^a. R^b, R^c, R^d and R^e are as defined in claim 1 : or a pharmaceutically acceptable salt thereof.

4. A product as claimed in claim 1, in which the flavonoid is of formula:

in which R⁵, R⁶, R⁷, R⁸, R^a, R^b, R^c, R^d and R^e are as defined in claim 1 ; or a pharmaceutically acceptable salt thereof.

5. A product as claimed in claim 1, in which the flavonoid is of formula:

in which R⁵, R°, R⁷, R⁸. R^a, R^b. R^c, R^d and R^e are as defined in claim 1 ; or a pharmaceutically acceptable salt thereof.

6. A product as claimed in any preceding claim, with the proviso that when X and

Y are hydrogen, then R^J represents a group OZ.

7. A product as claimed in any preceding claim, in which at least three of R³, R³,

R⁶, R⁷, R⁸, R^a, R^b, R^c, R^d and R^e represent a group OZ.

8. A product as claimed in any preceding claim, in which Z is hydrogen, lower alkyl or a glycosyl group of formula:

wherein X represents CH₂OH or COOH.

9. A product as claimed in any preceding claim, in which each group R^J, R\ R⁶, R⁸, R^a, R^b, R^c, R^d and R^e represents independently hydrogen or an OH group and R⁷ represents a group OZ in which Z is hydrogen or a group of formula:

10. A product as claimed in claim 9, in which the flavonoid is of formula:

-j j-

11. A product as claimed in any preceding claim, in which the flavonoid is present in such an amount so as to potentiate the action of the antibiotic.

12. A product as claimed in claim 11, in which the ratio of flavonoid to antibiotic is in the range of from 2:1 to 1 :25 by weight.

13. A product as claimed in any preceding claim, in which the flavonoid is present in such an amount so as to give plasma concentrations in the range of 0.1-100 μg mL^"1.

14. A product as claimed in any preceding claim, in which the antibiotic is an antibacterial agent.

15. A product as claimed in claim 14, in which the antibiotic is a β-lactam antibiotic.

16. A product as claimed in claim 15, in which the β-lactam antibiotic comprises penicillin, methicillin, ampicillin, cefotaxime, amoxicillin, cloxacillin. cefoperazone and piperacillin.

17. A product as claimed in any preceding claim, in which the flavonoid and antibiotic are present in a single formulation.

18. Use of a flavonoid of formula:

in which: each group R³, R⁵, R⁶, R⁷, R⁸, R^a, R^b, R^c. R^d and R^e represents independently hydrogen or a group OZ; Z is hydrogen, lower alkyl, a glycosyl group or a leaving group which in vivo is transformed into a hydrogen group; and

X and Y are each hydrogen or X and Y together represent a double bond; or a pharmaceutically acceptable salt thereof; in the manufacture of a medicament for combating microbial infection, said medicament comprising an antibiotic.

19. Use as claimed in claim 18, in which the flavonoid potentiates the action of the antibiotic.

20. Use as claimed in claim 19, in which the ratio of flavonoid to antibiotic is in the range of from 2:1 to 1 :25 by weight.

21. Use as claimed in any one of claims 18 to 20. in which the flavonoid is present in such an amount so as to give plasma concentrations in the range of 0.1-100 μg mL^"1.

22 Use as claimed in any one of claims 18 to 21, in which the antibiotic is an antibacterial agent.

23. Use as claimed in claim 22, in which the antibiotic is a β-lactam antibiotic.

24. Use as claimed in claim 23, in which the β-lactam antibiotic comprises penicillin, methicillin, ampicillin, cefotaxime. amoxicillin, cloxacillin. cefoperazone and piperacillin.

25. Use as claimed in claim 23 or claim 24, in the manufacture of a medicament for treating or preventing bacterial infections which are at least partially resistant to treatment by the β-lactam antibiotic alone.

26. A product as claimed in claim 1 , substantially as described herein.

27. Use as claimed in claim 18, substantially as described herein.

28. A method of treatment of a microbial infection by administering a medicament containing a flavonoid of formula:

in which: each group R³, R⁵, R°, R⁷, R⁸, R^a, R^b, R^c, R^d and R^e represents independently hydrogen or a group OZ;

X and Y are each hydrogen or X and Y together represent a double bond; or a pharmaceutically acceptable salt thereof; and an antibiotic to a patient afflicted with such an infection.