MICROBIOLOGICAL SCREENING OF

HERBAL EXTRACT

INTRODUCTION

Antimicrobial activity of plants can be detected by observing the growth response of various microorganisms to those plant tissues or extracts, which are placed in contract with them. Many methods for detecting such activity are available, but since they are not equally sensitive or even based on the same principle, the results obtained will also be influenced by the method selected and the microorganisms used for the test. Biological evaluation in general can be carried out much more efficiently on water soluble, nice crystalline substances than on mixtures like plant extracts.

In order to detect antimicrobial activity in plant extracts, three conditions must be fulfilled:
1.

2. 3.

The plant extract must be brought into contract with the cell wall of the microorganisms that have been selected for the test. Conditions must be adjusted so that the microorganisms are able to grow when no antimicrobial agents are present. There must be some means of judging the amount of growth, if any, made by the test organism during the period of time chosen for the test. The currently available methods for antimicrobial screening fall into three groups i.e. diffusion, dilution and bioautographic methods. These methods are influenced by several factors such as extraction method, inocula volume, culture medium composition, PH and incubation temperature.

PREPARATION OF PLANT SAMPLES FOR ANTIMICROBIAL SCREENING:

Plant extracts are usually prepared by maceration or percolation of fresh green plants or dried powered plant material with water or organic solvents. Sometimes, a fractionation of the total extract is carried out prior to testing in order to separate polar from non-polar compounds and acid and neutral from basic substances. In order to detect antimicrobial substances present in very small quantities in the plant extracts, testing is carried out on the extracts in the form in which they are prepared or on concentrated extracts, obtained by evaporation of the solvent in vacuo. This often results in the precipitation or co-precipitation of possibly active substances during the testing procedure.

It is advisable to extract the plants and to evaporate the extracts at low temperature in order not to destroy any thermolabile antimicrobial agents present in the extracts. Therefore, sterilization of the extracts by autoclaving or other sterilization methods should be avoided as well . In addition, sterilization by membrane filteration has its disadvantages since many antimicrobial agents can be adsorbed on the filter material, rendering the extracts inactive. The water insolubility of lipophilic samples, such as essential oils or non polars extracts, makes it necessary to use other solvents than water or to make aqueous dispersions or emulsions with a surface active agents./

Several solvents including alcohol, acetone,chloroform, dimethylsulphoxide,dioxane, glycerol and other different emulsifiers such as macrogol ethers, sorbitan and cellulose derivatives etc. have been used. In any case, solvents other than water should always be tested simultaneously with the extracts to make sure that they have no antibacterial properties in the test system. It should also be pointed out that homogeneous dispersions in water are principally only required in the liquid dilution techniques, but not in the diffusion and agar dilution methods.

Nevertheless, diffusion methods are not the best choice for testing non-polar or other samples, which are difficult to diffuse in the media, since there is no relation between diffusion rate and antimicrobial activity. Also, aqueous dispersions containing high molecular weight solubilisers ( mol. Wt. >100000) should be avoided in diffusion methods, since they cannot diffuse into 1% agar media. Besides the polarity, the PH of the samples should also be checked before testing , since microorganisms may not be able to grow in media which have been rendered too acidic or too alkaline by the sample. In practice extracts are best adjusted to neutrality ( between PH 6.0 & 8.0) or dissolved in buffer solutions such as physiological tris buffer or others.

General methods for antimicrobial screening:

The commonly used test methods can be classified by whether or not they require sterile samples. Sterilization by membrane filtration is excluded for aqueous dispersions or emulsions and can result in the loss of antimicrobial activity from aqueous solutions. Sterilization by gamma-irradiation is a very effective, inexpensive but rather time- consuming method A good alternative is to prepare all samples in an aseptic way sterilized tubes and flasks, laminar flow, etc) and to use aqueous ethanol as extraction solvent for the plant material. Since, one is never sure of having eliminated all microbes, the methods that require sterile samples ( e.g. the liquid dilution method ) should not be carried out on plant extract, which have not been sterilized by filtration or irradiation. Among the methods to be considered as valuable for the testing of plant extracts, which cannot be sterilized by filtration, are the hole- plate diffusion and the agar dilution techniques.

Diffusion method:

In the diffusion technique a reservoir containing the plant extract to be tested is brought into contract with an inoculated medium ( eg. Agar) and after incubation, the diameter of the zone around the reservoir (inhibition diameter) is measured. In order to lower the detection limit, the inoculated system is kept at a low temperature during several hours before incubation, which favors diffusion over microbial growth and thus increases the inhibition diameter This method was originally designed to moniter the amounts of antibiotic substances in fermentation cultures and has also been used for obtaining biograms and for testing essential oils The different types of reservoirs have been employed, including filter paper discs, porcelain or stainless steel cylinders placed on the surface and holes punched in the medium. It is not necessary to sterilize the test samples since any bacteria present will be confined to the reservoirs and will not therefore be able to spread and ruin the place.

The hole plate methods, however, is the only suitable diffusion technique for testing aqueous suspensions of plant extracts. In this method, the presence of suspended particulate matter in the sample being tested is much less likely to interfere with the diffusion of the antimicrobial substance into the agar than in the filter paper disc and the cylinder plate methods. Precipitation of water in soluble substances in the cylinder or the disc will indeed prevent any diffusion of antimicrobial substances into the agar. Nevertheless, in order to limit precipitation as much as possible in the hole plate method, pre-incubation should be carried out at room temperature (250C) rather than at 40C. Advantages of the diffusion methods are the small size of the sample used in the screening and the possibility of testing five or six compounds per plate against a single microorganism.

In most studies inhibition zones are compared with those obtained for antibiotics. This is useful in establishing the sensitivity of the test organism, but a comparison of the antimicrobial potency of the samples and antibiotics cannot be drawn from this, Since a large inhibition zone may be caused by a highly active substance present in quite small amount or by a substance of comparatively low activity but present in high concentration in the plant extract. On the other hand, the filter paper disc method is a satisfactory and acceptable method for assaying water soluble antibiotics. The zone diameter of inhibition is indeed inversely related to the minimum inhibitory concentration ( MIC), where as an appropriate MIC can be extrapolated from the zone diameter.

Dilution method:

In the dilution methods, samples being tested are mixed with a suitable medium that has previously been inoculated with the test organism. After incubation, growth of the microorganism may be determined by direct visual or turbidimetric comparison of the test culture with a control cultures. Usually a series of dilutions of the original sample in the culture medium is made and then inoculated with the test organism. After incubation, the end point of the test is taken as the highest dilution which will just prevent perceptible growth of the test organism (MIC value). These methods the best for assaying water soluble or lipophilic pure compounds and to determine their MIC values, which can be carried out in liquid as well as solid media. Also growth curves of a microorganism can be recorded using this method. Only the solid or agar dilution method, is suitable for testing nonsterile plant extracts, because aerobic organisms do not develop well under the solidified agar. The occasional contaminating culture, which develops on the surface of the agar, is no problem since it can be easily recognized.

Moreover, non-polar extracts, essential oils, suspensions of solids or emulsions and antimicrobial substances, which do not diffuse through agar media, can be tested directly by incorporating them with the agar media as if they were aqueous solutions. Unlike the diffusion methods, no concentration gradient occurs during the testing procedure In contrast with the dilution in a liquid medium however, emulsions of, for example an essential oil in the medium, may break down during the solid dilution assay. Several different test microorganisms may be tested simultaneously on the same dilution, which makes the agar dilution method very quick and time saving Taking into account all properties mentioned, the agar dilution method seems to be the most convenient method for routine testing of complex samples such as plant extracts. The method is also very useful to guide the isolation of antimicrobially active components from plant extracts.

Preparation of plant extract:

Fresh green plants (50.0g) or dried plant material (5.0g) Macerated with 80% ethanol & filtered Marc Exhaustively percolated with the same solvent 80% ethanol Filtrate + percolate combine Evaporated at 400C Thick residue (extract). The residue is suspended or dissolved in physiological Tris buffer ( PH 7.2) or in a mixture of polyethylene glycol 400 (PEG 400) & physiological Tris buffer (4.6) (10.0ml).

A per warmed ( 500C) solubilized plant extract ( 2.0ml) or its four- fold dilutions ( eg. & 1/6) are mixed with an equal amount of liquid double concentrated agar medium at 500C. This gives dilutions of , 1/8 & 1/32 respectively. The holes of the lanes B-D or F-H of a microtitre plate are filled with these dilutions (0.3 ml per hole) under an infra red lamp in order to prewarm the plate and to prevent solidification during filling. The holes of the lanes A & E are filled with a control consisting of a mixture of solubilizing buffer ( 2.0 ml) and culture medium (2.0ml ) ( 0.3 ml per hole ). After solidification at room temperature, all holes are inoculated with a 1:100 dilution (5 l) of over night cultures of test bacteria ( +_ 103 bacteria ). After incubation for 24h at 370C, inhibition of growth of the test organisms is judged by comparing it with the amount of growth in the control holes using a light microscope.

In this way, a concentration range of 7500-0.5 g of potential antimicrobial agents in fresh plants is covered, assuming that the fresh plant material contains 10% dry matter and 90% water and that the active compounds are present within a range of 1% to 0.001%. A prominent antibacterial effect, worthy of further investigation, is obtained if not only the , but also the 1/8 & 1/32 dilutions show inhibitory activities. The finding of inhibition for the dilution only mostly refers to synergistic effects of different plant components and is less promising for further investigation. Sterile plant extracts, obtained by membrane filtration of their aqueous solutions can be tested by the liquid dilution method. Thus, serial two- fold dilutions of the plant extracts in standard nutrient broth ( eg. Up to 1/32 dilutions) are made in small glass tubes or dispensed in the holes of microtitre plates. After inoculation and incubation of all dilutions, growth of the microbes is determined as previously mentioned.

Aqueous suspensions and emulsion of plant extracts, made sterile by gama- irradiation, can also be tested, provided that the stability of the emulsion remains constant and sedimentation of suspended particles of the extract in the extract in the liquid culture medium is avoided by shaking during the entire assay. Dilution in liquid medium is the most laborious, but also the most accurate technique. The method is very much appreciated for assay purposes of pure samples, particularly when a high degree of sensitivity is required, but estimated by many researchers as being less suitable than diffusion methods for qualitative work eg, the rapid screening of a large number of plant extracts.

It is, however, the only method to determine whether an antimicrobial agent is bactericidal or only bacteriostatic to the test organism at various concentrations. The minimum bactericidal concentration ( MBC) can be determined by plating out samples of completely inhibited dilution cultures on to solid or liquid media containing no antibiotic. When the germ does not grow, the sample is bactericidal. All the methods described for bacteria are equally suitable for yeasts and for certain other unicellular organisms.

Bioautographic methods:

Bioautography, as a method to localize antibacterial activity on a chromatogram, has found wide spread application in the search for new antibiotics from microorganisms. Most published procedures are based on the agar diffusion technique, whereby the antimicrobial agent is transferred from thin layer or paper chromatogram to an inoculated agar plate through a diffusion process. Zone of inhibition are then visualized by appropriate vital stains. The problems due to the differential diffusion of compounds from the chromatogram to the agar plate are simplified by direct bioautographic detection on the chromatographic layer. This method, however, requires more complex microbiological equipment and is, in contrast to the contact bioautographic methodology, easily affected by possible contamination from airborne bacteria. Although bioautographic methods are suitable for testing highly active antibiotic (MIC values of at least 10g/ml ), they did not prove very promising for testing plant extracts, which often contain much less potent antimicrobial agents than the currently available antibiotics.

Some phytoconstituents with antimicrobial activity:

Numerous investigation have been carried out in individual plants and the respective antimicrobial agents have been identified in a gratifying number of cases. Various studies, it is clear that the chemical structures of these agents belong to the most commonly encountered classes of higher plant secondary metabolites. In many cases, investigation with modern methodology has confirmed folkloric accounts of the use of higher plant preparations for the treatment of infections. Up till now, however all antimicrobial substances from higher plants have been found either to be toxic to animals or not competitive therapeutically with the products of microbial origin, due to their low potency and narrow spectrum. Therefore, no antimicrobial compound from a higher plant has yet come into significant clinical use.

Research, however continues in the hope of finding plant antimicrobials that are effective for the systematic or tropical treatment of human or agricultural infections.

Since the screening methodology for detection of such agents, their isolation from the plants and successive structure activity relation studies are rather easy to perform, there are grounds for cautions optimism to be successful in the future.

Antibacterial screening method:

The antimicrobial activity of any drug of natural origin is assayed separately using the agar diffusion method employing 24 h culture of test bacterial and fungi strains. For the antibacterial screening , bacterial strains used in this study are Bacillus subtilis, Bacillus coagulans, Bacillus megatorium, Pseudomonus cepacia, Staphyloccus aureus, Escherichia coli etc. The test organisms are seeded into sterile nutrient agar media for bacterial screening. 1l of inoculum is mixed uniformly with 20l of sterile melted nutrient agar and cooled to 48-500C in a sterile petridish.

When the agar solidifies, holes of uniform diameter are made using a sterile borer. 0.3l of each of the test solutions containing different extract solutions at varying concentration as well as the standard drug solutions ( e.g. tetracycline or other) and DMSO ( control- blank) are placed in each hole separately under aseptic conditions. The plates are then maintained at room temperature for 2h and allowed for diffusion of the solution into the specific medium. All the plates are then incubated at 250C for one week and the zone of inhibitions is measured. Usually for the screening of the antibacterial activity of different plant extracts of varying polarity like petroleum ether, acetone, chloroform and methanol, extracts of plant parts are screened for their activity by the above method.

After preliminary screening, the effective extracts are further investigated for their activity at different concentrations and the optimum activity of the specific extract is measured. By using this model several plant species like Nelumbo nucifera rhizome, leaf extract of Leucas lavendulaefolia , Drymeria cordata, Cryptostegia gradiflora, Moringa oleifera several species of the genus Hypericum- H. hookerianum, H. patulum and H. mysorense have been shown to produce antimicrobial constituents. The nature of the extracts of all the above species with reported potent antibacterial potential suggests the use of the plant extract as a source of active antimicrobial principles against infection caused by susceptible organisms.

Occurrence of infrequent variation in concentrations within species and related organisms suggest that resistance to the individual extracts, when it occurs, is due to the intrinsic properties of the species involved rather than acquired characters. For this reason, it would be more effective and much more useful if the plant extract or its active constituents can be exploited in the development of anti-microbial chemotherapeutic agents. This can be considered in the line with the current search for such substances to augment or replace the antibiotics in current clinical use, which because of the spread of resistance are less useful than before.

Assay for phytopharmaceuticals having antifungal activity

Phytoalexins appear to have a very broad range of fungi for which they are quite toxic in the 10-100 g/ml concentration range. In fact, their non-specificity could reduce their usefulness to medicine .

Biological estimation procedures for screening/ evaluation of antifungal agents:

Assays using sporelings in liquid media produced the most reliable indication of fungicidal activity. The results of other tests were less reliable and often, complete inhibition of growth did not occur. For most fungi, spores and 1-day-old sporelings were equally sensitive. However , the spores of some fungi were markedly resistant to compounds that inhibited many other fungi.Similarly, 3-day-old sporelings sometimes grew in relatively high levels of the fungicides tested. The growth, which was produced after prolonged incubation, arose from a few cells that survived the potentially fugicidal effects

Mycelial growth in agar media containing phytoalexins was quite variable and was rarely linear throughout the incubation period. Skipp and Bailey (1977) divided the growth response of fungi in their bioassays into three types:
a)

Growth started immediately and was essentially linear through out the assay. Produce a growth curve with a lag period , but then proceeded at a constant rate and The rate of growth increased progressively throughout the incubation period.

b)

c)

In general, assays using older mycelial inocula in liquid media, which may reflect the situation during lesion limitation, gave results similar to those often with 1-dayold sporelings . When prolonged incubation led to increased MIC values, this was probably due to survival of some cells within the inoculum and subsequent mycelial growth. Assays measuring inhibition of mycelial growth on agar media are widely used. The lag period in the growth of several fungi was probably caused by the death of most cells within the inoculum. Growth , which eventually occurs, arises from hyphae the survive for some reason. The end of the lag period consists with alteration in the sensitivity to the compound by adaptation and/or detoxification. Chemicals may not be equally active against closely related fungi or may be equally active against unrelated fungi. Pathogens differ appreciably in their sensitivity to particular chemicals and each disease reveals a number of chemicals that are not active against any of the other pathogens.

Media used: Solid agar and liquid broth culture media C and D as a specified in pharmacopoeia of India (1985) may be used for sensitivity, turbidity, spore germination and to determine the zone of inhibition. Cultures: Pure cultures of the organisims are to be procured from reputed laboratories/ organizations and to be cultured in the laboratory where the test is being performed . The fungalstrains usally used for this study are Aspergilus niger, Aspergillus flavus, Candida albicans, candida tropicalis, Cryptococcus neoformans and Trichophyton lignorum etc.

Sensitivity test :

This is performed by tube dilution technique. A series of test tubes (16x 125 mm)., containing 9ml of sterile culture medium, C and 1ml of various concentration of test drugs (ten tubes for each concentration) are taken.

All tubes are inoculated with microorganisms to be tested and than incubated at20-25 C for 48 h. Turbidity produced is observed there after by determine the absorbance at 530 nm.

Turbidity method :

This is performed as per the method describe in pharmacopoeia (Indian pharmacopoeia 1985 ). One ml of each concentration of the test drug to be screened is placed . To each tube 9ml of nutrient medium, C previously seeded with the appropriate test organism is added. One control containing the inoculated culture medium and another blank, which is identical with the control broth treated immediately with 0.5ml of dilute formaldehyde solution is used. All the tubes are incubated at the specified temperature form 48 h. The growth of the test organisms is measured by determining the absorbance at 530nm in spectrophotometer.

Spore germination methods:

Spore suspensions of 7 days old culture are prepared in the test compounds and standard drug for comparison griseofulvin (in specific concentration) . A control is prepared as identical with this but without using the test compounds. A drop of spore suspension is placed on a sterilized slide and incubated in humid chamber for 12h and scored the number of spores germinated to calculate the percentage of spore germination .

Disc diffusion method :

Filter paper disc agar diffusion method is used (Mukherjee et al. 1995). 20ml of the sterilized medium D is taken in each petridish. 2ml of 24h old broth culture of sub-cultured organisms is distributed evenly over the surface of the plates. Sterilized Whatman filter no.1 discs (6 mm diameter) thoroughly moistened with different concentration of test and standard drugs are placed on the surface of the plate. Discs moistened with sterile water are used as control. The test organisms are seeded into sterile SDA media for fungal screening. 1l of inoculum is mixed uniformly with 20l of sterile melted nutrient agar and cooled to 48-500C in a sterile petridish.

When the agar solidifies, holes of uniform diameter are made using a sterile borer. 0.3 l of each of the test solutions containing different extracts solutions at varying concentrations as well as the standard drug solutions ( e.g. griseofulvin or other ) and DMSO (control-blank) are placed in each hole separately under aseptic condition. The plates are then maintained at room temperature for 2h and allowed for diffusion of the solution into the specific medium. All the plates are then incubated at 250C for one week and the zone of inhibition is measured. By using these models several plant species like Nelumbo nucifera rhizome, leaf extract of cassia tora, several species of the genus Hypericum-H. hookerianum, H. patulum and H. mysorense have been show to produce antifungal potentials.