Ethnomedicinal Approach in Biological and Chemical Investigation of Phytochemicals as Antimicrobials

Amit Roy * and Shailendra Saraf +

* Address for Correspondence: GRY Institute of Pharmacy, Vidya Vihar, Borawan- 451228
,Dist Khargone, [MP], INDIA , Email: wakratund@gmail.com

+ Prof. and Head Institute of Pharmaceutical Technology, Ravishankar Shukla University , Raipur [Chhattisgarh]

The aim of this review is to show that phytochemicals have long been and will continue to be extensively important as sources of active agents and models for the design, synthesis and semi synthesis of novel antimicrobial substances.

We have tried to highlight some of the developments achieved over the recent years relating to extraction, isolation, characterization and biological properties of phytochemicals from plant kingdom, useful in antimicrobial therapy. It is an attempt to provide useful references regarding recent studies conducted. The literature surveyed includes original research papers, articles, and other communications etc, published in print and electronic media. Our emphasis is to highlight the information derived from research and studies based on ethnomedicine. The present report is not exhaustive but a representative overview.

Summary

Microbial diseases rank as number one cause for almost half of the deaths in underdeveloped and tropical countries. One of the greatest accomplishments of modern medicine has been the development of antimicrobials for the treatment of infectious diseases. In 1935, Domagk discovered synthetic antimicrobial chemical (sulfonamides), in 1942 penicillin was introduced in the market and its miraculous curative properties prompted discovery of more antibiotics, which is still going on. Several hundred plant species that have potential antibacterial properties have been studied. An expansive range of plants belonging to an equally wide variety of plant families, have yielded products with antibacterial properties. Phenols and polyhenols, alkaloids and glycosides are the most common classes of phytochemicals that have exhibited promising activity against a wide range of bacterial species. Some volatile essential oils of commonly used culinary herbs and spices have also shown a high level of antibacterial activity. In these studies, the plant species have been chosen, due to their ethnomedicinal or traditional use. Both human as well as phytopathogens have been subjected to antimicrobial studies and agar well or agar disk diffusion and agar broth dilution or macro/micro broth dilution methods are the most commonly employed methods for these studies. The phytochemicals/ plant extracts have shown more activity against gram-positive organisms, compared to gram-negative and in very few studies have exhibited stronger and or broader spectrum of activity, compared to typical antibiotics. There is little effort to investigate mechanism of action of phytochemicals and to translate the results of in-vitro studies into in-vivo and preclinical and clinical trials. The investigations targeted to solve the problem of antibiotic resistance has led to discovery of efflux pumps and Multi Drug Tesistance pumps (MDRs) and there are studies to indicate that several compounds present in plants having negligible or no antibacterial activity are capable of inhibiting the efflux pumps, thereby making the most resistance microorganisms susceptible to even those plant derived antibacterials, which otherwise show very poor or no activity in in-vitro. It is therefore essential to investigate further the presence of such compounds from plants that can hinder and destroy the MDRs, and potentiate the effect of antibiotics. This can be major breakthrough in the time when world is facing the threat of drug resistance. The success achived using medicinal plants and herbal formulations based on Ethnomedicinal and traditional use against a number of bacterial infections, therapeutically, raises optimism about the future of phyto-antibiotics. But, for future development of safe and effective antimicrobials based on ethnomedicine our approach should be multidisciplinary. Plants based antimicrobials represent a rewarding and vast untapped source and have enormous potential for developing antimicrobial agents based on their indigenous and local knowledge. A continued exploration of medicinal plants is needed today so that those plants that have shown promising antibacterial activity can be investigated further.

Introduction (Microbial diseases/ pathogenesis)

Microbes are microscopic organisms
living with, in and on human beings from the very beginning of mankind. They have
played a very significant role in shaping human existence in this planet. The
microbial population includes potential pathogens. The most used examples of
microorganisms are bacteria and fungas. Bacteria are a major group of
microorganism that appeared on earth billions of years ago. They are the most
abundant of all life forms and are omnipresent. There are several evidences to
indicate that many of the bacterial infections are as old as humankind itself,
and they have affected man since antiquity. Bacterial diseases rank as number
one cause for almost half of the deaths in underdeveloped and tropical
countries, hence the need to find a safe and highly effective cure for
microbial diseases remains a major challenge for modern science even today¹.
Although a number of concepts were put forward from time to time, man knew
nothing reliable about the nature of infectious diseases until the 1800’s. Only
after the proposal of ‘Germ Theory’ by Louis Pasteur and Kosch’s postulate it
could conclusively be proved that the microbes were the cause behind infectious
diseases². Further evidence given by old and modern
theories demonstrated that only very few of the millions of existing
microorganisms were pathogenic in nature. And even these too could get
established and cause infections only after they overcame and compromised our
immune system. This capability depends upon their virulence and pathogenicity³.

Problem statement (Present scenario): The 21^st century has become an age of emerging new microbes with stunning virulence, newer microbial diseases are seen and deaths from infections are alarmingly rising in developed countries too. Out break of diphtheria in Eastern Europe, plague in India , Staphylococcal infections in the West are some of the happenings that have denied our belief that the infectious organisms would be eradicated completely by the end of 20^th century. But, this is only half of the story, as
many of these diseases have also been the cause of permanent disabilities and
deformities^{4, 5}.

Some factors that aids to the
development of present day’s microbial pathogenesis are: ^5-7

Identification of emerging pathogens (HIV, Hantavirus, rotavirus etc)

Microbial diseases of unknown etiology

Emergence of previously uncommon infections

Reemergence of older pathogens with severe virulence

Diseases due to uncultivable bacteria

Implication of microbial infections in some diseases that were thought to be of physiological origin (like Helicobacter pylori to gastric ulcers and some cancers of stomach and Hepatitis B infections to cancer of liver)

Opportunistic infections in aging and immunocompromised patients

The reasons for above factors are change in socio-economic setup, globalization and increase in antimicrobial resistance / multi drug resistance amongst common pathogens throughout the world (eg multi drug resistant tuberculosis) due to irrational and overuse of antibiotics, failure to finish an antibiotic prescription, genetic versatility of microbes and horizontal transfer of resistant genes among bacterial species. And all these are diminishing the
clinical usefulness of antibiotics^5-7. .

Antibiotics- the Magic Bullet! (The past and present): One of the greatest accomplishments of modern medicine has been the development of antimicrobials for the treatment of infectious diseases. The discovery and development of antibiotics have led to a dramatic improvement in the ability to treat infectious diseases and is among the major advances of the 20 th century. In 1928, British scientist, Alexander Fleming, accidentally found bactericidal potential of a mould Penicillium notatum , and thus the first antibiotic Penicillin was discovered.Thereafter the whole course of drug discovery and antibiotic
therapy changed completely^{8, 9}. In 1935, Domagk discovered synthetic
antimicrobial chemical (sulfonamides) ¹⁰; in 1942 penicillin was introduced in the market and its miraculous curative properties prompted discovery of more antibiotics viz: streptomycin and bacitracin (1943), cephalosporins (1945), chloramphenicol and chlortetracycline (1947), neomycin (1949), erythromycin (1952), vancomycin (1956), quinolones (1962) and and so on^11,
12. It is estimated that about 5000 to 10,000 natural antibiotics have been isolated and characterized and 50,000to 100,000 analogues have been synthesized till date, but most of them could not be realized for medicinal use due to toxicity, adverse effects or other practical problems¹³.

Initially most of the antibiotics came
from streptomyces and other bacteria and fungi, but for some time now, rare
microorganisms are used for isolation of novel antimicrobiological agents¹⁴.
In search of newer antibiotics, scientists have directed their research towards
bacteriophage genomics¹⁵, genetic engineering¹⁶,
mutasynthesis¹⁷, integrated combinatorial and medicinal chemistry
approach¹⁸, promoter-inducible reporter assays for high-throughput
screening¹⁹. Sea organisms are also being explored ²⁰,
and the search is still on. Some fruitful results have started coming due to
such extensive studies, like discovery and characterization of a novel ribosome
inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial
activity. Compounds in this class inhibit growth of many gram-positive and
gram-negative bacteria, including the common respiratory pathogens Streptococcus
pneumoniae, Haemophilus influenzae, Staphylococcus aureus,
and Moraxella catarrhalis, and are even nontoxic to human cell lines²¹.

So, at present when more than 50 years
have passed, after the conception of modern antibiotic era that started with
the first clinical trial of penicillin in early 1941, the medical scenario has
completely changed. Even the severe most infection can be treated effectively
with a variety of antibiotics. We also find that the availability and use of
antibiotics has grown into enormous proportions. However, a continuing search
for new antimicrobials remains indispensable because most of the major
antibiotics have considerable drawbacks in terms of serious side effects and
limited antimicrobial spectrum^{7, 22}.

Now, when the main focus is shifting
towards making the antibacterial drug therapy safe, effective and affordable in
any given situation²³, we find that, even though pharmacological
industries have produced a number of new antibiotics in the last decades, most
of the antimicrobials are known to exhibit serious adverse reactions leading to
systemic toxicity and other undesirable effects, and at the same time,
resistance to these drugs by microorganisms is also being reported with
increasing frequency, making it necessary to redesign drug discovery^{24, 25}.

Plants as source of antimicrobials: Plants have always been source of various effective anti-infective agents. The use of higher plants for the treatment of infections predates written records and even at this date we find that plant products provide models for number of modern drugs. Bacteriostatic and fungicidal properties of Lichens , antimicrobial action of allicin in garlic ( Allium sativum ), or berberines in Hydrastis canadensis are a few of common examples of age-old antibacterial therapy^26,
27. Even after the discovery of microbial and synthetic antibiotics and
miraculous cure provided by penicillin and sulfa drugs, in the 1940s, screening
and evaluation of plant-derived antimicrobials continued^{28, 29}. Infact organized and systematic research for plant-derived antimicrobials, in laboratories had started since 1926 and is prevalent even today ³⁰ .

Co-evolution between plants and their natural enemies inc
luding man animal and microorganisms is considerably more far reaching than current theories of reciprocal interaction suggests. Counter-resistance, genetic adaptability, polymorphic immune capacity, and pleomorphism among microbial agents allow for immense diversity of species and endless biochemical possibilities. Plants produce a vast number of natural compounds (phytochemicals) with diverse antimicrobial potential in order to adapt to these environmental threats. Phytochemicals are products of secondary metabolism, and flavonoids, alkaloids, phenols; terpenes resin acids are among the many classes of secondary metabolites. The array of compounds astonishes, as they possess number of chemical, physical and biological properties. A number of these phytochemicals were extracted & isolated even in ancient times for use in infectious diseases ^31-34 .

In adition, there are a number of parallels between plant immunological activity and the immune system of mammals, including adaptive mechanisms for microbial resistance; hence search of new molecules with antimicrobial properties is no longer restricted to customary sources. Plants, the oldest source of pharmacologically active compounds that has provided man with many medicinally useful substances for centuries, are now the most important source for prospecting of new bioactive molecules ^{25, 31, and 35} .

Herbal medicine against microorganisms

In recent times, due to several intricacies of modern antibiotics, there has been significant shift towards alternative treatment and herbal remedies 36 . Antibiotic screening of plants and natural products used in alternative systems of medicines like Ayurvedic and Unani is a major thrust of R&D, in the Indian pharmaceutical sector today ^{37, 38} . This is the reason why we find research being carried out to characterize and investigate commercial herbal preparations and plants prescribed in alternative system of medicines ^{30, 36 and 39-44} . These studies have confirmed antimicrobial potential of herbal combinations and extracts of medicinal plants used in infection control against a broad spectrum of pathogenic organisms ^{30, 42} ; strong activity against multi-drug resistant Salmonella typhi too has been reported ⁴⁰ . The efficacy has also been established for control of gingivitis ⁴¹ , against food borne pathogens 43 and also the medicinal importance of chewing sticks in oral hygiene, the use of which is so deeply rooted in many cultures ⁴⁴ . Results have shown that many herbs actually help the growth of indigenous microflora that in turn combats with the pathogenic organisms and thus aid in prevention and control of infectious microorganisms ³⁶ . The findings
have also confirmed that many Indian plants viz., black pepper⁴⁵,
clove⁴⁶, garlic⁴⁷, neem⁴⁸, terminalia chebula⁴⁹,
tulsi⁵⁰, and turmeric⁵¹ among others, possess significant
antimicrobial activity.

Ethnomedicinal approach: Throughout the human history mankind has accumulated a rich body of empirical knowledge of the use of medicinal plants for the treatment of various diseases. Therefore in an effort to discover new antimicrobials, scientists have started exploring folk and traditional medicines more exhaustively. There is also a strong belief ingrained in minds of most of the population, that, plant drugs have enormous health benefits with little side effects that is so common with synthetic drugs. This has further fuelled the study of folklore and herbal medicines ⁴⁷ .

Today, investigations based on ethnopharmacological and taxonomic/ chemosystematic information are being carried out in all the parts of world (Table 1). During the course of chemotaxonomic studies on medicinal plants of Indigenous Systems of Medicine (ISM), investigations have afforded many phytochemicals with promising antibacterial properties (Table 3).

Recent findings: In contemporary research, a host of scientists have made important contribution in the area of developing anti-infective agents from plants. Results show that evaluation of plants from traditional system of medicines has afforded diverse phytoproducts and phytochemicals. Some of these findings have been described below and are even summarized in tables (1-3) and figures (A-E).

Major plants and families: An expansive range of plants belonging to an equally wide variety of plant families, have yielded products with antibacterial properties. It is difficult to specify any particular plant species for being superior, as several of them have generated extracts and/or compounds that can be the antimicrobial of future. But, we find that Asteraceae and Euphorbiaceae are the most important plant families followed by Apocynaceae, Fabaceae/ Leguminoceae/ Papilionaceae, Labiateae/ Lamiaceae, Rubiaceae, Rutaceae and Zingiberaceae. The major proportion of plants mentioned herein belongs to one of these families (Table 2).

Plant derived substances

In maximum number of experiments, the results of which we have summarized here, crude extracts and/ or their different fractions obtained from different parts of plants have been evaluated to screen for antibacterial activities. In a good number of studies essential oils too have been used. In many of these experiments active constituents have been identified and even been isolated, but very few pure compounds have been actually subjected to antibacterial screening. Compounds belonging to a number of groups have been reported/ suggested to be active against bacteria and other microorganisms (Table 2 and 3). We shall describe a few of those classes of phytochemicals that have been mentioned in several studies.

Essential oils: These are odorous, volatile principles of plants and animals, used as flavoring and perfuming agents and also as therapeutic substances. They are mostly terpenoid in origin, while a few are principally benzene derivatives mixed with terpenes. The essential oils are generally mixtures of a large number of compounds, which may be hydrocarbons and/ or oxygenated compounds derived from these hydrocarbons. The oxygenated compounds are responsible for the odor of the essential oils and the other components are usually credited with their therapeutic properties ^{26, 27 .}

Plant essential oils and their individual components have been used in traditional systems of medicines for a variety of bacterial infections for centuries. It has also been demonstrated that antibacterial properties of these oils can be attributed to their hydrocarbon and terpene constituents 307-309 . In the course of our review we too have come across many compounds present in essential oils that have been reported or suggested to be inhibitors of bacteria and other microorganisms, and this is in accordance to previous reviews and experiments. We have therefore presented structures of a few of representative components of essential oils (Fig A). These belong to different chemical classes like terpene (eg. a and b -pinene), sesquiterpene (eg. d -cadinene), esters and alcohol (eg. artemisia alcohol, borneol, bornyl acetate), aldehyde (eg. cinnamoldehyde), ketone (eg. artemisia ketone, camphor, carvone, thujone), phenols (eg. eugenol, thymol), and ethers (eg. anethole, 1,8-cineole)

Phenolics and polypheno
lics: The largest group of plant secondary metabolites found in most classes of natural compounds having aromatic moieties is phenols. They range from simple structures with one aromatic ring to highly complex polymeric substances. The medicinally important phenolics are simple phenols, flavonoids, tannins, coumarins, anthraquinones, naphthaquinones, lignans and derivatives of these ^{26, 27} .

Many reviews and
articles reporting antibacterial activities of flavonoids^{310, 311},
anthraquinones³¹², polyphenols and phenols^{313, 314}, and
tannins³¹⁵ have been published in recent years. Several phenolic compounds have been identified and isolated from plants and they have shown promising bacterial inhibiting property against specific and broad spectrum of cultured as well as clinical bacterial strains including MRSA and multidrug resistant bacteria (Table 2 and 3).

Some important compounds of this class are eugenol (simple phenol), hyperforin (phloroglucinol derivative), ellagic acid (hydrolysable tannin), epicatechin (condensed tannin), gallic acid (pseudotannin), quercetin, kaempferol and its derivatives (flavonoids), bakuchiol, sabinene (monoterpene), nasimulin A, totarol (diterpene), oleanolic acid, betulin (triterpene), muzigadil, sugikurojinol B (sesquiterpene), taxusin, baccatin III and IV (polycyclic diterpene), andrographolide (diterpene lactone), americanin (neolignan), xanthorrhizol, assiguaxanthone (xanthone derivatives), psoralidin, bakuchicin (coumarin), etc. (Figs. B-E).

Other class of compounds: Although essential oil and its components and phenolics have been reported in literatures, multiple times, as important antimicrobial phytoconstituents, other classes of compounds too have been found to show activity against microbes. Alkaloids and glycosides are such two classes that have number of
biological activities and strong antibacterial potential too^26-28.
Alkaloids have exhibited promising activity against H.pylori³¹⁶
and a number of other bacterial strains^{104, 115, 137, 161, and 317,318}.
Similarly a few glycosides too have presented with antibacterial potency^{252,
298, and 319}. Other classes of compounds exhibiting antimicrobial
properties are amines¹²⁶, amino acid (cystine) derivative⁸⁸,
anionic components²⁶³, aromatic acids^{177, 213, 225},
chromanone acids¹¹⁹, fatty acids^{182, 272}, germacranolide¹²⁵,
lactones¹⁶⁸, monocyclic diaryl ether¹¹⁰, proteins^94,
320, steroids^{80, 99, 136, 172, and 281}, and seven membered
ring compounds²⁹⁰ among others (Table 3).

Antimicrobial screening

In large number of experiments, plants are chosen, based on their traditional and previous reported uses. In traditional medicines, majority of plants are attributed with a number of uses (Table 2), hence for in-vitro screening of extracts/ essential oils/ pure compounds, a wide spectrum of laboratory cultured bacterial species, including gram positive (g +ve) and gram negative (g-ve) bacteria are used.

Bacterial species: The commonly screened g +ve bacteria are Bacillus anthracis , B.cerus , B.megaterium , B. subtilis , B. thuringiensin , Clostridium sp., Enterococcus sp., Sarcina lutea , Staphylococcus aureus , S.epidermides , S.albus , Streptococcus fecalis , Strept.pyogenes , and S.viridans . And the g –ve bacteria includes Bacter roides fragilis , Enterobacter sp., Escherichia coli , Klebsiella pneumoniae , Micrococcus luteus , M.roseus , Proteus vulgaris , Pseudomonas aeruginosa , Salmonella enteriditis , Sal.typhi , Sal.paratyphi , Shigella boydii , Sh.dysenteriae , Sh.flexneri , Sh.sonnei , and Vibrio cholerae and other Vibrio species.

In some works Mycobacterium tuberculosis^{130, 175,268,293},
methicillin resistant Staphylococcus aereus^{117, 164, 165, 178, 189,
213, 254}, methicillin sensitive S.aureus¹¹⁷, penicillin
resistant E.coli¹²⁶, and multiresistant S.aureus¹⁸⁷
have been used. In other studies clinical isolates of enteric pathogens ^128,
184, oral pathogens^{150, 177, 229, 263}, were used for
screening. Even phytopathogens responsible for causing diseases in crops and
vegetables were subjected to antimicrobial studies¹⁰⁸.

Assay methods: The potency and activity of antimicrobials
is usually determined by minimum inhibitory concentration (mic) and zone of
inhibition they produce when they act upon bacteria. Agar well or agar disk
diffusion methods and agar broth dilution or macro/microbroth dilution methods
are the most frequently applied assay methods for these. Agar overlay assay
method¹³³, alamar blue bioassay^{174, 268}, cup-plate assay^{146,
194, 226, 234, 273, 305}, microtitre plate method^{224, 272},
poison food assay technique¹⁸⁰, pour plate method^{97, 160},
TLC bioautography assay^{101, 140, 163, 168, 272}, and time kill
studies^{203, 224} are the other methods that are employed at times. In
several experiments brine shrimp lethality test was also performed to determine
bioactivity and cytotoxicity of the plant material^{113, 121, 126, 129, 205,
240, 256, and 304}.

Mechanism of action: The
mechanism of action by which the phytochemicals exert their antibacterial
activity has not been mentioned/ studied in the reviewed articles. We came
across only three reports in which mechanism was determined, viz. bacterial
enzyme, sortase inhibitory effect¹⁷², DNA replication and bacterial
toxin and enzyme inhibitory action¹⁷⁷, and causing lysis of
bacterial cells²⁴³.

Activity, potency and spectrum: The phytochemicals display concentration dependant activity and their potency depends also on the solvent used for extraction and fractionation and fractionation invariably results in increased activity. Further, extracts and other phytoproducts though exhibit mild to moderate activity in large number of experiments, but they show a broad spectrum of activity in most of these studies. However, g+ve bacteria are more susceptible to these substances than g-ve organisms. In some cases higher activity has been reported against g-ve
bacteria^{79, 247, and 274}. Many agents have displayed strong activity
comparable to positive control, and some have even shown more potent activity^{92,
106, 108, 116, 166, 92, 106, 108, 116, 1692, 106, 108, 116, 166, 169, 172, 231,
243, 246, 297} or a broader spectrum of activity^{108, 113, 114, 134,
166, 186}, compared to positive control (Table 2).

{mospagebreak title=Bacterial resistance and phytochemicals}

Bacterial resistance and phytochemicals

Antimicrobial resistance is a natural phenomenon, but it becomes a significant public health threat when it is amplified by mishandelling our precious arsenals of antibiotics by overuse, misuse, and underuse and halfhearted use. Resistance to antimicrobials, therefore, in hospitalized patients and in general is becoming a common feature, today. Organisms like MRSA, Staphyllococci with decreased susceptibility to vancomycin, vancomycin-resistant Enterococci (VRE), multidrug resistant Pseudomonas spp., Enterobacter spp., Acinetobacter spp. and Streptococcus pneumoniae are the main culprits for it. There is also decreased susceptibility to penicillin and other antibiotics. All this has resulted in wound infections, increased mortality and morbidity, and longer duration of treatment, world-wide^{321, and 322}.

Bacteria are able to resist a broad spectrum of chemically unrelated antibiotics due to presence of drug transporters or efflux pumps ore Multidrug Resistance Pumps (MDRs). These are membrane translocases with “drug sensors”, proteins, which bind a range of structurally unrelated antimicrobial agents, and pump them out from the cell. Today the function of microbial MDRs has become a hotly debated subject. Its first description in bacteria resulted from the study of resistance to tetracyclines and later on when same mechanism was revealed for resistance towards other classes of antibiotics like macrolides, fluoroquinolones, ß-lactam and aminoglycosides. This led eventually to the concept that efflux must be considered as a common and basic mechanism of resistance, and it is more ubiquitous in nature than to target modification or production of antibiotic-inactivating enzyme. It has further been demonstrated that such efflux pumps are present in virtually all cell types from prokaryotic to superior eukaryotes and antibiotics are subjected to efflux because most of them have a combination of an amphiphatic moiety with an ionizable group, which are easily recognized by the efflux system ^323-326

We find that plant antimicrobials have poor activity (MICs 100 to 1000 µg/ml) compared to typical antibiotics produced by bacteria and fungi (0.01 to 10 µg/ml) against almost all microorganisms, hence, in number of experiments, phytochemicals exert very weak antimicrobial activity in in-vitro (Table 2). But, still, in nature the plants are able to defend themselves from a multitude of pathogens. This is because plants produce many unrelated compounds with or without antimicrobial properties that have the capability to inhibit the MDRs. These MDR inhibitors are capable of dramatically increasing the efficiency of putative plant antimicrobials against even the highly resistant microbes. The microorganisms thus become highly sensitive to phyto-antimicrobials, which otherwise show negligible or weak activity in in-vitro studies. This phenomenon of synergistic interaction among different compounds (antimicrobials or not), makes plant extracts and phytochemcal combinations to effectively inhibit resistant bacteria and toxins produced by them, which is seldom demonstrated by single, pure isolated compounds from the same extracts ^{323, 325-331} .

Recommendations for the coming time

On the basis of above narration, we propose a course of action for development of safe and effective antimicrobials based on ethnomedicine-

Our approach should be multidisciplinary, where collaborators from diverse field such as pharmacy, biochemistry, botany, chemistry, parasitology, physiology, plant production, and veterinary sciences should come together and concentrate on identifying plants with highest potential for selected diseases

Promising agents should be subjected for advanced studies like in-vivo assays, clinical trials, and toxicological studies

Candidate molecules identified, from plants, need to be extracted in significant quantities and high purity. These should be analysed retro synthetically in order to identify readily available and/ or accessible starting material

For the furure of chemotherapy efflux mechanisms should now be taken fully into account in the evaluation of new antimicrobials

At the level of clinical microbiology laboratory, suspicion of efflux as a cause of resistance should be confirmed either phenotypically or genotypically so that cross-resistance during therapy can be anticipated

We also need to investigate full potential of MDR inhibitors present in plants along with their mechanism of action so that rational synergistic combinations can be formulated to efficiently deal with ever increasing menace of drug resistance in antimicrobial therapy

New technologies like combinatorial chemistry, high throughput screening, proteomics, and microbial genomics among others should be the prime focus for future antimicrobial drug discovery.

Conclusion

Many traditional medicinal plants and herbs were reported to have various levels of antibacterial activity. In general, plant extracts have yielded promosing results hence it seems reasonable to conclude that there are probably plentiful antimicrobial compounds present in plants, but we need to isolate and identify these active constituents. Further characterization will reveal the identity of these compounds. They belong to a range of structural classes like alkaloids, flavonoids, glycosides, polysaccharides, sterols, tannins, terpenes, etc. Other compounds may turn out to be identical or structurally related to compounds listed in table 3 and ellustrated in figs A-E. There may also be novel phytochemicals. But, there recognition by efflux pumps should now be in the forefront of their activity assessment studies. Identification and isolation of MDR inhibitors and their use as adjuvant therapy also presents an interesting area of drug discovery. This approach is very critical because efflux transporters are also present in eukaryotic cells due to which several compounds can become unusable in clinical practice. It is therefore essential to identify and design efflux pump inhibitors that are specific for prokaryotic transporters; it is also of essence that the pharmacokinetic/dynamic properties of pump inhibitors need to match closely with those of the companion antibiotic. The present review also shows that traditional and folkloric use of number of medicinal plants and their products for the treatment of infectious diseases of bacterial origin is justified because of their ability to exhibit interesting antibacterial properties in-vitro and in some cases, in pre-clinical and clinical trials.

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{mospagebreak title=Tables and Figures}

Table 1: A brief overview of study on antbiotic plants, carried-out in different parts of world

*Methicillin Resistant Staphylococcus aureus; 1 Methicillin Sensitive Staphylococcus aureus; 2 Multi Drug Resistant Pseudomonas aeruginosa

Table 2: A summary of ethenomedicinally important plants demonstrating antimicrobial properties

* Common name

1[W= Weak, M= Moderate, S= Strong, C= Comparable Activity to Positive Control, Mp= More Potent Activity than Positive Control, Mbr= Broader Spectrum of Activity than Positive Control, Abr= Active Against Bacteria Resistant To Positive Control]

Table 3: Phytochemicals reported to posses antimicrobial property