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Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 93-101 Review Article Could the products of Indian medicinal plants be the next alternative for the treatment of infections? B Nandagopal, S Sankar, M Ramamurthy, S Sathish, G Sridharan Division of Biomedical Research, Sri Narayani Hospital and Research Centre, Thirumalaikodi, Sripuram, Vellore - 632 055, Tamil Nadu, India Correspondence Address: B Nandagopal Division of Biomedical Research, Sri Narayani Hospital and Research Centre, Thirumalaikodi, Sripuram, Vellore - 632 055, Tamil Nadu India director@snhrc.org Date of Submission: 14-Dec-2010 Code Number: mb11026 PMID: 21654101 DOI: 10.4103/0255-0857.81775 Abstract Indian medicinal plants are now recognized to have great potential for preparing clinically useful drugs that could even be used by allopathic physicians. Traditionally, practitioners of Indian medicine have used plant products in powder, syrup or lotion forms, without identification, quantification and dose regulation, unlike their allopathic counterparts. The present review explores the immense potential of the demonstrated effect of Indian medicinal plants on microbes, viruses and parasites. In the present context, with the available talent in the country like pharmaceutical chemists, microbiologists, biotechnologists and interested allopathic physicians, significant national effort towards identification of an "active principle" of Indian medicinal plants to treat human and animal infections should be a priority.Keywords: Active principle, bacterial infections, fungal infections, medicinal plants, viral infections Introduction The treatment of human diseases has been attempted with varying degrees of success by various peoples of the world through the history of humankind. Records of specific treatment for certain conditions are a few thousand years old, maintained by different global populations. In what is now India, the ancients have practiced herbal medicine through thousands of years and even attempted surgery. There was a boost to allopathic medicines with the dominance of Western civilization and relative decline of Indian heritage in the field of medicine. This has changed now and there is growing recognition of the value of traditional Indian medicines and herbal-based treatment modalities. More important in treating infectious diseases is the fact that allopathic physicians are encountering several problems with the emergence of drug-resistant pathogens. Drug discovery is not a fast-enough process to overcome this crunch. It hence makes immense sense to explore the empirical wisdom of the ancients with modern research technology. For example, the Tulsi (Ocimum sanctum Linn) plant, a sacred plant revered by the Hindus, is known for its medicinal properties. Presently, it is recognized that there is some scientific evidence for several of its properties, including its antimicrobial activity. However, the evidences are from in vitro effects and animal experimentation, but only a few human studies have been performed. [1] The purpose of this review is to highlight the value of Indian medicinal plants. Unfortunately, many of them have not been tested for antimicrobial activity and host toxicity by the methodology that would be acceptable today. The present review aims to point out the need to exploit the potential with an approach that would be acceptable to allopathic practitioners. Medicinal Plant Products Versus ′Active Principle′ Concept of Allopathic Medical Formulations Traditional practitioners of the Indian medicine system generally categorized as alternate medicine by westerners have used an approach that is contrary to the basic tenet of allopathic medicine, viz use of whole plant pulp, decoctions or dry powders of plant leaves, stems, roots or whole plants. Often times, it could even be a herbal mix. The "active principle", meaning the molecule that brings about the direct effect, has to be identified for several preparations used in traditional medicine. Hence, understandably, the products by themselves show several variations in the effect they produce as the active principle content of the preparation may vary with the cultivation conditions and preparation methods. This limitation is an inherent weakness in the case to be made out for use of such Ayurvedic/Unani/Siddha and homeopathic formulations. The allopathic medical system relies entirely on purified, quantified ingredient with a widely approved dosage system after the rigor of careful experimental and clinical evaluation. Importantly, "selective toxicity" is the cornerstone of the allopathic system and careful analysis of risks and benefits by due testing process. In the development and use of chemotherapeutic drugs for infectious agents, the mandated steps are use of purified drug (active principle), in vitro testing of drug against the agent in statistically acceptable designed studies, non-toxic dose, animal toxicity studies and clinical trials. This approach is required for herbal products; there is some progress in this regard but, essentially, there is still a long way to go. In India, the Government has an agency named AYUSH (Department of Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homeopathy) under the Department of Health Research, Government of India, for fostering research. The strong link up between allopathic physicians, biomedical researchers and practitioners of traditional medicine will allow the use of modern scientific experimentation methods to unlock this invaluable treasure. An illustrative example is the use of the decoction of the root of Trichodesma indicum, commonly in India for diarrhoea, dysentery and fever in Indian traditional medicine. A study was undertaken to verify the anti-diarrhoeal property of this folk remedy in animal models. The researchers found that the extract significantly inhibited castor oil-induced diarrhoea and decreased propulsion of charcoal meal through the gastrointestinal tract. Also, the castor oil-induced small intestinal fluid accumulation was reduced. This property of the herb was retained in the ethanol extract of T. indicum root. [2] As is obvious, the need would be to identify the chemical nature of the ingredient(s) that had this property and, specifically, to explore the biological effects of the ingredient. It seems likely that the effect may be on the host and not the infectious agent, but it is difficult to comment specifically without data. The in vitro activities of the extracts of 18 South African medicinal plants were tested against certain gastrointestinal pathogens. Acetone and methanol extracts from the 18 plant species were tested against 110 clinical isolates of Campylobacter spp. The extracts from eight of the plant species were also tested against a standard strain (HM-1:IMSS) of Entamoeba histolytica. Several of the extracts were also tested for cytotoxicity on Vero cell cultures. The authors found that at least one extract of each plant species was active against some of the Campylobacter isolates. Extracts of Lippia javanica and Pterocarpus angolensis had the highest antibacterial activity, with a minimum inhibitory concentration (MIC) of 90 μg/ml. Of the extracts tested against E. histolytica, only those of P. angolensis (MIC: 1.2 mg/ml) and Syzigium cordatum (MIC: 7.5 mg/ml) were found to have an inhibitory activity. The active extracts showed little cytotoxicity against Vero cells. [3] Emanating from this approach of the need to identify the active principle is the establishment of the concept of selective toxicity of medicinal plant products. There is a clear need for demonstrating the ability of the therapeutic product to eradicate the microbial or parasitic agent without any long-term residual damage to the host. Presently, several allopathic agents are licensed with known side-effects, but there are strict rules and regulations for their use and understanding and handling the side-effects. Also, there is careful risk-benefit analysis. The products are carefully certified and monitored by regulatory bodies like the Federal Drug Agency (FDA) in the US, the Drugs Controller of India (DCI), etc. Assays for Active Principle The most important modern approach to drugs of herbal origin will be the purification of the specific ingredient that shows the biological effects (active principle). Its effect against the infectious agent should be tested in vitro and toxicity tests carried out in appropriate cell culture systems or animal models. The chemical purification, characterization, toxicity testing and establishment of bioavailability would require significant expertise in pharmaceutical chemistry and pharmacology. Efficacy testing against infectious agents will require well-trained microbiologists and molecular biologists. Chemical assay for active principle identification Globally, several thousands of plants and herbs have been identified for their medicinal property. Indigenous populations often have information from empirical usage. Much of this potential is yet to be harnessed and the benefits opened up for all. What is of cardinal importance is the preparation of the purified product. An example of the purification and characterization steps is shown for the handling of extracts from fresh rhizomes of Zingiber officinale (ginger). Ginger oil is obtained by steam distillation of rhizomes in which curcumene was found to be the major constituent. The thermally labile zingiberene-rich fraction is obtainable from its diethyl ether extract. The structures of the compounds are established by nuclear magnetic resonance (NMR) and mass spectral (MS) analysis. [4] Biotoxicity measurement and genotoxicity of medicinal plants A systematic attempt at developing herbal-derived drug(s) for the treatment of infections has to address the issue of toxicity to the hosts. Some allopathic drugs are shown to have an inhibitory effect on bone marrow and renal toxicity with prolonged use. The examples are chloramphenicol and gentamicin, which are widely used. Such information on herbal drugs should be obtained and may be studied in appropriate systems that reflect the biological toxic effects. Tea tree (Melaleuca) oil has become popular for its antiseptic and anti-inflammatory actions. An example of carrying out toxicity testing is well described in a review by Carson et al. [5] Artemisinins have anti-malarial activity and are a class of compounds that include artesunate, artemether and artemisinin. Some of these compounds are the first-line treatment recommended by the World Health Organization for infections with Plasmodium falciparum. In some instances, hepatitis might be associated with consumption of the herbal supplement containing artemisinin. [6] The effect of plant products on the chromosomal DNA of the human or animal hosts is an important concern. Even more emphasis should be given to understand the effect of the drug in question on the foetus of the pregnant mothers. Such information is vital to prevent long-term damage to the child if pregnant women were treated. We have the example of allopathic antibiotic tetracycline(s) that are forbidden for pregnant women. Tinospora cordifolia (TC) products are widely used as folk medicine for animals and ayurvedic treatment for various diseases. It is considered to improve the immune system. Chandrasekaran et al.[7] evaluated the genotoxic risk of the aqueous extract of this plant. The tests applied for evaluating genotoxicity were in vitro chromosome aberration, rodent bone marrow micronucleus and Comet assay, and the results were confirmed in Ames test for mutagenic effect (S. typhimurium model). The authors concluded that TC exposure did not cause clastogenicity or DNA damaging effect in bone marrow erythrocytes and peripheral blood lymphocytes, respectively. Opportunistic infections are a problem in human immunodeficiency virus (HIV)-infected patients in Tanzania. Certain plant products have been used to treat oral candidiasis. van den Bout-van den Beukel et al.[8] investigated the cytotoxicity, genotoxicity and cytochrome P450 interaction potential of these medicinal plants. The researchers used Hoechst 33342 dye, Alamar Blue, calcein-AM, glutathione depletion and O 2 -consumption assays for cytotoxicity assay and Vitotox assay for genotoxicity testing. Importantly, both genotoxicity and cytotoxicity were observed for several of the products. Immunobiological effects observed for medicinal plants Many plant products have been shown to have the immuneregulatory/stimulatory effect on the host′s immune system. Such agents are used in combination with certain other products for treating infections by practioners of alternative medicine. One clear example is the recognized enhancement of immunity in infected bovine mammary gland by the hydromethanolic extract of stems of Tinospora cordifolia. Using assays like somatic cell count, total bacterial count, phagocytic activity and leukocyte lysosomal enzymes like myeloperoxidase and acid phosphatase activity and interleukin-8 (IL-8) level estimation, immunomodulatory properties and antibacterial properties were documented. [9] Appropriate bioassays for testing antimicrobial activity It is imperative that standardized internationally accepted protocols for in vitro testing of the activity of the plant products are undertaken. Only if the testing methods are according to established standards would the results be acceptable. Presently, we recommend adopting standards according to the provisions of international bodies like Central Laboratory Standards Institute (CLSI), European Committee for Antifungal Susceptibility Tests (EUCAST) and British Society for Antimicrobial Chemotherapy (BSAC). The current document of CLSI for performance standards for antimicrobial susceptibility testing is M100-S20 Vol 30(1): 2010. The anti-fungal property of the fruits of Embelia ribes native to India was tested using standard methodology as recommended by the above-mentioned international organizations. The authors determined the MIC 50 values (MIC required to inhibit the growth of 50% of the organisms) of the purified active principle "embelin", a compound that resembles a natural Coenzyme Q10 (ubiquinones). The molecule was shown to be active against different Candida spp., with MIC 50 values below 700 μg/ml. [10] The concept of "breakpoint" refers to a discriminating concentration used in the interpretation of the results of susceptibility testing of strains of organisms as susceptible, intermediate or resistant. The whole rationale for determining a breakpoint is predicated by the fact that an organism designated as "susceptible" should respond to the standard dose of the agent. The term susceptible indicates that infection due to the bacteria will probably respond to the antibiotic. The term intermediate refers to an indeterminate or uncertain response that is likely. In some situations, increased dose of the drug may be effective. The term resistant refers to the infection which will not respond to the antibiotic against the bacteria. The three features of both the antimicrobial agent and the infectious organism to be considered when determining the breakpoint include: the distribution of susceptibilities, pharmacological properties (bioavailability) of the antimicrobial and clinical outcome data. The distribution of MIC of an antimicrobial for a given bacterial strain (population) is either unimodal (bacteria are either susceptible or resistant) or bimodal (a population that possesses a mechanism for drug resistance, e.g. Escherichia coli with and without the TEM-1 enzyme). Overall, an understanding of the pharmacokinetics to a given drug is important. For instance, the antibiotic gentamicin is given at 80 μg/kg body weight at 8-h intervals. This concentration reaches 8 μg/ml of plasma at the "peak" level, with "trough" levels of 2 μg/ml. About one-fourth of the "trough" level (0.5 μg) reaches the tissues. Thus, for gentamicin to be efficacious against a given strain of bacteria, its MIC should be less than 0.5 μg/ml. The BSAC recommendation for breakpoint concentration estimation is shown below: Breakpoint concentration = C max f x s / et where, C max = maximum serum concentration following a stated state and, usually, at 1 h post-dose. e = factor by which the C max should exceed the MIC. Normally, a value of four is used, but this may be less for compounds that achieve high tissue concentrations in relation to their serum levels. f = factor to allow for protein binding, which affects both an antimicrobial′s in vitro activity and its activity in serum. For protein binding <70%, f = 1; for protein binding 70-90%, f = 0.5; and for protein binding >90%, f = 0.2. t = factor (normally 1) to allow for the serum elimination half-life. For a serum elimination half-life of between 1 and 3 h, t = 1; if it is >3 h, t = 0.5; or if it is <1 h, t = 2. s = shift (or reproducibility) factor mentioned above. Typically, s = 1 and should not normally be <0.5 or >2. [11] In the quest for antiviral agents, the choice of methodologies is equally important. Viruses that grow in cell cultures are checked for their susceptibility to antiviral agents by different phenotypic assays. These include the Dye Uptake (DU) assay using vital stains such as neutral red. This is used to determine the inhibitory concentration (IC 50 ) values. The other alternative is the plaque assay. Here, each infectious virus particle multiplies under conditions that result in a localized area of infected cells or "plaque". The plaques are seen as areas of dead/destroyed cells detected by neutral red or as areas of infected cells detected by immunostaining. More recently, drug susceptibility is directly determined by genotypic assays (nucleotide sequencing of the viral genome), which predict drug resistance like for HIV and the pandemic H1N1 strain of influenza virus. Agents of Infections and the use of Medicinal Plant Products In a survey of the published literature as indexed in the PubMed, several formulations derived from herbal products have been reported with properties that could be exploited for treating infections. A majority of products have direct inhibitory activity on several species of bacteria and fungi. Some products have been shown to have anti-viral and anti-parasitic properties. What is lacking is clear identification of the active principle and its evaluation against infectious agents in well-designed clinical trials. Because much of the published literature on Indian medicinal plants is on extracts (aqueous/alcoholic solvents) with inadequate characterization of the active ingredient (molecule), it is difficult to glean information on the mechanism of action. Anti-bacterial effects including anti-tuberculous activity of medicinal plants This decade has seen some interesting developments in the testing and evaluation of herbal products for antimicrobial activity. In this section, some significant papers are reviewed. Early on, the crude hexane extract of Arnebia hispidissima DC. (Boraginaceae) was shown to have an antimicrobial activity against Gram positive and Gram negative bacteria and fungi. [12] Ravindranath et al.[13] purified deoxypreussomerins, palmarumycins CP1, JC1 and JC2 from the stems of Jatropha curcas with antibacterial activity. Methanol extracts of the leaves of Tamarindus indica, Lawsonia inermis and Hibiscus rosasinensis, the rhizome extracts of Curcuma longa and the seeds of Vigna radiata were tested for activity against Burkholderia pseudomallei by the disc diffusion method. Among these, only the methanol leaf extracts of T. indica exhibited anti-B. pseudomallei activity. [14] The authors did not perform animal experiments and toxicity testing. The ethanolic extracts of the plants from Camellia sinensis (leaves), Delonix regia (flowers), Holarrhena antidysenterica (bark), Lawsonia inermis (leaves), Punica granatum (rind), Terminalia chebula (fruits) and Terminalia belerica (fruits) were tested for their antibacterial activity against beta-lactamase-producing methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). The extracts from the leaves of Ocimum sanctum showed best activity against the MRSA strains. Extracts from Allium sativum (bulb) and Citrus sinensis (rind) exhibited little or no activity against the MRSA strains. Further, the acetone and methanol extracts of Punica granatum and Delonix regia showed antibacterial activity. The authors demonstrate in vitro synergistic interaction of crude extracts with certain antibiotics. The antimicrobial activity was associated with phytochemicals like alkaloids, glycosides, flavonoids, phenols and saponins. TLC-bioautography indicated phenols and flavonoids as the majorly active compounds. [15] The aqueous extract from the bark of mango (Mangifera indica L.), contains polyphenols whose major ingredient is mangiferin [C-glucosylxanthone (1, 3, 6, 7-tetrahydroxyxanthone-C2-β-D-glucoside)]. Mangiferin is a normal metabolite also found in mango leaves. Stoilova et al. [16] reported the antimicrobial effect of the polyphenol mangiferin obtained from the leaves of mango trees and showed it to have an activity against bacterial agents such as Bacillus spp., Staphylococcus aureus, Escherichia coli, Salmonella spp and Klebsiella pneumoniae and fungi like Aspergillus flavus and Aspergillus fumigatus. Crude extracts of Dorema ammoniacum, Sphaeranthus indicus, Dracaena cinnabari, Mallotus philippensis, Jatropha gossypifolia, Aristolochia indica, Lantana camara, Nardostachys jatamansi, Randia dumetorum and Cassia fistula exhibited significant antimicrobial activity by the agar dilution method against Bacillus cereus var mycoides, Bacillus pumilus, Bacillus subtilis, Bordetella bronchiseptica, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus faecalis, Candida albicans, Aspergillus niger and Saccharomyces cerevisiae. [17] Further tests for active principle identification, toxicity and animal experimentation were not performed. A study was carried out by Srikumar et al.[18] on the aqueous and ethanol extracts of Terminalia chebula, Terminalia belerica and Emblica officinalis for their antibacterial activity against Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, S. flexneri, Staphylococcus aureus, Vibrio cholerae, Salmonella paratyphi-B, Escherichia coli, Enterococcus faecalis and Salmonella typhi using the Kirby-Bauer′s disk diffusion and MIC methods. T. chebula was found to possess a high phytochemical content, followed by T. belerica and E. officinalis in both aqueous and ethanol extracts. Antimicrobial activity of the extracts from several plants was screened against the pathogenic bacterial and fungal isolates using a microbroth dilution assay. The extracts obtained from Phyllanthus urinaria, Thevetia neriifolia, Jatropha gossypifolia, Saraca asoca, Tamarindus indica, Aegle marmelos, Acacia nilotica, Chlorophytum borivilianum, Mangifera indica, Woodfordia fruticosa and Phyllanthus emblica were shown to have an antimicrobial activity. The aqueous extracts of Acacia nilotica, Justicia zelanica, Lantana camara and Saraca asoca showed good activity against all the bacteria tested. [19] Tuberculosis is caused by Mycobacterium tuberculosis in humans and is presently a major public health problem in developing countries and deprived sections of developed countries as well. Two problems have complicated the control of tuberculosis: the global HIV pandemic has increased the susceptible individuals many-fold and emergence of drug resistance of M. tuberculosis. A few hundred plant species have been tested and have shown to have an antimycobacterial activity. [20] The alcoholic extracts of Acorus calamus, Hemidesmus indicus, Holarrhena antidysenterica and Plumbago zeylanica were shown to inhibit the growth of extended spectrum beta-lactamases (ESBL) producing multidrug-resistant enteric bacteria. The ethyl acetate fraction of Plumbago zeylanica was reported to be most active. The acetone fraction showed higher activity compared with the ethyl acetate and methanol fractions for other plants. Synergistic interactions of crude extracts with antibiotics were demonstrated in the 12 different combinations against ESBL-producing E. coli. The active principle (molecules) includes alkaloids, phenols and flavonoids as active phytoconstituents. [21] Extracts of six plants, namely Plumbago zeylanica, Hemidesmus indicus, Acorus calamus, Punica granatum, Holarrhena antidysenterica and Delonix regia, contained active phytocompounds obtained primarily in the acetone and ethyl acetate fractions that were found to be active against multidrug-resistant Gram positive and negative bacteria. [22] The authors investigated the ethanolic extracts of 12 plants and found them to be non-toxic to sheep erythrocytes and non-mutagenic as determined by the Ames test using Salmonella typhimurium test strains. The authors concluded that identification of active constituents and identification of their additive and/or synergistic interactions are needed to exploit them in evaluating their efficacy and safety in vivo against MDR bacteria. A polyherbal formulation called BASANT had been tested against Chlamydia trachomatis in an in vitro cell culture (HeLa 229 cells) and has been found to be effective. [23] This cream (BASANT) has been constituted with diferuloylmethane (curcumin), purified extracts of Emblica officinalis (Amla), purified saponins from Sapindus mukorossi, Aloe vera and rose water. It was shown to be effective in inhibiting Neisseria gonorrhoeae, Candida glabrata, Candida albicans and Candida tropicalis.[24] A study has been reported on the antimicrobial properties of Soymida febrifuga (Maliaceae) leaves. Hexane, methanol and aqueous extracts were tested for their antimicrobial activity against six bacterial and five fungal strains. The technique employed was the agar diffusion method, MIC and minimum microbicidal concentration (MMC) for the extracts against different test organisms. The extracts showed antimicrobial activity; the methanol extract was more potent against Aspergillus fumigatus and Candida tropicalis. [25] Further, evaluation of the plant extracts were not performed. The ethanol extracts of Zingiber officinale, Punica granatum, Terminalia chebula, Ocimum sanctum, Cinnamomum cassia and Azadirachta indica have been tested by the disc diffusion method and have variably shown to have a strong antibacterial activity against Gram negative bacilli like Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and a sturdy Gram positive coccus: Enterococcus faecalis. [26] Antiviral properties of medicinal plants Presently, the number of antiviral agents available is over 65. But, still, several viral infections are not treatable; there are over 500 different viruses that have the potential to cause human infections. The picture, however, is a little bleak as viruses also demonstrate drug resistance, which complicates therapy. Therefore, there is a need for a search for newer agents for chemotherapy. There is exciting potential to achieve success in this regard by exploring the medicinal plants as a source of antivirals. About two decades ago, there was excitement regarding the possibility of using some herbal products in the case of hepatitis virus infection. Certain herbs have been used by traditional Indian practitioners for the treatment of acute infections of the liver. The earliest report testing this herbal product (Phyllanthus amarus) showed promise in the treatment of chronic carriers of hepatitis B. [27] Subsequently, the study on two such extracts (Phyllanthus amarus and Phyllanthus maderaspatensis) in an experimental model of duck hepatitis B virus (DHBV) infection did not show efficacy of the herbal medication. [28] In an in vitro study in Vero cells, a crude aqueous seed extract of Pongamia pinnata Linn. was shown to have anti-HSV-1 and anti-HSV-2 activity by showing absence of any cytopathic effect. However, clinical evaluation was not done. [29] Phyllanthus niruri, which was previously shown to have activity against hepatitis B virus, was shown to have an inhibitory effect against HIV-1. The activity was in an alkaloidal extract of P. niruri. The in vitro testing showed inhibition of virus-induced cytopathogenecity in MT-4 cells. Importantly, the extract showed a clear selective toxicity for the viral cells. [30] Notka et al.[31] showed the inhibitory effect of the aqueous and alcohol extracts of P. amarus against HIV-1 attachment and the HIV-1 enzymes integrase, reverse transcriptase and protease. On characterization of the extracts, it was found that the gallotannin-containing fraction, specifically ellagitannins, geraniin and corilagin, was shown to be mediating these antiviral activities. The authors concluded that the preparations blocked the interaction of HIV-1 gp120 with its primary cellular receptor CD4. This finding in a cell culture system was extended to testing the sera of uninfected volunteers who were orally fed the herbal preparation; the sera of the volunteers showed an HIV-inhibitory activity. Phyllanthus urinaria L (Euphorbiaceae), a close relative of P. amarus, was reported to have an inhibitory activity against HBV, EBV, HIV and HSV. [32] In this study, the anti-HSV-1 and HSV-2 activities of different extracts from P. urinaria were investigated in vitro by the plaque reduction assay. The extracts did not show a cytotoxic effect against Vero cells at concentrations of 10.0 μg/ml or below. The "time-of-addition" study showed that the extracts were effective only when added during the HSV-2 infection. The previously shown BASANT preparation was also shown to have a virucidal action against HIV-1. This property was shown for HIV-1 NL4.3 in the cell culture systems CEM-GFP reporter T and P4 (Hela-CD4-LTR-betaGal). [24] The authors found that the BASANT preparation was safe in pre-clinical testing for toxicity carried out on rabbit vagina after application for up to 3 weeks. Very recently, extracts of Swertia chirata were reported to have activity against Herpes simplex virus (HSV) type- 1. The authors used multiple approaches like cytotoxicity in Vero cells, plaque reduction in Vero E6 monolayer cells, virus infectivity, antigen expression and polymerase chain reaction (PCR) assays. [33] The authors carried out HSV antigen expression and time kinetics experiments using the indirect immunofluorescence (IFA) test. They found that the characteristic pattern of small foci of single fluorescent cells was present in the Swertia chirata extract-treated HSV-1 infected cells at 4 h post-infection. Compared with acyclovir, which showed 100% inhibition, this plant product at a non-toxic concentration showed 70% inhibition. The authors concluded that this finding suggested inhibition of viral dissemination. Anti-parasitic potential of medicinal plants Singh et al.[34] showed the inhibitory activity of the crude ethanolic extract of Desmodium gangeticum and its fractions against visceral leishmaniasis in hamsters. The butanol fraction showed moderate anti-leishmanial activity in hamsters but the hexane, ethanol and aqueous extracts did not show any significant activity against L. donovani multiplication. The fraction produced significant non-specific resistance of peritoneal macrophages against Leishmania infection. This compound showed moderate anti-leishmanial activity when tested against established infection of Leishmania donovani in hamsters. Sharma et al.[26] reported the anti-leishamial activity of the methanolic extract from Withania somnifera Dunal (ashwagandha) and Allium sativum Linn. (garlic). The active principle of Ashwagandha was identified as withaferin A. The cytotoxic effects were investigated in the murine macrophage model and reported to be safe. Certain compounds like artemisinins, including artesunate, artemether and artemisinin, are used to treat malarial infections caused by Plasmodium falciparum.[5] Anti-fungal effects of certain medicinal plants The essential oils extracted from the Eucalyptus species was shown to inhibit fungi like Fusarium solani, F. oxysporum, F. pallidoroseum, F. acuminatum and F. chlamydosporum. [35] The bulb extracts of A. cepa and A. sativum exhibited activity against both filamentous and non-filamentous fungi. [36] Broad-spectrum antimicrobial activity was observed for the alcoholic extracts of L. inermis, Eucalyptus sp., H. antidysentrica, H. indicus, C. equistifolia. T. belerica, T. chebula, E. officinalis, C. sinensis, S. aromaticum and P. granatum, including Candida albicans. The active compounds were phenols, tannins and flavonoids. [37] Further experiments on toxicity testing and animal testing were not performed. Extracts of three South Indian plants, Celastrus paniculatus, Eriodendron anfractuosum and Ficus glomerata, showed inhibitory activity against fungi like Trichophyton mentagrophytes, T. rubrum, T. soudanense, Candida albicans, Torulopsis glabrata and C. krusei. [38] The methanol extract of D. metel was shown to have a significant activity against Aspergillus. Extract of S. xanthocarpum were shown to exhibit a similar activity. [39] Tulsi (Ocimum sanctum Linn.) essential oil (TEO) was found to be effective against Candida spp. Linalool was found to be a major active constituent of TEO. [40] The choloroform extract of the leaves from Wrightia tinctoria showed in vitro activity against dermatophytic and non-dermatophytic fungi. The authors opined that the indole compound, indirubin, may be the active ingredient. [41] Further experimentation was not reported. It could be concluded that the information on anti-fungal activity is limited in terms of purified product and mechanism of action. Herbal Medications in Controlled Clinical Trials on Human Subjects There is a recent review on plant extracts or phytochemicals that have been shown to inhibit the growth of oral pathogens. They help reduce the development of biofilms and dental plaques. Of importance is the fact that the review has chosen studies that clearly demonstrate adequate statistical power, blinding and standardization of extracts or purified compounds. What is of extreme importance is the quality control of such products. [42] Martin and Ernst [43] have reviewed seven randomized clinical trials of herbal products with antibacterial activity. Among the studies, trials of garlic and cinnamon treatments for Helicobacter pylori infections did not show promise. Success was seen for an ointment containing tea leaf extract for impetigo contagiosa infections. Additionally, trials of tea tree oil preparations used for acne and MRSA and a trial of Ocimum gratissimum oil for acne showed comparable results to conventional treatments like benzoyl peroxide. A summary table of potentially efficacious plant products is shown in [Table - 1]. Also, classification of the chemical nature of plant-derived anti-infectives and their proposed mechanism of action is shown in [Table - 2]. It needs to be emphasized here that these products have to be subjected to clinical testing on humans with appropriate study design and ethics consideration. Conclusions The present scenario of the treatment of infections is a mixed one. While there has been significant progress in the identification of specific allopathic drugs for treating an overwhelming majority of the infections, there are many bumps along the road. The first major problem is the emergence of organisms and viruses that have evolved to be drug resistant. It is vital to explore the medicinal properties of herbal products used traditionally by practitioners in Indian medicine. Importantly, to get greater insight into the drugs of herbal origin, an attempt should be made to identify, purify and evaluate the active principles in the herbal products. This has to be followed by carefully carried out controlled clinical trials. It is important for pharmaceutical chemists, microbiologists and practitioners of traditional and allopathic medicine to come together and facilitate discovery of what would be vital drugs for the treatment of infections in the coming decades.[44] References
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