1H nuclear magnetic resonance-based metabolite profiling of guava leaf extract: an attempt to develop a prototype for standardization of plant extracts.

BACKGROUND: Herbal medicines are fast gaining popularity. However, their acceptability by modern practitioners is low which is often due to lack of standardization. Several approaches towards standardization of herbals have been employed. The current study attempted to recognize key peaks from 1H NMR spectra which together would comprise of a spectral fingerprint relating to efficacy of Psidium guajava (guava) leaf extract as an antidiarrhoeal when a number of unidentified active principles are involved. METHODS: Ninety samples of guava leaves were collected from three locations over three seasons. Hydroalcoholic (water and ethanol, 50:50) extracts of these samples were prepared and their 1H NMR spectra were acquired. Spectra were also obtained for quercetin, ferulic acid and gallic acid as standards. Eight bioassays reflecting different stages of diarrhoeal pathogenesis were undertaken and based on pre-decided cut-offs, the extracts were classified as 'good' or 'poor' extracts. The bioactivity data was then correlated with the 1H NMR profiles using Regression or Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA). RESULTS: OPLS-DA showed seasonal and regional segregation of extracts. Significant models were established for seven bioassays, namely those for anti-bacterial activity against Shigella flexneri and Vibrio cholerae, adherence of E. coli, invasion of E. coli and S. flexneri and production and binding of toxin produced by V. cholerae. It was observed that none of the extracts were good or bad across all the bioassays. The spectral analysis showed multiple peaks correlating with a particular activity. Based on NMR and LC-MS/MS, it was noted that the extracts contained quercetin, ferulic acid and gallic acid. However, they did not correlate with the peaks that segregated extracts with good and poor activity. CONCLUSIONS: The current study identified key peaks in 1H NMR spectra contributing to the anti-diarrhoeal activity of guava leaf extracts. The approach of using spectral fingerprinting employed in the present study can thus be used as a prototype towards standardization of plant extracts with respect to efficacy.

A randomized open label efficacy clinical trial of oral guava leaf decoction in patients with acute infectious diarrhoea.

BACKGROUND: Diarrhoea is amongst the first ten causes of death and its treatment faces an increased threat of drug resistance. Previous studies on the guava leaf decoction (GLD) revealed its suitability for use in infectious diarrhoea of unknown etiology. OBJECTIVE: The objective of this trial was to establish efficacy, dose and safety of GLD prepared from the Indian Sardar variety in adults with acute infectious diarrhoea. METHODS: The current trial was an open efficacy randomized 5-day, parallel group multi-arm interventional study. Amongst 137 adults (18-60 years) suffering with acute diarrhoea, 109 were included (57% females, 43% males). Three doses of GLD (6-leaf, 10-leaf and 14-leaf) were compared with controls receiving oral rehydration solution. Decrease in stool frequency and improvement in consistency were the outcomes measured. The data was analyzed using ANOVA, Tukey's post-hoc test, Kruscal-Wallis test and Chi-Square test where applicable. RESULTS: The trial showed that the 14-leaf (7.4 g) decoction was the most effective. Administration of the decoction, thrice daily helped the patients regain normalcy in 72 h as opposed to 120 h in controls. Safety of the intervention was reflected by normal levels of haemoglobin, liver and kidney parameters. No adverse events were reported. CONCLUSION: The 14 leaves decoction was a safe treatment for adult acute uncomplicated diarrhoea of unknown etiology. Moreover due to component synergy and divergent mechanisms of action, it could possibly combat the generation of drug resistance and destruction of gut microbiota. Hence GLD has the potential for development as a first line treatment for diarrhoea. TRIAL REGISTRATION: Trial was registered with Clinical Trials Registry - India (CTRI registration number: CTRI/2016/07/007095). The trial was retrospectively registered.

Psidium guajava: A Single Plant for Multiple Health Problems of Rural Indian Population.

The rural population in India faces a number of health problems and often has to rely on local remedies. Psidium guajava Linn. (guava), a tropical plant which is used as food and medicine can be used by rural communities due to its several medicinal properties. A literature search was undertaken to gauge the rural health scenario in India and compile the available literature on guava so as to reflect its usage in the treatment of multiple health conditions prevalent in rural communities. Towards this, electronic databases such as Pubmed, Science Direct, google scholar were scanned. Information on clinical trials on guava was obtained from Cochrane Central Register of Controlled Trials and Clinicaltrial.gov. The literature survey revealed that guava possesses various medicinal properties which have been reported from across the globe in the form of ethnobotanical/ethnopharmacological surveys, laboratory investigations and clinical trials. Besides documenting the safety of guava, the available literature shows that guava is efficacious against the following conditions which rural communities would encounter. (a) Gastrointestinal infections; (b) Malaria; (c)Respiratory infections; (d) Oral/dental infections; (e) Skin infections; (f) Diabetes; (g) Cardiovascular/hypertension; (h) Cancer; (i) Malnutrition; (j) Women problems; (k) Pain; (l) Fever; (m) Liver problems; (n) Kidney problems. In addition, guava can also be useful for treatment of animals and explored for its commercial applications. In conclusion, popularization of guava, can have multiple applications for rural communities.

Antimycobacterial Efficacy of Andrographis paniculata Leaf Extracts Under Intracellular and Hypoxic Conditions.

The inhibition of the growth of Mycobacterium tuberculosis by the extracts of Andrographis paniculata has been studied using intracellular and axenic hypoxic conditions. The inhibition (confirmed using the gold standard colony forming unit assay) was found to increase with "double stimuli" or higher concentration of the extract. Organic solvent extracts were found to inhibit bacterial growth more than the aqueous extracts under microaerophilic conditions mimicked through axenic and intracellular assays. This could be further explored to evaluate the potential of the plant to be used against nonreplicating/dormant bacilli.

Approaches in fostering quality parameters for medicinal botanicals in the Indian context.

India is among the important megabiodiversity centers of the world with nearly 45,000 known plant species. This diversity coupled with a rich heritage of traditional knowledge has made India a home to several important time-honored systems of health care such as Ayurveda, Siddha and Unani. Herbal medicines, however, are associated with a number of shortcomings including uniform efficacy and lack of appropriate quality control measures at various stages of product development. The review intends to outline the importance of fostering quality parameters towards standardization and manufacturing of botanicals for India to emerge as a leader in global market of herbal products. Literature survey was carried out on important parameters for processing and manufacturing of botanicals. The review highlights that there have been constant efforts for developing state of the art technologies in the field of herbal research. It also reflects that Government authorities have also taken a number of initiatives to formulate appropriate guidelines from standardization of raw materials to obtaining botanical products. However, in the Indian context, there exist certain lacunae in the current regulatory mechanisms which need to be strengthened and stringently implemented to ensure safety, purity and efficacy of herbal medicines. Towards this the approaches being developed globally can be adopted. Based on the literature reviewed, in our opinion, four areas viz., benefit sharing, investment by industry, standardization and national/international networking structure need immediate attention for strengthening Traditional Systems of Medicine in India.

Bactericidal Effect of Selected Antidiarrhoeal Medicinal Plants on Intracellular Heat-Stable Enterotoxin-Producing Escherichia coli.

Diarrhoeal diseases due to enterotoxigenic Escherichia coli continue to be a cause of global concern. Medicinal plants have been gaining popularity as promising antidiarrhoeal agents. In the present study, four antidiarrhoeal plants, viz. Aegle marmelos, Cyperus rotundus, Psidium guajava and Zingiber officinale were screened against a heat-stable toxin-producing enterotoxigenic E. coli strain. Decoctions of these plants were studied for their effect on intracellular killing of the bacterial strain using murine monocytic cell line, J774. [(3)H] thymidine release assay was used to evaluate the apoptotic/necrotic effect. All plants at concentrations <1% enhanced intracellular killing of the bacteria by J774 cells. However, at higher concentrations, the decoctions induced apoptosis in J774 cells. The study demonstrates that these plants could control diarrhoea caused by heat-stable toxin-producing enterotoxigenic E. coli through their immunomodulatory effect.

Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays.

BACKGROUND: In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. METHODS: Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 µg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. RESULTS: 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. CONCLUSION: A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy.

Studies on the activity of Cyperus rotundus Linn. tubers against infectious diarrhea.

To study the antidiarrheal activity of the decoction of Cyperus rotundus Linn. tubers using representative assays of diarrheal pathogenesis and understand its mechanism of action.Antibacterial, antigiardial and antirotaviral activities were studied. Effect on adherence of enteropathogenic Escherichia coli (EPEC) and invasion of enteroinvasive E. coli (EIEC) and Shigella flexneri to HEp-2 cells was evaluated as a measure of effect on colonization. Effect on enterotoxins such as enterotoxigenic E. coli (ETEC) heat labile toxin (LT), heat stable toxin (ST) and cholera toxin (CT) was also assessed. The decoction showed antigiardial activity, reduced bacterial adherence to and invasion of HEp-2 cells and affected production of CT and action of LT. The decoction of C. rotundus does not have marked antimicrobial activity and exerts its antidiarrheal action by mechanisms other than direct killing of the pathogen.

Newer insights into the mechanism of action of Psidium guajava L. leaves in infectious diarrhoea.

BACKGROUND: Psidium guajava L., Myrtaceae, is used widely in traditional medicine for the treatment of diarrhoea, dysentery, gastroenteritis, stomachaches, and indigestion. However, the effect of the leaf extract of P. guajava on the pathogenesis of infectious diarrhoea has not been studied. The present study evaluates the effect of a hot aqueous extract (decoction) of dried leaves of P. guajava on parameters associated with pathogenicity of infectious diarrhoea. The aim was to understand its possible mechanism(s) of action in controlling infectious diarrhoea and compare it with quercetin, one of the most reported active constituents of P. guajava with antidiarrhoeal activity. METHODS: The crude decoction and quercetin were studied for their antibacterial activity and effect on virulence features of common diarrhoeal pathogens viz. colonization of epithelial cells and production and action of enterotoxins. Colonization as measured by adherence of enteropathogenic Escherichia coli (EPEC) and invasion of enteroinvasive E. coli (EIEC) and Shigella flexneri was assessed using HEp-2 cell line. The production of E. coli heat labile toxin (LT) and cholera toxin (CT) and their binding to ganglioside monosialic acid (GM1) were studied by GM1-ELISA whereas the production and action of E. coli heat stable toxin (ST) was assessed by suckling mouse assay. RESULTS: The decoction of P. guajava showed antibacterial activity towards S. flexneri and Vibrio cholerae. It decreased production of both LT and CT and their binding to GM1. However, it had no effect on production and action of ST. The decoction also inhibited the adherence of EPEC and invasion by both EIEC and S. flexneri to HEp-2 cells. Quercetin, on the other hand, had no antibacterial activity at the concentrations used nor did it affect any of the enterotoxins. Although it did not affect adherence of EPEC, it inhibited the invasion of both EIEC and S. flexneri to HEp-2 cells. CONCLUSION: Collectively, the results indicate that the decoction of P. guajava leaves is an effective antidiarrhoeal agent and that the entire spectrum of its antidiarrhoeal activity is not due to quercetin alone.

Studies on the antidiarrhoeal activity of Aegle marmelos unripe fruit: validating its traditional usage.

BACKGROUND: Aegle marmelos (L.) Correa has been widely used in indigenous systems of Indian medicine due to its various medicinal properties. However, despite its traditional usage as an anti-diarrhoeal there is limited information regarding its mode of action in infectious forms of diarrhoea. Hence, we evaluated the hot aqueous extract (decoction) of dried unripe fruit pulp of A. marmelos for its antimicrobial activity and effect on various aspects of pathogenicity of infectious diarrhoea. METHODS: The decoction was assessed for its antibacterial, antigiardial and antirotaviral activities. The effect of the decoction on adherence of enteropathogenic Escherichia coli and invasion of enteroinvasive E. coli and Shigella flexneri to HEp-2 cells were assessed as a measure of its effect on colonization. The effect of the decoction on production of E. coli heat labile toxin (LT) and cholera toxin (CT) and their binding to ganglioside monosialic acid receptor (GM1) were assessed by GM1-enzyme linked immuno sorbent assay whereas its effect on production and action of E. coli heat stable toxin (ST) was assessed by suckling mouse assay. RESULTS: The decoction showed cidal activity against Giardia and rotavirus whereas viability of none of the six bacterial strains tested was affected. It significantly reduced bacterial adherence to and invasion of HEp-2 cells. The extract also affected production of CT and binding of both LT and CT to GM1. However, it had no effect on ST. CONCLUSION: The decoction of the unripe fruit pulp of A. marmelos, despite having limited antimicrobial activity, affected the bacterial colonization to gut epithelium and production and action of certain enterotoxins. These observations suggest the varied possible modes of action of A. marmelos in infectious forms of diarrhoea thereby validating its mention in the ancient Indian texts and continued use by local communities for the treatment of diarrhoeal diseases.

A community-based bacteriological study of quality of drinking-water and its feedback to a rural community in Western Maharashtra, India.

A longitudinal study of the bacteriological quality of rural water supplies was undertaken for a movement towards self-help against diseases, such as diarrhoea, and improved water management through increased community participation. Three hundred and thirteen water samples from different sources, such as well, tank, community standpost, handpumps, percolation lakes, and streams, and from households were collected from six villages in Maharashtra, India, over a one-year period. Overall, 49.8% of the 313 samples were polluted, whereas 45.9% of the samples from piped water supply were polluted. The quality of groundwater was generally good compared to open wells. Irregular and/or inadequate treatment of water, lack of drainage systems, and domestic washing near the wells led to deterioration in the quality of water. No major diarrhoeal epidemics were recorded during the study, although a few sporadic cases were noted during the rainy season. As a result of a continuous feedback of bacteriological findings to the community, perceptions of the people changed with time. An increased awareness was observed through active participation of the people cutting across age-groups and different socioeconomic strata of the society in village activities.