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.

Suspensión oral antidiarreica de Psidium guajaba, L / Oral antidiarrheal suspension from Psidium guajaba L

Se efectuó la estandarización de la droga cruda de Psidium guajaba, L., así como el diseño y elaboración de una suspensión oral antidiarreica a partir de dicho material vegetal previamente secado y tamizado. En la estandarización de la droga cruda, se encontró que todos los parámetros de calidad evaluados estaban dentro de los límites establecidos por la Norma Ramal de Salud Pública (NRSP) 308,1992. A la formulación diseñada se le determinó su estabilidad física, química y microbiológica, con resultados satisfactorios durante los 270 d de evaluación. Finalmente se desarrolló un estudio preclínico preliminar de la suspensión teniendo en cuenta las pruebas farmacológicas en ratas, y se obtuvo como resultado que la formulación presentaba efecto antidiarreico a las dosis de 3, 6 y 9 mg/kg de peso, bajo condiciones experimentales en los animales evaluados

The impact of face-to-face educational outreach on diarrhoea treatment in pharmacies.

Private pharmacies are an important source of health care in developing countries. A number of studies have documented deficiencies in treatment, but little has been done to improve practices. We conducted two controlled trials to determine the efficacy of face-to-face educational outreach in improving communication and product sales for cases of diarrhoea in children in 194 private pharmacies in two developing countries. A training guide was developed to enable a national diarrhoea control programme to identify problems and their causes in pharmacies, using quantitative and qualitative research methods. The guide also facilitates the design, implementation, and evaluation of an educational intervention, which includes brief one-on-one meetings between diarrhoea programme educators and pharmacists/owners, followed by one small group training session with all counter attendants working in the pharmacies. We evaluated the short-term impact of this intervention using a before-and-after comparison group design in Kenya, and a randomized controlled design in Indonesia, with the pharmacy as unit of analysis in both countries (n = 107 pharmacies in Kenya; n = 87 in Indonesia). Using trained surrogate patients posing as mothers of a child under five with diarrhoea, we measured sales of oral rehydration salts (ORS); sales of antidiarrhoeal agents; and history-taking and advice to continue fluids and food. We also measured knowledge about dehydration and drugs to treat diarrhoea among Kenyan pharmacy employees after training. Major discrepancies were found at baseline between reported and observed behaviour. For example, 66% of pharmacy attendants in Kenya, and 53% in Indonesia, reported selling ORS for the previous case of child diarrhoea, but in only 33% and 5% of surrogate patient visits was ORS actually sold for such cases. After training, there was a significant increase in knowledge about diarrhoea and its treatment among counter attendants in Kenya, where these changes were measured. Sales of ORS in intervention pharmacies increased by an average of 30% in Kenya (almost a two-fold increase) and 21% in Indonesia compared to controls (p < 0.05); antidiarrhoeal sales declined by an average of 15% in Kenya and 20% in Indonesia compared to controls (p < 0.05). There was a trend toward increased communication in both countries, and in Kenya we observed significant increases in discussion of dehydration during pharmacy visits (p < 0.05). We conclude that face-to-face training of pharmacy attendants which targets deficits in knowledge and specific problem behaviours can result in significant short-term improvements in product sales and communication with customers. The positive effects and cost-effectiveness of such programmes need to be tested over a longer period for other health problems and in other countries.

PIP: Controlled trials in Kenya and Indonesia documented the efficacy of face-to-face educational outreach in improving the response of private pharmacists to childhood diarrhea. Previous research had indicated that pharmacists in developing countries often lack scientific information about diarrhea and its treatment and face pressure from drug companies to sell specific products, including antidiarrheals and antibiotics. The World Health Organization Program for the Control of Diarrheal Diseases (WHO-CDD) Guide for Improving Diarrhea Treatment Practices of Pharmacists and Licensed Drug Sellers was used in one-to-one meetings between owners and employees in 194 pharmacies and diarrhea program educators. Pharmacists and counter attendants were provided with materials promoting oral rehydration solution (ORS), instruction on the indications for medication, and training in effective communication techniques. Trained surrogate patients posing as mothers of a young child with diarrhea were used to validate pharmacist self-reports. Before training, 67% of staff in Kenya but only 16% of those in Indonesia considered fluid replacement to be the most essential aspect of diarrhea treatment. At baseline, 66% of pharmacists in Kenya and 53% of those in Indonesia reported they sold ORS to the last customer having a child with diarrhea; however, only 33% of surrogate patients in Kenya and 5% in Indonesia were actually sold ORS. Moreover, 48% of surrogate patients in Kenya and 74% in Indonesia were sold antidiarrheal medications. Focus group discussions indicated that both pharmacists and their customers felt something stronger than ORS was needed to stop diarrhea and acknowledged that drug sales netted higher profits than ORS sales. After training, however, ORS sales increased by an average of 30% in Kenya and 20% in Indonesia and there was a trend toward increased questioning of customers, especially about signs of bacterial diarrhea.