In vitro antigiardial and antirotaviral activity of Psidium guajava L. leaves.

Sir,

Psidium guajava L., family Myrtaceae, is used widely in traditional medicine throughout the world for a number of gastrointestinal ailments.[1] The antidiarrhoeal activity of the aqueous extract of the leaves has been studied and various mechanisms of action, namely antimicrobial activity, antispasmodic activity, inhibition of acetylcholine release, inhibition of bacterial colonization, and production/action of bacterial enterotoxins have been proposed.[12] The antigiardial and antirotaviral studies with P. guajava leaves from different geographical locations have been previously reported.[1] Because of known differences in the biological efficacy of medicinal plants due to differences in geographical locations we screened an Indian variety of P. guajava leaves for its in vitro antigiardial and antirotaviral activity.

Leaves of P. guajava variety Sardar collected from Parinche valley, Maharashtra, India were used. A hot aqueous extract (decoction) was used for the assays based on its field use as revealed during an ethnobotanical survey carried out by us.[3] The decoction was prepared by boiling 1 g of shade dried leaves in 16 mL double distilled water until the volume became 4 mL.[4] To replicate field conditions a fresh decoction was prepared every time. The decoction was centrifuged and filtered through a membrane of 0.22 µm pore size before use. The percent yield with respect to the starting dried plant material was 10.8% ± 0.5% (w/w); the dry weight was (27.0 ± 1.25 mg/mL) (n = 25). For the biological assays a 0.1% (0.027 ± 0.001 mg/mL), 1%, (0.270 ± 0.013 mg/ml), 5% (1.350 ± 0.063 mg/mL) and 10% dilutions (2.7 ± 0.125 mg/mL) prepared in appropriate media were used for each experiment.

For the antigiardial assay, Giardia lamblia P1 trophozoites (kindly provided by Dr. P. Das, National Institute for Cholera and Enteric Diseases, Kolkata, India) were incubated in the absence (control) and presence of different dilutions of the decoction in Diamond's TYI-SS medium. After 24 h, viability was estimated using trypan blue stain. Three independent experiments were carried out with duplicate tubes for control and each dilution of the decoction. Metronidazole (10 μg/mL) was used as the antigiardial control.

The entry and subsequent survival of simian rotavirus SA-11 (kindly provided by Dr. S. Kelkar, National institute of Virology, Pune, India) in MA-104 cells was assayed by the neutral red uptake assay.[4] Three independent experiments were carried out with triplicate wells for control as well as each dilution of the decoction.

The results are expressed as the mean ± standard error of the percent values from three independent experiments. The percentage in each experiment was calculated using the formula (C or T)/C × 100, where C is the mean value of the duplicate/triplicate readings of the control group and T is mean value of the duplicate/triplicate readings of the test (dilutions of the decoction) groups. Hence, the values of test groups have been represented as percentages relative to control (100%). Data were analyzed by ANOVA and Dunnett's post-test using Prism 4.0 (GraphPad, Inc., San Diego, CA, USA). P ≤ 0.05 was considered as statistically significant.

The decoction affected the viability of G. lamblia trophozoites at 5% and 10% concentrations [Figure 1a]. The antigiardial activity though statistically significant, was lesser than that of metronidazole. Rotavirus, following entry, causes death of MA-104 cells. The decoction decreased cell death in virus infected cells at 5% and 10% concentrations indicating that it had antirotaviral activity at these concentrations [Figure 1b]. The decoction at these concentrations was not cytotoxic to MA-104 cells (data not shown).

Figure 1

Antigiardial and antirotaviral activity of a decoction of Psidium guajava L. leaves (a) Anti-giardial activity; C: Control, trophozoites in medium alone; M: Trophozoites incubated with metronidazole (10 μg/ml). Values represent mean ? standard error (n= 3) of percentage viable trophozoites relative to control (100%); *P< 0.05. (b) Anti-rotaviral activity; C: Control, rotavirus infected MA-104 cells in medium alone. Values represent mean ? standard error (n= 3) of percentage dead MA-104 cells relative to control (100%); *P< 0.05.

Results of the study thus showed that the decoction of P. guajava leaves has antigiardial and antirotaviral activity. Hence, combining this data with other reported studies,[12] it can be suggested that P. guajava leaves have a broad spectrum of antimicrobial action and thus could be effective in controlling diarrhoea due to a wide range of pathogens.