In Vitro Effect of Punica granatum and Nigella sativa Extracts on Schistosoma mansoni Cercariae, Miracidia and Adult Worms

Abstract

Schistosomiasis, is a disease caused by a blood borne flukes. It is the second most prevalent tropical disease after malaria and it causes great public health and socio-economic problems in developing countries. More than 200 million people suffer from the disease and about 500-600 million people in 74 countries are at risk of infection. Synthetic compounds have been used to interrupt the life cycle of Schistosoma. However, right concentrations, these compounds are harmful to the environment. Also some other synthetic compounds, the most popular of which is praziquantel are used in treatment of infection with bilharzia; however, there are some reports of tolerance of the worm to higher concentrations of the drug. Therefore, many medicinal plants have been screened to disrupt one or more stage of Schistosoma life cycle, as they are safer to the environment than synthetic compounds.In this study, tannins and alkaloids of different parts of pomegranate as well as saponins and alkaloids of black seeds were tested for their biocidal activity against miracidia, cercariae and adult worms. All tested tannins and alkaloids were lethal to miracidia, cercariae and adult worms. Miracidia were the most sensitive to the lethal effect of the tested compounds. A concentration as low as 0.39 ppm of tannins was enough to kill 100% of miracidia after 50-150 min and to kill 50% of miracidia within 25-50 min. At a higher concentration (50 ppm) the lethal time for 100% of miracidia ranged between 5 and 15 min and the lethal time for 50% mortality of miracidia ranged between 0.5 and 6 min. Placenta tannins were the most potent tannins of the tested pomegranate tannins. Fifty ppm pomegranate root and stem alkaloids killed 100% miracidia after 0.6 and 50 min respectively and 0.39 ppm killed 50% miracidia after 0.6 and 21.5 min respectively. Nigella sativa alkaloids and saponins were lethal to 100% miracidia at 50 ppm after 5 and 25 min respectively, while 50% miracidia were killed after 0.6 and 16.6 min respectively. At a low concentration of 0.39 ppm, 100% of miracidia were killed after 150 and 180 min respectively and 50% miracidia were killed after 41 and 51 min. Amongst the 6 tested compounds purified from pomegranate stem alkaloids were the most potent cercaricides, followed by, placenta tannins, rind tannins, root tannins, stem tannins and root alkaloids respectively. The LT100% was achieved in a time range of 50 to 700 min at 0.39 ppm and 5 to 60 min at 50 ppm. On the other hand, the LT 50% was achieved after 47 to 300 min at 0.39 ppm and 0.7 to 40 min at 50 ppm. Stem alkaloids which were the most potent tested compounds of pomegranate killed 50% of cercariae after 0.7 and 37 min at concentrations levels of 50 and 0.39 ppm and killed 100% cercariae after 5 and 50 min at concentrations of 50 and 0.39, respectively. Black seeds extract killed 100% of the tested cercariae after 35-330 min in case of alkaloids and 50-400 min when saponins were used in concentrations ranging between 50-0.39 ppm were tested. On the other hand, the LT50% was achieved after 19.3-127.1 min when alkaloids were used and 27.8-312.1 min when saponins were used at concentrations ranging between 0.39 and 50 ppm. The eight compounds extracted from pomegranate and black seeds were tested in vitro for their lethal effect on adult worms and were compared with praziquantel at three concentrations (25, 50, and 100 ppm). All tannins were lethal at 100 ppm to effect 100% mortality in worms after 24-120 hr. The most powerful tannin on S.mansoni adult worm was rind tannin. After 120 hours, 40% mortality was observed at 25ppm. At a concentration of 50 ppm, 20, 60 100% of worms were killed after, 72, 96 and 120 hours respectively. At 100 ppm, 20, 60 100% of worms were killed after 24, 48 and 72 hr respectively. It should be mentioned that worms looked paralyzed and deformed, even at non-lethal low concentrations of pomegranate tannins. Alkaloids from the root of P. granatum caused 50% and 100 % mortality of S. mansoni after 24 h and 48 h, respectively at 100 ppm concentration. None of the tested worms died at the other lower concentrations till the fifth day of the experiment. N. sativa alkaloids were less potent than pomegranate stem alkaloids. None of the tested concentrations had any effect after the first 72 h of the experiment. After 96 and 120 h at 100ppm, 25% and 50 % mortality of the worms was observed respectively. On the other hand 25 and 50 ppm were lethal to 25 % of the worms after 120h. Nigella sativa saponins also showed a higher lethal effect than alkaloids on worms. Fifty percent mortality of the worms was observed after 24 h and 100% mortality was observed after 48 h. At 50 and 25 ppm, 25% and 50% of the worms died, respectively after 120 h. Praziquantel which is the drugs of choice for treatment of Schistosomiasis was used as a positive control. One hundred percent mortality of S. mansoni adult worms was observed after 48 h for all the concentrations used (25 ppm, 50 ppm, and 100 ppm). After 24 h 50% of worms were killed at 25 ppm and 75% of worms were killed by both 50 ppm and 100 ppm. Saponins of black seeds and rind tannins of pomegranate exist abundantly in both plants and they were found to have a potent lethal effect on adult schistosomes. Therefore, they were chosen for further investigation of their effect on the tegument using scanning electron microscope. Both drugs were found to cause changes in the topography of the worms and the effect of both drugs was dependent on the dose used. Saponins caused, swelling of the tegument, wrinkling and fusion of tegumental ridges, loss of tubercles, peeling. Peeling caused by saponins was severe enough to produce large ulcers in both male and female worms. On the other hand, rind tannins of pomegranate caused, erosion of tegument, though not to the extent of ulceration as in the case of N. sativa saponins. It also caused wrinkling, swelling, loss of spines and degeneration and rupture of tubercles and this was associated with blebbing. The detrimental effect of tegumental damage was discussed in the thesis. Severe damages to suckers of the worms were observed by both rind tannins of pomegranate and saponins of black seeds which might result in a loss of the ability of worms to ingest blood and to attach themselves to blood vessels. Histopathological study of worms treated with P. granatum tannins and N. sativa saponins revealed a severe damage of sub-tegumental layers including longitudinal and circular muscle fibers, which suffered from focal lysis, vacuolization, and degenerations. Total protein profile of adult worms (both males and females) treated with rind tannins and of P. granatum and saponins of N. sativa was investigated by SDS- polyacrylamide gel electrophoresis. Changes in 6 proteins of different molecular weights were detected. Proteins of molecular weights, 59, 18 and 13 kDa decreased in amounts compared to untreated worms, while a protein of a molecular weight, 55 kDa, increased in amounts. Possible effect of level of protein expression observed energy production, viability and morphology of worms was discussed.