Collection and identification of the plant
The P. minima plant was collected from the medicinal plant garden Khwaja Yunus Ali University, Enayetpur, Sirajgonj, Bangladesh. The plant was identified and verified by Professor Dr. AHM Mahbubur Rahman (Taxonomist), Department of Botany, University of Rajshahi, Bangladesh. The voucher specimen (no. 317) was preserved and deposited in the herbarium of the Department of Botany, University of Rajshahi, Bangladesh.
The leaves and steam of P. minima were sun-dried for one week and leaves and stem were pulverized in a mortar and pastel to produce fine powder materials. The powder materials were screened though a 50 mesh screen and stored for experimental purposes.
Preparation of the plant extract
The air-dried fine powder of the leaves and stem of P. minima were homogenized with ethanol using soxhlet’s apparatus. The 200 gm powdered leaves and stem were defatted with petroleum ether (60–80 °C) and then subjected separately in a soxhlet extraction apparatus with ethanol (1L) for extraction. The ethanolic extract was collected and concentrated at 30 °C under reduced pressure in a rotary evaporator. 0.125, 0.25, 0.50 and 1gm of extracts were taken in four separate test tubes and mixed with 0.2% v/v of Tween 80 which was used as a suspending agent. The suspension of ethanol extract of P. minima at the concentrations of 12.5, 25, 50 and 100 mg/ml was prepared. After that, the final volume was adjusted to 10 ml for each concentration through phosphate-buffered saline.
Phytochemical analysis was performed using standard procedures available in the literature (Trease and Evans 1989; Govind and Madhuri 2006). Phytochemical screening was carried out by testing of different chemical groups present in P.minima fruits extract (Sunitha et al. 2018). Presently our research focused on the presence of chemical groups individually in leaves and stem extract of this plant for detection of alkaloids, steroids, tannins, flavonoids, glycosides, reducing sugar, saponins, terpenoids, quinine, phenols, proteins and gum in P. minima extract.
Test for reducing sugars (Fehling’s test)
Two milliliters of plant extract was added with 1 ml of a mixture of equal volumes of Fehling’s solutions A and B and boiled for some minutes for detection of reducing sugars. No precipitate was found which indicates the absence of reducing sugar (Ramakrishna et al. 1994).
Tests for tannins
Five milliliters of plant extract was taken in a test tube and added 1 ml of 5% ferric chloride solution for the detection of tannins. The black color appeared that identify the presence of tannins (Mace 1963).
Test for flavonoids
Each 2 milliliters of leaves and stem extracts was taken in a test tube and added a small amount of dilute NaOH, yellow colour has appeared. Now, a few drops of 70% dilute HCl was added and yellow colour was disappeared. This point out the presence of flavonoids in the leaves and stem extracts (Evans 1970).
Test for saponin
One milliliter of extract was diluted with 20 ml of distilled water and shaken in a measured-off test tube for 10–15 min. Foam layer has not appeared which specifies the lack of saponin properties (Kokate 1999).
Test for steroids
One milliliter of chloroform extract solution was taken and then added 2 ml Libermann-Burchard reagent. A greenish color appeared that specifies the presence of steroids (Finar 1986).
Tests for alkaloids
Two milliliters of each extract was taken in test tubes and added 0.2 ml of dilute HCl. In these two portions of the solution, 1 ml of Mayer’s reagent was added to one portion and 1 ml of Dragendorff’s reagent was added to the other. The formation of a yellow color precipitate (with Mayer’s reagent) or orange brown precipitate (with Dragendorff’s reagent) was regarded as positive for the presence of alkaloids (Evans 1970; Waldi 1965).
Tests for proteins
Take from each extract (2 ml) and add 1 ml of 40% caustic soda and a few drops of 1% copper sulfate. The formation of violet color indicates the presence of peptide bond molecules within the sample extract (Gahan 1984).
Tests for cardiac glycosides (Keller-Killiani test)
Take from every extract (1 ml) and add 0.5 ml of glacial acetic acid and 1–2 drops of 1% aqueous ferric chloride solution. The creation of a brown ring at the edge confirms the existence of cardiac glycosides in the solution of extract (Mace 1963).
Tests for terpenoids
Take from each extract (1 ml) and add 0.5 ml of chloroform and a few drops of concentrated sulphuric acid, reddish-brown precipitate to confirm the presence of terpenoids in the extract (Mace 1963).
Test for quinone
One milliliter of extract was taken and 1 ml of conc. H2SO4 was added. No color was formed which indicates the absence of quinone (Peach and Tracoy 1955).
Test for gums
Five milliliters from the extract was taken and then Molish reagent and sulphuric acid were added with them. A red or reddish-violet ring was formed at the junction of the two layers representing the presence of gums (Whistler and BeMiller 1993).
Anthelmintic activity tests
Collection of parasite samples
Collection Paramphistomum cervi from freshly slaughtered cattle were supplied from slaughter houses and confirmed by experts. After cleaning, parasites were stored in 0.9% of PBS of pH 7.4 at 37 ± 1 °C.
Standard drug used
Albendazole (collected from Square Pharmaceuticals Ltd., Bangladesh) suspension (concentration of 50 mg/ml) was used as the standard anthelmintic drug.
In vitro tests
For the present study, live parasites P. cervi of cattle were selected randomly (Dash et al. 2002; Szewezuk et al. 2003; Shivkar and Kumar 2008). Before experimentation, the flatworms were kept in the laboratory environment. The flatworms were divided into four groups and of three flatworms for each one. Albendazole suspension was used as a standard drug at a concentration 50 mg/ml, and it poured into a petri dish. The sample extracts were prepared in different concentrations (12.5 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml). Control group was treated with 0.1% tween-80 in Phosphate-buffered saline. Three parasites nearly equal in size were placed in each petri dishes and observed at room temperature (Kaushik et al. 1974). The time taken for complete paralysis and death as compared to control were recorded (Williams et al. 2014; Islam et al. 2015). The paralysis time and death time were examined according to every sample. That time was mentioned as a paralysis time when the worms to became motionless. For ensuring the death external stimuli were applied continuously to the each worm which indicates that if alive the worm, its movement would be encourage (Lal et al. 1976).
All values are reported as mean ± S.E.M. The statistical differences among groups were assessed using the Duncan multiple range test and analysis of variance (ANOVA). A value of P < 0.05 was considered significant. Statistical analysis was performed using the SAS for Windows software.