Collection of plant materials The Root, leaf and stem of Luisia zeleynica Lindl. were used for the qualitative estimation of alkaloids and other secondary metabolites viz. flavonoid, steroid, saponin, phlobatannin, terpinoid, tannin, glycoside, anthroquinone, quinine and coumarin. The plant was collected from Kaptai National Park, Rangamati. Samples were thoroughly washed with water and dried in oven at 650C for 48 hours. It was then ground into coarse powder by using grinding machine and stored in airtight container for further investigation. Mixing of one part with another was carefully avoided. The voucher specimen of the orchid species preserved at the herbarium of Chittagong University.
Preparation of plant extract 25 gm of sample from each of part were taken for further analysis. 50 ml of Methanol was added to the 25 gm of samples in a conical flask. Shaken very well for 30 minutes and then kept overnight and then shaken again and sonnicated for 10 minutes and then filtered using Whatman filter paper No 1. The process was repeated for three times with Methanol and the extract was then rotavaporated. The dried sample was kept as crude sample for each part. The concentrated crude extract was separated into four different solvent systems (Methanol, n-Hexane, Butanol-1, Dichloromethane) by following kupchan modified method.
Phytochemical screening Qualitative tests were carried out on the fresh sample, powdered specimens and methanol extracted crude using standard procedures to identify the constituents as described.
Test of alkaloids: For qualitative test of alkaloid, the most reliable and rapid testing method was developed and the method was slightly modified. For the qualitative test of alkaloid, five alkaloid detecting reagents were used. These are Dragendroff’s reagent (D), Hager’s reagent (H), Mayer’s reagent (M), Wagner’s reagent (W) and Tannic acid reagent (T). These reagents were prepared following the methods. 5 gm fresh finely chopped and pasted plant material was mixed up to moisten with 10 ml 2% HCL and heated in water bath of 60º C for one hour. After cooling the extract was filtered through Whatman No. 1 filter paper. Two drops of extract were put on a microscopic groove slide with one drop of the alkaloid detecting reagent. The relative abundance of precipitate, if any formed in the plant extract with the reagent was considered as an index of the quality of the presence of alkaloid and was expressed by‘+’, ‘++’ and ‘+++’ signs which mean slight, moderate, substantial to heavy amount respectively. No precipitate was indicated by ‘ - ‘ (negative sign) and stood for the absence of alkaloid in the plant extract. Phytochemical screening of Luisia zeleynica Lindl. Orchid species for secondary metabolites were analyzed following standard methods.
Test for phlobatannins: Deposition of a red precipitate when an aqueos extract of each plant sample was boiled with 1% aqueous hydrochloric acid (HCL) was taken as evidence for the presence of phlobatannins.
Test for flavonoids: A portion of the crude powdered plant sample was heated with 10 ml of ethyl acetate over a stem bath for 3 min. The mixture was filtered and 4 ml of filtrate was shaken with 1 ml of dilute Ammonia solution. A yellow coloration was observed indicating a positive test for flavonoids.
Test for saponins: About 2 gm of crude powder was boiled with 20 ml of distilled water in a water bath and filtered. 10 ml of filtrate was mixed with 5ml of distilled water and shaken vigorously for a stable persistent froth. The persistent of froth indicates the presence of saponins.
Test for tannins: About 0.5 gm of the crude powdered samples boiled in 10ml of distilled water in a test tube and filtered. A few drops of ferric chloride reagent added to the filtrate. A blue-black precipitate was taken as evidence for the presence of tannins.
Test for terpenoids: 0.5 gm of crude powder was dissolved in 5 ml of methanol. 5 ml of the extract was treated with 2 ml of chloroform in a test tube. 3 ml of concentrated sulphuric acid carefully added to the mixture to form a layer. An interface with a reddish brown coloration formed if terpenoid constituent is present.
Test for steroids: 0.5 gm of crude powder was dissolved in 5 ml of methanol 1 ml of the extract was dissolved in 10 ml of chloroform and equal volume of concentrated sulphuric acid was added by sides of the test tube. The upper layer turns red and sulphuric acid layer showed yellow with green fluorescence. This indicated the presence of steroids.
Test for glycosides: 0.5 gm crude powder was dissolved in 5 ml of methanol. 10 ml of 50% HCL was added to 2 ml of methanolic extract in a test tube. Then it was heated in a boiling water bath for 30 minutes. 5 ml of Fehling’s solution was added to the mixture and the mixture was boiled for 5 minutes. A brick-red precipitate was taken as evidence for the presence of glycosides
Antioxidant activities The antioxidant activity of the Methanolic, n-Hexane, Butanol-1 and DCM extract of the root, leaf and stem of Luisia zeleynica Lindl. and the standard antioxidant ascorbic acid were assessed on the basis of the free radical scavanging effect of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH, MWt.394.32) free radical activity according to the method described [28] with slight modification.