Saponins

The total phenolic content was determined by employing the methods given in the literature (Slinkard and Singleton, 1977 (link)) with some modification. Sample solution (1 mg/mL; 0.25 mL) was mixed with diluted Folin–Ciocalteu reagent (1 mL, 1:9, v/v) and shaken vigorously. After 3 min, Na₂CO₃ solution (0.75 mL, 1%) was added and the sample absorbance was read at 760 nm after a 2 h incubation at room temperature. The total phenolic content was expressed as milligrams of gallic acid equivalents (mg GAE/g extract) (Vlase et al., 2014 ).
The total flavonoids content was determined using AlCl₃ method (Zengin et al., 2014 (link)). Briefly, sample solution (1 mg/mL; 1 mL) was mixed with the same volume of aluminum trichloride (2%) in methanol. Similarly, a blank was prepared by adding sample solution (1 mL) to methanol (1 mL) without AlCl₃. The sample and blank absorbances were read at 415 nm after a 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. Rutin was used as a reference standard and the total flavonoid content was expressed as milligrams of rutin equivalents (mg RE/g extract) (Mocan et al., 2015 (link)).
The total saponins content of the extract was determined by the vanillin-sulfuric acid method (Aktumsek et al., 2013 (link)). Sample solution (1 mg/mL; 0.25 mL) was mixed with vanillin (0.25 mL, 8%) and sulfuric acid (2 mL, 72%). The mixture was incubated for 10 min at 60°C. Then the mixture was cooled for another 15 min, followed by the sample absorbance measurement at 538 nm. The total saponin content was expressed as milligrams of quillaja equivalents (mg QAE/g extract).
The total triterpenoids content of the extracts was determined according to Zhang et al. (2010) (link) method with some modifications. Briefly, sample solution (1 mg/mL; 500 μL) was mixed with the vanillin–glacial acetic acid (5%, w/v, 0.5 mL) and 1 mL of perchloric acid. The mixture was incubated at 60°C for 10 min, cooled in an ice water bath for 15 min and then 5 mL glacial acetic acid was added and mixed well. After 6 min, the absorbance was read at 538 nm. Oleanolic acid was used as a reference standard and the content of total triterpenoids was expressed as oleanolic acid equivalents (mg OAE/g extract) through a calibration curve with oleanolic acid.
HPLC-PDA analyses were performed on a Waters liquid chromatograph equipped with a model 600 solvent pump and a 2996 photodiode array detector, and Empower v.2 Software (Waters Spa, Milford, MA, United States) was used for acquisition of data. A C18 reversed-phase packing column (Prodigy ODS (3), 4.6 × 150 mm, 5 μm; Phemomenex, Torrance, CA, United States) was used for the separation and the column was thermostated at 30 ± 1°C using a Jetstream2 Plus column oven. The injection volume was 20 μL. The mobile phase was directly on-line degassed by using Biotech DEGASi, mod. Compact (LabService, Anzola dell’Emilia, Italy). Gradient elution was performed using the mobile phase water-acetonitrile (93:7, v/v, 3% acetic acid) (Zengin et al., 2016 (link)). The UV/Vis acquisition wavelength was set in the range of 200–500 nm. The quantitative analyses were achieved at maximum wavelength for each compound.

A small portion of the dry extract was used for the phytochemical tests for compounds which include tannins, flavonoids, alkaloids, saponins, and steroids in accordance with the methods of [17 ,18 ] with little modifications. Exactly 1.0 g of plant extract was dissolved in10 ml of distilled water and filtered (using Whatman No 1 filter paper) A blue colouration resulting from the addition of ferric chloride reagent to the filtrate indicated the presence of tannins in the extract. Exactly 0.5 g of the plant extract was dissolved in 5 ml of 1% HCl on steam bath. A millilitre of the filtrate was treated with few drops of Dragendorff's reagent. Turbidity or precipitation was taken as indicative of the presence of alkaloid. About 0.2 g of the extract was dissolved in 2 ml of methanol and heated. A chip of magnesium metal was added to the mixture followed by the addition of a few drops of concentrated HCl. The occurrence of a red or orange colouration was indicative of the flavonoids. Freshly prepared 7% blood agar plate was used and wells were made in it. The crude extract dissolved in 10% methanol was used to fill the wells bored in the blood agar plates. Ten percent methanol was used as a negative control while commercial saponin solution was used as a positive control. The plates were incubated at 35°C for 6 h. complete haemolysis of the blood around the extract was indicative of saponin. About 0.5 g of the extract was dissolved in 3 ml of chloroform and filtered. Concentrated H₂SO₄ was carefully added to the filtrate to form lower layer. A reddish brown colour at the interface was taken as positive for steroid ring.

In the search for new anti-Alzheimer’s and neuroprotective drugs from Polygonacae, P. hydropiper L. was identified, collected and processed for fractionation as previously reported from our laboratory (Ayaz et al., 2014b (link), 2016 (link)). A plant specimen was deposited with voucher no H.UOM.BG.107 at the herbarium of the University of Malakand, Chakdara, Dir (L), KP, Pakistan. Solvent based fractions and saponins were initially subjected to in vitro anti-Alzheimer’s studies. The solvent fractions, especially chloroform and ethyl acetate were selected for the isolation of pure compounds due to their prominent activities in preliminary assays. Several compounds were isolated using gravity column chromatography eluting with n-haxane and ethyl acetate. The purified compounds were rotary evaporated to remove any remaining solvent. Initially, ¹H NMR spectra was obtained to reveal the chemical structure by comparing the spectra with those reported in the literature. The ¹³C NMR spectra were used to ascertain the carbon skeleton of the compounds. The spectral data was supplemented by using mass spectrometry to confirm the structures. Among the isolated compounds, β-sitosterol was most active in the preliminary analysis and was evaluated in detail (Figure 1).

Senna occidentalis roots were collected from Migori county (0.9366^o S, 34.4198^o E), Kenya in the months of August and September. This plant was identified by Mr. Jonathan Ayayo, a taxonomist of the National Museums of Kenya (NMK), and a voucher specimen (38/81) deposited at East African Herbarium of NMK for future reference. Further, the plant name was verified with http://www.theplantlist.org on 10/05/2022.
The collected plant roots were air dried in the shade, ground into powder and stored in airtight plastic containers at 4 °C until extraction.
Hexane, chloroform, ethyl acetate and methanol, in their absolute forms, as well as distilled water were used for extraction by maceration. For organic solvents, the plants root powder was macerated separately with the solvents for 48 hours in an orbital shaker and a filtrate obtained (Whatman No. 1 filter paper). For water, the roots powder was soaked in double distilled water for 24 hours. In addition, a decoction was prepared by boiling the roots powder in double distilled water for 30 minutes at a mean temperature of 95 °C [38 (link)]. Filtration of aqueous extracts was done through a cotton wool plug followed by filtration (Whatman No. 1 filter paper). The aqueous decoction was included to mimic the local’s traditional method of preparing the antimalarial therapy. The filtrates were concentrated by rotary vaporization (BÜCHI R-200 rotary evaporator) at 50 °C and reduced pressure for organic solvents, and lyophilization (NANBEI freeze dryer: NBJ-10-1, Zhengzhou, China) for aqueous solutions. Upon drying, the extracts were stored in sealed sample bottles at 4 °C until needed.
Plant secondary metabolites are excellent predictors of their bioactivity potential [39 (link)]. In order to predict the bioactivity of S. occidentalis roots extract, .standard procedures were used to screen the extracts for the presence of saponins, tannins, alkaloids, flavonoids and sterols [40 (link)].