General
NMR analysis was performed using JOEL GX-400 (400 and 100 MHz for 1H and 13C NMR), NMR Laboratory, Faculty of Pharmacy, Cairo University, Cairo, Egypt. All samples were analyzed in DMSO-d6 and CD3OD-d4 solvent. Thin layer chromatography (TLC) was performed on silica gel 60 F254 precoated aluminium sheets (20 × 20, 0.2 mm thikness) and cellulose precoated aluminium sheets (20 × 20, 0.2 mm thikness), (E. Merck, Darmstadt, Germany). Paper Chromatograpy (PC) was performed by Whatmann No.1 (Whatmann Ltd., Maidstone, Kent, England). Aluminium chloride reagent (1% in ethanol) for flavonoids and Ferric chloride reagent (1% in ethanol) were used for spots visualization. Solvent systems S1: chloroform: methanol: water ((70:30:5) & (70:30:2) v/v/v), S2: Chloroform: methanol ((80:20),(70:30)&(50:50) v/v), S3: n-butanol: acetic acid: water (BAW) (4:1:5 v/v/v, upper layer) and S4: Acetic acid: water (15:85 v/v). HR-ESI/MS analyses were carried out using a Bruker LC micro-Q-TO-F mass spectrometer, Faculty of Pharmacy, Ain Shams University, Egypt. Additionally, Microplate reader (SunRise, Tecan, USA), 96-well microtiter plates (Greiner, Germany), inverted microscope (Olympus 1 × 70, Tokyo, Japan) and Jouan® centrifuge, 1,000 – 10,000 r.p.m., France for biological studies which were performed at theMycology and Biotechnology Reginal Center, Al-Azhar University.
Plant material
Ludwigia adscendens subsp. diffusa (Forssk.) P.H.Raven syn. Ludwigia stolonifera (Guill. & Perr.) P.H.Raven (Onagraceae) aerial parts were collected from the Nile River et al.-Qanater Al-Khayriyah, El Qulyoubia governorate, Egypt, at 54VM + 52 in September 2019. The plant was botanically identified by Prof. Dr. Rim Hamdy, Botany Department, Faculty of Science, Cairo University, Egypt. A voucher specimen has been deposited at Pharmacognosy Department, Faculty of Pharmacy, Helwan university No = 31Lus1/2022. The air dried coarsely divided aerial parts (1050 g) were macerated in 5 L of 100% methanol with occasional stirring at room temperature and the process was repeated three times (3 × 5 L) till exhaustion. The methanolic extract was concentrated and dried under reduced pressure at 50 οC to give dry total extract (170 g).
Chemical reagents
Quercetin, myricetin and monosaccharaides standards were obtained from Sigma/Aldrich, USA. Dimethylsulphoxide (DMSO) was provided from Sigma, St. Louis, CA, USA. Acarbose, silymarin, doxorubicin, PC-3 cells (human prostate cancer cell line) were obtained from The Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt.
Extraction and isolation
The dried aerial parts (1050 g) of L. adscendens were macerated in 100% methanol (3 × 3L), to yield concentrated methanol extract (170 g). The dried residue (150 g) was reconstituted in 250 ml distilled water and sequentially partitioned and fractionated using different immiscible solvents (petroleum ether, chloroform, ethyl acetate and n-butanol solvents). The ethyl acetate fraction (35 g) was subjected to silica G 60 in a glass column (3 × 1.5 mm dimensions) using a step gradient chloroform and methanol mixtures. The fractions were investigated and collected according to their similarities on TLC cellulose plates using S4 solvent system and ammonia spray reagent to afford 10 main collective fractions. Fraction-III (15 g) eluted with 80% CHCl3/MeOH, was added on sephadex sub-column and eluted with BAW to afford five main sub-fractions-(1–5) according to TLC cellulose plates. Sub-fraction-3 (3 g) was further purified on sephadex sub-column to afford one pure compound 1 (15 mg). Sub-fraction-4 (6 g) was subjected to more purification on sephadex sub-column to afford two pure compounds 2 (30 mg) and 3 (20 mg). Fraction-IV (12 g) eluted with 70% CHCl3/MeOH, was further purified on sephadex column and eluted with BAW to yield five main sub-fractions according to similarity on TLC cellulose plates. Sub fractions-a (4 g) was subjected to more purification on sephadex sub-column to afford two pure compounds 4 (15 mg), and 5 (10 mg). Sub-fraction-b (3 g) was purified using sephadex LH-20 sub-column to afford one pure compound 6 (10 mg). The n-butanol fraction (35 g) was mixed with 50 ml MeOH and poured on 250 ml acetone to yield acetone precipitate (25 g) which was subjected to silica gel G 60 in a glass column using step gradient chloroform and menthol mixtures with increasing polarity from 100% CHCl3 to 100% MeOH for elution. Fractions were investigated and collected according to TLC silica plates using different solvent system and spray reagents. Fraction-III (9 g) eluted with 80% CHCl3/MeOH, was subjected to silica gel sub-column and elution with CHCl3/MeOH to afford five main sub-fractions which were collected according to TLC silica plates. Sub-fraction-A (2 g) was purified using a silica gel sub-column to afford one pure compound 7 (10 mg). Sub-fractions-B (5 g) was subjected to more purification by silica gel sub-column to afford two pure compounds 8 (15 mg) and 9 (10 mg). Fraction-IV (11 g) eluted with 70% CHCl3/MeOH, was further purified on silica sub-column and elution with CHCl3/MeOH to afford four main sub-fractions. Sub-fraction-C (4 g) was then purified by another silica sub-column to yield two pure compounds 10 (15 mg) and 11 (10 mg).
Antidiabetic activity
The antidiabetic activity of L. adscendens aerial parts fractions against acarbose was investigated in vitro using α-glucosidase-inhibitory assay as previously mentioned [11]. Briefly, in a 96-well plates a mixture of 50 μL phosphate buffer (100 mM, pH = 6. 8), 10 μL α-glucosidase (1U/mL), and 20 μL of each concentration (sample and standard) was pre-incubated for 15 min at 37 ºC. 20 μL of p-Nitrophenol (5 mM) was further added with incubation for 20 min at 37 ºC. 50 μL of Na2 CO3 (0.1 M) was added and absorbance was measured at 405 nm using Multiplate Reader. The percentage inhibition was calculated using the formula.
$$ {\text{Inhibitory activity }}\left( \% \right) \, = \, \left( {{1 } - {\text{ As}}/{\text{Ac}}} \right) \, \times {1}00 $$
Where, (As) the absorbance in the presence of extract, (Ac) is the absorbance of control.
Hepatoprotective activity
The hepatoprotective effect of L. adscendens aerial part was tested in vitro [12]. In brief, 50 μL of MTT (5 mg/mL) was added to each well containing 100 μL rpm hepatocyte suspension. The plates were incubated in the dark at 37 ˚C for an additional 4 h in 5% CO2 atmosphere. 150 μL DMSO was added and absorbance was measured at 570 nm with a microplate reader. The results were expressed as percentage of viability calculated as [(ODt/ODc)] × 100%. The 50% Effective concentration (EC50) was estimated from graphic plots of the dose–response curve for each conc using Graphpad Prism software and the following equation.
$$ {\text{Hepatoprotective percentage}}\, = \,\% {\text{ Viability of treatment group}}{-}\% {\text{ Viability of negative control}} $$
Cytotoxicity activity
Cytotoxic activity of L. adscendens aerial part fractions were tested in vitro using MTT cell viability assay as previously described [13]. Briefly, in each well plate 100 µL of fresh culture RPMI 1640 medium without phenol red then 10 µL of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) were added to each well. An 85 µL aliquot of the incubated media was replaced by 50 µL of DMSO and incubated at 37 ºC for 10 min. The optical density was measured at 590 nm with the microplate reader to determine the number of viable cells and the percentage of viability was calculated as the percentage of cell survival was calculated as follows:
$$ {\text{Surviving fraction }} = \, \left( {{\text{O}}.{\text{D}}. \, \left( {\text{treated cells}} \right)} \right)/\left( {{\text{O}}.{\text{D}}. \, \left( {\text{control cells}} \right)} \right) \, *{ 1}00 $$
The 50% inhibitory concentration (IC50) was estimated from graphic plots of the dose response curve for each concentration using Graphpad Prism software [14].