Antioxidant and Anti-inflammatory Potential of Cissus quadrangularis L. Stem Extracts

• Cite this article as: Latif M^1*,
• Umar MI¹,
• Rahim A¹,
• Khan SA¹,
• Fatima N¹,
• Hussain I¹

1. ¹ et al. Isolation and biological evaluation of some secondary metabolites from seeds of Silybum marianum. Phcog Mag 2020;16:S93-8. MUHAMMAD LATIF, et al.: Phytochemical Evaluation of Silybum marianum S94 Pharmacognosy Magazine, Volume 16, Issue 68, January-March 2020 (Supplement 1) INTRODUCTION Silybum marianum (SM), previously known as Cardus marianus, is a member of Asteraceae family frequently recognized as Daisy family. It is a biennial or an annual plant indigenous to North Africa, North America, Mediterranean region, Europe, Middle East, and Australia.[1] It is also common in India at a height of 1800–2400 m.[2] Milk thistle is the common name of the plant, which is given due to the presence of “milky white” veins on the surface of leaves. The seeds of SM have been used as a therapeutic source from thousands of years, and Theophrastus was the first who reported this plant as a source of remedy and cure. The medicinal value of SM is well known for over 2000 years, and frequent use of its seeds has been reported in the West (European countries) as a therapeutic agent in the treatment of several diseases such as hepatic ailments (to remove gall stones), for pregnant women (as a bitter tonic), anxiolytic issues, stomach acidity, varicose veins, splenic congestions, uterine hemorrhage, amenorrhea, and menstrual disorders.[3‑5] It has shown promising results in ameliorating pesticide‑induced hepatotoxicity.[6] Similarly, its extract has exhibited tremendous cardioprotective potential by improving healing after a myocardial infarction.[7] When used in combination, SM extract has potentiated the antidiabetic and antibacterial activities of zinc oxide nanoparticles.[8] SM has a variety of natural products with promising biological potential. Among them, antioxidants are those compounds which can stop or slow down the oxidation of biomolecules such as lipid oxidation by hindering the chain reactions of oxidation and have the ability to give protection or reconstruct cellular damage that can occur in the body because of oxygen.[9] The present study was designed to investigate the phytochemical and cytotoxic potential of SM seed extract with special focus on its antibacterial, antifungal, and antileishmanial effects. MATERIALS AND METHODS Collection and extraction of plant seeds Plant material of SM was collected from Haripur district of Khyber Pakhtunkhwa (610 m above the see level), followed by identification by a taxonomist at Quaid‑i‑Azam University, Islamabad, Pakistan (herbarium voucher no. QAU/Bot‑Herb‑14328). The seeds were dried, crushed, pulverized into powdered form by using a heavy‑duty blander, and weighed (~2.5 kg). The powdered material was macerated in methanol for 9 days with occasional shaking to achieve maximized extraction of the seed constituents and kept in a dry shaded place at room temperature. The filtrate was evaporated under reduced pressure by using a rotary evaporator at 30°C to complete dryness to yield crude seed extract. The dried seed extract was preserved till further experimentation. Fractionation via column chromatography A slurry of crude methanolic extract (20 g) was prepared by dissolving the extract in minimum volume of 10% (MeOH in CHCl3) and adsorbed on silica gel by keeping the sample to adsorbent (1:1.5) proportion. The sample was loaded on the top of a chromatographic column packed with silica gel as stationary phase and eluted with 100% (CHCl3) to 10:1 (CHCl3: MeOH) followed by 50% (CHCl3: MeOH) in gradient manner. Forty fractions were collected and further subjected to normal‑phase thin‑layer chromatography (TLC) analysis. Based on TLC investigation, the fractions with similar Rf values were pooled up to obtain a total of ten fractions from SA1 to SA10, having % yield of 12.5, 18, 11.25, 5, 7.5, 10, 12.5, 12.75, 5, and 4, respectively. The fractions were used for further phytochemical and biological evaluation. Antioxidant assays 2,2‑diphenyl‑1‑picrylhydrazyl (DPPH) free radical scavenging activity of fractions (SA1–SA10) was determined by a previously reported method with slight modification.[10‑12] Briefly, 3.2% of DPPH reagent, 4% of ascorbic acid, and 1% of each sample fraction was prepared as stock solution. A volume of 20 μL of each fraction to be tested was taken in 96‑well plate followed by the addition of 180 μL of DPPH reagent in each well to make the final volume of 200 μL and incubated for 1 h at 37°C. Ascorbic acid and ethanol were used as positive and negative controls, respectively. The DPPH reduction activity was measured by reading the absorbance at 517 nm. The experiment was performed in triplicate, and the percentage of final scavenging was calculated by the following formula: DPPH scavenging activity (%) = (A0 – A1/A1) ×100. The antioxidant capacity of all fractions (SA1–SA10) was determined using reported methodology with slight modifications.[13] Premeasured 1.63 mL H2SO4 (conc.), NaH2SO4 (1.679 g), and ammonium‑molybdate (0.247 g) were dissolved in a 100‑mL volumetric flask and finally, the volume was made up to 100 mL. In 1 mL of dimethyl sulfoxide (DMSO), 4 mg of ascorbic acid was dissolved to prepare a stock solution of ascorbic acid. First, 1 mL of the reagent was taken in an Eppendorf tube, in which 0.1‑mL sample was added and mixed properly. The mixture was then placed in an incubator at about 95°C for 1.5 h. The mixture was cooled to 28°C, and the absorbance of the mixture was measured at 695 nm with the help of a microplate reader. For calibration curve, ascorbic acid was tested at various concentrations (125, 100, 75, 50, and 25 μg/mL). The reducing power of the plant extracts was determined by using the method reported previously with slight modifications.[10] Phosphate buffers (0.2 M), potassium ferric cyanide (1%), trichloroacetic acid (10%), and ferric chloride (0.1%) were used as stock solutions. Each fraction (200 μL) was taken in the Eppendorf tube and was added with buffer (500 μL). Then, potassium ferric cyanide (500 μL) was added and incubated for 20 min at 50°C. After incubation, trichloroacetic acid (500 μL) was added, and the mixture was then subjected to centrifugation for 10 min at 3000 rpm. A volume of 100 μL of the upper layer was removed and carefully poured into the assigned well. Ferric chloride (1%) was further added to each well followed by the addition of distilled water (20 μL) in each well. The absorbance was measured by a microplate reader at 630 nm wavelength. Phytochemical analysis The total phenolic content (TPC) was determined by using Folin–Ciocalteu assay.[14] Folin–Ciocalteu reagent (FCR) and distilled water (in 1:10 v/v ratio), 6% sodium carbonate, and 4% gallic acid in methanol were used as stock solutions. In 96‑well plate, the sample (20 µL) was taken followed by the addition of FCR (90 µL) and incubated at 40°C for 5 min. After an interval of 5 min, 6% sodium carbonate solution (90 µL) was added and the reaction mixture was incubated at 40°C for 60 min. Gallic acid and DMSO were used as positive control and negative control, respectively. The absorbance was measured at 630 nm on a microplate reader. The total flavonoid content (TFC) was measured by aluminum chloride colorimetric assay protocols as described earlier.[14] The stock solutions of aluminum chloride (10%), potassium acetate (1.0 M), and quercetin (4 mg/mL) in DMSO were prepared. To a sample (20 µL) placed in 96‑well plate, aluminum chloride (10 µL), potassium acetate (10 µL), and distilled water (160 µL) were added to make a final volume to 200 µL and incubated for 30 min at room temperature. Quercetin and DMSO were used as positive control and negative control, respectively. The absorbance was measured at 405 nm using a microplate reader. For preliminary phytochemical screening and identification of bioactive components in MESM, several phytochemical investigations were carried out by using the standard procedures described previously with slight modifications.[15‑17], Pakistan.

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