Sennoside a

The standard compounds sennoside A (≥96.0%), sennoside B (≥90.0%), aloe-emodin (≥95%), emodin (≥97.0%), and chrysophanol (≥98.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HPLC-grade methanol and acetonitrile were procured from WINLAB (Market Harborough, UK). A Millipore Milli-Q^® (Bedford, MA, USA) assembly was used to obtain highly pure water. A Millipore-Millex-HV^® filter unit with a membrane filter (0.45 µm pore size) was used for the filtration of solvents, and a syringe filter of 0.22 µm was used for sample preparation. For quantitative analysis, an Alliance 2695 separation module equipped with a 2487 dual wavelength absorbance detector (Waters Instruments, Inc., Milford, MA, USA) was used. DPPH and ABTS reagents for antioxidant assays were procured from Sigma-Aldrich (St. Louis, MO, USA).

Sephadex G-200 (discontinued product) was originally obtained from Pharmacia Biotech (now GE Healthcare). The Microsource Spectrum Collection and small-molecule library containing 2560 compounds included in the US and International Drug Collections, together with Natural Product and Discover libraries, was purchased from Microsource Discovery Systems, Inc. DMSO and H₂O₂ (30%) were from Thermo Fisher Scientific. DTPA and all other chemicals were from Sigma–Aldrich and were from the highest available purity grade. Specialty chemicals (>95% purity guarantee by manufacturer) present in the Microsource library that were purchased in powder form included the following: gambogic acid (drug G) from Shanghai Tauto Biotech Co, closantel (drug C), hematoporphyrin (drug H), hexachlorophene (drug X), and docusate sodium (drug DS) from MCE (MedChemExpress), dihydrogambogic acid (drug D), chaulmoogric acid (drug M) and hematein (drug I) from MicroSource, Inc, bixin (drug B), sennoside A (drug S), tetradecyl sulfate sodium (drug T), agaric acid (drug A) and escin (drug E) from Sigma, avocadene (drug V) and all other drugs were taken directly from the Microsource microtiter plate library as 10 mM solutions in DMSO.

Baicalin, baicalein, chrysophanic acid, emodin, aloe-emodin, and wogonoside were purchased from Tokyo Chemical Industry Co., Ltd., (Tokyo, Japan). Rhein, wogonin, palmatine chloride, coptisine chloride, and berberine chloride were purchased from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). Sennoside A was purchased from Sigma-Aldrich Co. LLC (Darmstadt, Germany). Calphostin C and N^G-nitro-L-arginine methyl ester hydrochloride (L-NAME) were purchased from Abcam PLC (Cambridge, England). Rottlerin was purchased from Enzo Life Science, Inc. (Farmingdale, USA).
Methanol, acetonitrile, and dimethyl sulfoxide (DMSO) for sample preparation and liquid chromatography were of HPLC grade. All other chemicals and reagents were of analytical reagent grade.

Sennoside content was estimated using High-performance liquid chromatography (HPLC) method. Reference standards of sennoside-A (purity 96%) and Sennoside-B (purity 94.5%) (Sigma-Aldrich, Bangalore, India), and HPLC grade methanol (Merck Specialties Pvt. Ltd, Mumbai, India) andultra pure distilled water with resistivity greater than 18 MΩ were used. Samples and solutions were filtered with 0.45µ membrane filters, while solvents were degassed prior to use. Leaf samples were collected at flowering from Cassia angustifolia (var. sona) plants grown in the experimental farm at the ICAR-Directorate of Medicinal and Aromatic Plants Research (ICAR-DMAPR), Anand, Gujarat in the year 2013. The pure and homozygous seed of ‘sona’ variety was maintained through inbreeding. The ICAR-DMAPR, Anand is located between 22.5^o N latitude and 73.0^o E longitude, having about 800 mm annual rainfall. Top 25 fresh leaves were collected in triplicates, dried, and powdered separately were used for estimation of sennoside content.

The tested compounds were purchased from commercial sources. Hydroquinone, aloin and emodin were purchased from Merck, Türkiye, while arbutin, lawsone, lapachol, juglone, chrysophanol, physcion, rhein, cascaroside A, chrysophanol-8-O-β-D-glucoside, frangulin A, sennoside A, sennoside B were commercially obtained from Sigma, Türkiye. Aloe-emodin, alkannin, and doxorubicin were provided from Cayman, Türkiye. On the other hand, emodin-8-O-β-D-glucoside was previously isolated from the roots of Rumex acetosella L. in our laboratory [35 (link)].