A Box-Behnken Extraction Design and Hepatoprotective Effect of Isolated Eupalitin-3-O-ß-D-Galactopyranoside from Boerhavia diffusa Linn.

The objectives of this study were to optimize and quantify the maximum percentage yield of eupalitin-3-O-ß-D-galactopyranosidefrom Boerhavia diffusa leaves using response surface methodology (RSM), as well as to demonstrate the hepatoprotective benefits of the bioactive compound. The Box-Behnken experimental design was utilized to optimize the eupalitin-3-O-ß-D-galactopyranoside extraction procedure, which also looked at the extraction duration, temperature, and solvent concentration as independent variables. Boerhaviadiffusa leaves were extracted, and n-hexane, chloroform, ethyl acetate, and water were used to fractionate the dried extracts. The dried ethyl acetate fraction was thoroughly mixed in hot methanol and stored overnight in the refrigerator. The cold methanol was filtered, the solid was separated, and hot methanol was used many times to re-crystallize the solid to obtain pure eupalitin-3-O-ß-D-galactopyranoside (0.1578% w/w). The proposed HPTLC method for the validation and quantification of eupalitin-3-O-ß-D-galactopyranosidewassuccessfully validated and developed. The linearity (R2 = 0.994), detection limit (30 ng), and quantification limit (100 ng) of the method, as well as its range (100-5000 ng), inter and intraday precision (0.67% and 0.991% RSD), specificity, and accuracy (99.78% RSD), were all validated as satisfactory. The separation of the eupalitin-3-O-ß-D-galactopyranoside band was achieved on an HPTLC plate using toluene:acetone:water (5:15:1 v/v) as a developing system. The Box-Behnken statistical design was used to determine the best optimization method, which was found to be extraction time (90 min), temperature (45 °C), and solvent ratio (80% methanol in water v/v) for eupalitin-3-O-ß-D-galactopyranoside. Standard silymarin ranged from 80.2% at 100 µg/mL to 86.94% at 500 µg/mL in terms of significant high hepatoprotection (cell induced with carbon tetrachloride 0.1%), whereas isolated eupalitin-3-O-ß-D-galactopyranoside ranged from 62.62% at 500 µg/mL to 70.23% at 1000 µg/mL. More recently, it is a source of structurally unique flavonoid compounds that may offer opportunities for developing novel semi-synthetic molecules.

Analgesic, Anti-Inflammatory, Cytotoxic Activity Screening and UPLC-PDA-ESI-MS Metabolites Determination of Bioactive Fractions of Kleinia pendula.

Kleinia pendula (Forssk.) DC. is a prostrate or pendent dark green succulent herb found in the southwestern mountain regions of Saudi Arabia. The literature survey of the plant reveals a lack of phytochemical and pharmacological studies, although traditional uses have been noted. The objective of the present work was to assess the in vivo analgesic and anti-inflammatory activities, as well as, the in vitro cytotoxic potential of the fractions of Kleinia pendula, and correlate these activities to the plant metabolites. The methanolic extract of Kleinia pendula was subjected to fractionation with n-hexane, ethyl acetate, chloroform, n-butanol, and water. The fractions were screened for their analgesic and anti-inflammatory activities, as well as cytotoxic activity against breast, liver, and colon cancer cell lines. The n-hexane and chloroform fractions of Kleinia pendula showed significant cytotoxic activity against all three cancer cell lines tested. The ethyl acetate and chloroform fractions showed significant analgesic and anti-inflammatory activities. The metabolites in these three active fractions were determined using UPLC-PDA-ESI-MS. Thus, the analgesic and anti-inflammatory activities of the plant were attributed to its phenolic acids (caffeoylquinic acid derivatives, protocatechuic, and chlorogenic acids). While fatty acids and triterpenoids such as (tormentic acid) in the hexane fraction are responsible for the cytotoxic activity; thus, these fractions of Kleinia pendula may be a novel source for the development of new plant-based analgesic, anti-inflammatory, and anticancer drugs.

Repeated Testing With the Hypertonic Saline Assay in Mice for Screening of Analgesic Activity.

BACKGROUND: In vivo animal assays are a cornerstone of preclinical pain research. An optimal stimulus for determining the activity of potential analgesics would produce responses of a consistent magnitude on repeated testing. Intraplantar (i.pl.) injection of hypertonic saline (HS) in mice produces robust nociceptive responses to different analgesics, without evidence of tissue damage. Here, we investigated whether the nociceptive response is changed by repeating the injection at different times and sites in a mouse and whether it is attenuated by morphine. METHODS: We conducted randomized and blinded experiments to assess responses to repeated i.pl. 10% HS in female CD-1 mice. An injection of HS was followed by a second injection into the same hind paw at 4 hours, 24 hours, or 7 days. A separate group of mice each received i.pl. injections at 5, 10, and 15 days. In 2 independent experiments, 30 minutes after initial HS injections in the ipsilateral hind paw, mice received HS injection into the contralateral hind paw or ipsilateral forepaw. The ability of morphine to block the nociceptive responses was examined by injecting morphine at 5-day intervals. RESULTS: Repeated injection of HS did not alter the responses at 4 hours (84 vs 75 seconds; mean difference [95% CI], -9 [-40 to 23]; P = .6), 24 hours (122 vs 113 seconds; -6 [-24 to 12]; P = .5), or 7 days (112 vs 113 seconds; -0.3 [-12 to 11]; P = .95) or at multiple injections (day 0, 122 seconds vs day 5, 121 seconds; -0.3 [-28 to 27], P > .99; day 10, 118 seconds; 2.5 [-36 to 41], P = .99; day 15, 119 seconds; 2 [-36 to 38], P = .99). A previous hind paw injection did not change the responses of the contralateral hind paw (right, 93 seconds versus left, 96 seconds; -3 [-20 to 13], P = .7) or of the ipsilateral forepaw (forepaw after HS, 146 seconds versus forepaw after 0.9% saline, 149 seconds; -3 [-28 to 22], P = .8). Morphine dose-dependently attenuated HS responses (control, 94 seconds vs 4 mg/kg, 66 seconds; 29 [-7 to 64], P = .12; vs 10 mg/kg, 27 seconds; 67 [44-90], P < .0001; 4 vs 10 mg/kg, 67 [44-90], P = .03). CONCLUSIONS: The repetition of i.pl. HS produces consistent reproducible responses without tissue damage. This results in efficient, rapid detection of analgesic activity, reducing the number of animals required.