Evaluation of the influence of green extraction solvents on the cytotoxic activities of Crinum (Amaryllidaeae) alkaloid extracts using in-vitro-in-silico approach.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional use of Amaryllidaceae plants to treat many disease have been known for a very long period of time. The chemical analysis of these plants has yielded a diversity of alkaloids with analgesic, anticholinergic, antitumor and antiviral activities. Crinum bulbispermum (Burm.f.) Milne-Redh. & Schweick in particular has been used by Zulu, Sotho and Tswana people to treat tumors as a form of chemotherapy, while in Madagascar, Crinum powellii Baker Handb. was used in the treatment of abscesses and tumors. Many of the alkaloids spawned by genus Crinum will surely take part in the production of anticancer drugs but their further clinical development is restricted by their limited commercial availability. An emerging area of research is the establishment of green extraction techniques of different targeted compounds. AIM OF THE STUDY: Our comparative study has investigated the possibility of getting improved biological responses by changing extraction solvent to a better and greener one. This study aimed to assess the cytotoxic activity of Crinum powellii and Crinum bulbispermum bulbs, when extracted by different green solvents. MATERIALS AND METHODS: The green solvents Genapol X-80 (a surfactant-aided extraction), DES-3 (Choline chloride: fructose 5:2) mixture (a natural deep eutectic solvent) and purified distilled water were used for extraction of the bulbs. Extracts were tested against two cell lines HEPG-2 and HCT 116, with doxorubicin as a positive reference. Molecular docking studies were carried out to illustrate binding orientations of the alkaloids in the active site of several molecular targets for treatment of hepatic and colorectal cancer. RESULTS: DES aided extraction showed highest cytotoxicity against the two cell lines, followed by surfactant aided extracts and finally aqueous extracts. There is an obvious relationship between alkaloidal content and antiproliferative potency of extracts. Multivariate statistical analyses were performed to aid the prediction of the alkaloids responsible for the activity. The alkaloid crinine showed high correlation coefficient value against HCT colon cancer cell line in the orthogonal projection to latent structures (OPLS) model, suggesting that it could operate with a selective mode of action on this cell line. In addition, the alkaloid lycorine had almost no correlation to anti-proliferative activity against HCT colon cancer cells. Molecular docking studies confirmed the same conclusions. CONCLUSION: Herein, it was demonstrated that natural deep eutectic solvents (NADES) components and surfactant solutions could be chosen to enhance biological activity of extracts prepared.

Green techniques in comparison to conventional ones in the extraction of Amaryllidaceae alkaloids: Best solvents selection and parameters optimization.

An undisputed trend in sample preparation at present is to meet the requirements of green chemistry especially in the field of natural products. Green technology continuously pursues new solvents to replace common organic solvents that possess inherent toxicity. Over the past two decades, non-ionic surfactants have gained enormous attention from the scientific community. The micelle-mediated extraction and cloud-point preconcentration (CPE) methods offer a convenient alternative to the conventional extraction systems. Recently, natural deep eutectic solvents (NDESs) have emerged as green and sustainable solvents for efficient extraction of bioactive compounds or drugs. They are generally composed of neutral, acidic or basic compounds that form liquids of high viscosity when mixed in certain molar ratio. The presented work aimed to comprehensively compare and evaluate the potential and effectiveness of NDES as well as non-ionic surfactants (Genapol X-080, Triton X-100 and Triton X-114) for extraction of Amaryllidaceae alkaloids from Crinum powellii bulbs as representative example of plant material, in comparison to the conventional solvents (methanol, ethanol and water).A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of three alkaloids markers, lycorine, crinine and crinamine, in the bulbs of C. powellii. Extraction efficiency of the targeted alkaloids from the bulb matrix with organic and ecofriendly (green) solvents were studied. Results revealed that NDES and surfactants were significantly more efficient in alkaloid extraction than previous methods requiring the consumption of organic solvents and water. Genapol X-80 demonstrated 138%, 149% and 145%, while choline chloride: fructose (5:2): H2O (35%) NDES mixture demonstrated 243%, 225% and 238% of the total alkaloidal extraction capacity of ethanol, methanol and water, respectively at 50 °C for extraction time 1 h using ultrasonication for all experiments. Furthermore, Box-Behnken response surface design combined with the overall desirability value were successfully employed to optimize and study the individual and interactive effect of process variables such as extraction temperature, time and surfactant %, for Genapol X-80, and sonication extraction temperature, time and water concentration, for choline chloride: fructose: H2O NDES mixture, on the alkaloidal yield from C. powellii. It was evident that parameters interacting together can act in synergism if adjusted properly according to the optimized conditions to obtain maximum alkaloids extractability. It is for the first time that the efficiency of micelle-mediated extraction has been compared to that of natural deep eutectic solvents for the extraction of alkaloids and the results thoroughly discussed.

HPTLC and GC/MS Study of Amaryllidaceae Alkaloids of Two Narcissus Species.

In this article, we report on the alkaloid profile and dynamic of alkaloid content and diversity in two Narcissus plants at different stages of development. The alkaloid profile of the two Narcissus species was investigated by GC/MS and HPTLC. Fifty eight Amaryllidaceae alkaloids were detected, and 25 of them were identified in the different organs of N. tazetta and N. papyraceus. The alkaloid 3-O-methyl-9-O-demethylmaritidine is tentatively identified here for the first time from the Amaryllidaceae family, and four alkaloids (tazettamide, sternbergine, 1-O-acetyllycorine, 2,11-didehydro-2-dehydroxylycorine) are tentatively identified for the first time in the genus Narcissus. The different organs of the two species analyzed showed remarkable differences in their alkaloid pattern, type of biosynthesis, main alkaloid and number of alkaloids. Lycorine-type alkaloids dominated the alkaloid, metabolism in N. papyraceus, while alkaloids of narciclasine-, galanthamine- and homolycorine-types were found only in the species N. tazetta L.

In vitro cytotoxicity of some Narcissus plants extracts.

This study compares the chloroform extracts of bulbs and roots of Narcissus papyraceus Ker Gawl. and Narcissus tazetta L. The cytotoxicity of the plant extracts was evaluated against human hepatocellular carcinoma cell line (HEPG2) and colon carcinoma cell line (HCT116) in comparison to doxorubicin. The extracts from the after-flowering (AF) bulbs of N. tazetta L. and N. papyraceus exhibited strong cytotoxic activity against HEPG2 (IC50: 2.2, 3.5 µg mL(-1)) and HCT116 (IC50: 4.2, 3.9 µg mL(-1)) cell lines, respectively. N. tazetta L. bulbs exhibited the least cell viability percentage in HepG-2 cell line (5.32%), while the AF root extracts of N. papyraceus exhibited the least cell viability percentage in HCT116 cell line (4.93%), when applied at a concentration of 50 µg mL(-1), thereby being more active than doxorubicin at the same concentration.

A new approach to develop a standardized method for assessment of acetylcholinesterase inhibitory activity of different extracts using HPTLC and image analysis.

A new, validated, sensitive and cheap method for preliminary quantitative evaluation of acetylcholine esterase inhibitory activity is presented. The proposed method combines HPTLC with data analysis by means of image processing software. An in-situ TLC autobiographic method was employed in which regions of the TLC plate which contain acetylcholinesterase inhibitors show up as white spots against the yellow background. Bleaching of the yellow color, caused by substances with acetylcholinesterase inhibitory activity was observed and recorded using a digital camera. ImageJ, JustTLC and Sorbfil, three image processing programs were evaluated for quantitative measurements. For evaluation of the assay efficiency, acetylcholinesterase inhibitory activity of different Amaryllidaceae plant extracts was expressed as Standard Activity Coefficients (SACs), which are relative measures of the activity to the well known acetylcholinesterase inhibitor eserine. We attempted to validate the method according to the ICH guideline. Different statistical data revealed that all image analysis software are able to detect the acetylcholine esterase inhibitory activity at very low concentration levels with the ImageJ program being the best of all three tested software regarding sensitivity, linearity and precision.

Two new alkaloids of the crinane series from Pancratium sickenbergeri.

Two new alkaloids; ent-6alpha/6beta-hydroxybuphanisine, (-)-8-demethylmaritidine and seven known alkaloids were isolated from Pancratium sickenbergeri grown in Egypt. Three of the known alkaloids were tested in the NCI cytotoxicity screen, but were found to be inactive.