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Data on cytotoxic activity of an Artemisia annua herbal preparation and validation of the quantification method for active ingredient analysis

The data in this article contain supporting information for the research manuscript entitled “Antitumor activity of an Artemisia annua herbal preparation and identification of active ingredients” by Lang et al. [1]. Momundo Artemisia annua extract and an acetonitrile fraction thereof induce apoptosis in MDA-MB-231 triple negative breast cancer cells as shown by XTT viability assay and induction of the subG0/G1 cell population by flow cytometric analysis. Furthermore, the HPLC-DAD method used to characterize the Artemisia annua herbal preparation as well as UHPLC-MS/MS method used to quantify the most abundant compounds in the extract and its validation are presented.


Induction of apoptosis by Artemisia annua herbal preparations
Induction of apoptosis was analyzed by DNA-staining with propidium iodide followed by flow cytometry [2]. MDA-MB-231 triple negative breast cancer cells were treated with Momundo (100 mg/ ml) or the acetonitrile fraction of Momundo (Momundo-ACN) (30 mg/ml) for 24 h; apoptotic cells with reduced DNA-fluorescence were quantified by using FlowJo software. Paclitaxel (100 nM) served as positive control. Momundo and Momundo-ACN, but not paclitaxel, slightly increased levels of apoptotic cells after 24 h (Fig. 1). We have previously shown that Artemisia annua extracts are not toxic to normal mammary epithelial cells and PBMC at concentrations 30 mg/ml, nor do they inhibit lymphocyte proliferation, or induce any overt adverse effects in an in vivo model [1].
Further, involvement of the extrinsic pathway and caspase 8 in apoptotic cell death induced by Momundo extracts was addressed. The caspase 8 inhibitor (IETD) partially antagonized the toxicity of Momundo-ACN extract towards MDA-MB-231 triple negative human breast cancer cells, when the highest concentration of the Momundo-ACN extract (50 mg/ml) was used (Fig. 2).
By semipreparative HPLC, 6,7-dimethoxycoumarin, chrysosplenol D, casticin, arteannuin B, and arteannuic acid have been purified from Momundo extract as described in the research manuscript entitled "Antitumor activity of an Artemisia annua herbal preparation and identification of active ingredients" [1]. Retention times of standard compounds are shown in Fig. 3. The gradient elution system of the respective semipreparative HPLC-DAD system is described in details in the experimental design, material and methods section.  The quantification of 6,7-dimethoxycoumarin, chrysosplenol D, casticin, arteannuin B, and arteannuic acid, the most abundant compounds identified in the Momundo extract, was analyzed in terms of linearity, precision, accuracy, limit of detection and limit of quantification. Data are shown in Table 1.

Statistical analysis
Results are expressed as mean ± SEM of at least three independent experiments if not indicated otherwise. In case of two-group comparison, results were analyzed with the two-tailed Student's t-test. Multi-group analysis was performed using the one-way analysis of variance, followed by Newman-Keuls post hoc test using SigmaPlot software. Significance levels were set as *p < 0.05, **p < 0.01, ***p < 0.001.

Validation of analytical HPLC-MS/MS analysis for quantification of most abundant compounds identified in Artemisia annua Momundo extract
The method was validated in terms of linearity, precision, accuracy, limit of detection, and limit of quantification. To obtain the linearity and to determine the limit of detection (LOD) and the limit of quantification (LOQ), standard solutions in the range from 10 ng/ml to 5000 ng/ml (9 levels) were analyzed, each in triplicates. The regression, LOD and LOQ were calculated with Valoo software (Applica, Bremen, Germany) based on the standardization criteria of DIN 32645. To evaluate the accuracy, the recovery was determined by using the standard addition method. Hence, a real sample was spiked at three levels and was analyzed, each in triplicates. Precision was determined by analysis of standards in two levels with six replicates at four different days yielding the intraday variations and the interday variations.

Gradient elution of semipreparative HPLC-DAD system
Gradient elution consisted of eluent A (ultrapure water þ 0.05% formic acid) and eluent B (acetonitrile þ 0.05% formic acid), starting with 30% acetonitrile and 70% water until 95% acetonitrile by 18 min, after 24 min the system was returning to initial conditions. The photodiode-array detector was set at 210 nm for acquiring of chromatograms.