Phytochemical Analysis and In Vitro Antileukemic Activity of Alkaloid-Enriched Extracts from Vinca sardoa (Stearn) Pignatti

Vinca sardoa (Stearn) Pignatti, known as Sardinian periwinkle, is widely diffused in Sardinia (Italy). This species contains indole alkaloids, which are known to have a great variety of biological activities. This study investigated the antileukemic activity against a B lymphoblast cell line (SUP-B15) of V. sardoa alkaloid-rich extracts obtained from plants grown in Italy, in Iglesias (Sardinia) and Rome (Latium). All the extracts showed a good capacity to induce reductions in cell proliferation of up to 50% at the tested concentrations (1–15 µg/mL). Moreover, none of the extracts showed cytotoxicity on normal cells at all the studied concentrations.


Introduction
Vinca sardoa (Stearn) Pignatti syn. Vinca difformis subsp. sardoa Stearn (Apocynaceae) is a perennial herbaceous widely diffused in Sardinia (Italy) where it is called Sardinian periwinkle [1]. From the morphological point of view, V. sardoa is very similar to the species Vinca difformis Pourret with the exception of the leaves: V. difformis has ovate and glabrous leaves, while V. sardoa has ovate-acuminate leaves with hairs up to 0.2 mm on the leaf margin [2]. The two species differ in the phytochemical analysis of the alkaloids, as we described previously [3]. The roots and leaves of V. sardoa contain indole alkaloids e.g., norfluorocurarine, akuammigine, conoflorine, and venalstonine [3][4][5]. Indole alkaloids are widely distributed in Angiosperm, including Apocynaceae, Rubiaceae, and Loganiaceae [6], as well as in the fungi kingdom [7]. The use of indole alkaloids has a long history, both in shamanic ceremonies [7] and in the pharmaceutical field [8]. Currently, indole alkaloids are known to show a variety of biological activities such as antimicrobial [9], anti-depressant [8,10], antiviral [11], and anticancer ones [12,13]. Some of them, vincristine and vinblastine (Figure 1), are even used for the treatment of some kind of cancers [14] such as lymphomas and acute lymphoblastic leukaemia (ALL) [15]. vincristine and vinblastine (Figure 1), are even used for the treatment of some kind cancers [14] such as lymphomas and acute lymphoblastic leukaemia (ALL) [15]. ALL is a paediatric malignancy that rarely affects adults. Patients affected by A show proliferation and accumulation of malignant and immature lymphatic blasts in bone marrow, peripheral blood, and lymphatic and non-lymphatic tissue. If untreat the disease is fatal within a few months from the diagnosis [16]. Despite the availa drugs, the side effects associated with chemotherapy, mainly neurotoxicity, cardiotoxic [17], and hepatotoxicity [18], together with drug resistance make new therapeutic optio necessary [19].
Due to the presence of indole alkaloids in V. sardoa, we decided to investigate the vitro activity of the alkaloid-enriched extract from V. sardoa on a B lymphoblast cell l (SUP-B15) and a normal cell line (fibroblast), by studying the effects on cell viability.

Extraction of V. sardoa Aerial Parts
For this study, we considered the aerial parts of V. sardoa collected in Iglesias (Ita in 2019 and 2021 and in the experimental botanical garden of Sapienza University of Ro in 2019. The three samples were treated with a diluted solution of acetic acid. The aci solutions were made alkaline with sodium bicarbonate and finally extracted w dichloromethane. The organic extracts gave a positive result to Dragendorff s reag indicating the presence of alkaloids. The extracts obtained from the plants collected Iglesias in 2019 and 2021 were named IG2019 and IG2021, respectively, while the extr obtained from the plant collected in Rome was named RM2021. All the extracts w analysed by LC-MS and NMR spectroscopy, as discussed below.

LC-PDA-ESI-MS and DI-ESI-MS/MS Analysis of the Extracts
The previously obtained [5]   ALL is a paediatric malignancy that rarely affects adults. Patients affected by ALL show proliferation and accumulation of malignant and immature lymphatic blasts in the bone marrow, peripheral blood, and lymphatic and non-lymphatic tissue. If untreated, the disease is fatal within a few months from the diagnosis [16]. Despite the available drugs, the side effects associated with chemotherapy, mainly neurotoxicity, cardiotoxicity [17], and hepatotoxicity [18], together with drug resistance make new therapeutic options necessary [19].
Due to the presence of indole alkaloids in V. sardoa, we decided to investigate the in vitro activity of the alkaloid-enriched extract from V. sardoa on a B lymphoblast cell line (SUP-B15) and a normal cell line (fibroblast), by studying the effects on cell viability.

Extraction of V. sardoa Aerial Parts
For this study, we considered the aerial parts of V. sardoa collected in Iglesias (Italy) in 2019 and 2021 and in the experimental botanical garden of Sapienza University of Rome in 2019. The three samples were treated with a diluted solution of acetic acid. The acidic solutions were made alkaline with sodium bicarbonate and finally extracted with dichloromethane. The organic extracts gave a positive result to Dragendorff's reagent indicating the presence of alkaloids. The extracts obtained from the plants collected in Iglesias in 2019 and 2021 were named IG2019 and IG2021, respectively, while the extract obtained from the plant collected in Rome was named RM2021. All the extracts were analysed by LC-MS and NMR spectroscopy, as discussed below.

LC-PDA-ESI-MS and DI-ESI-MS/MS Analysis of the Extracts
The previously obtained [5]  Although they had been previously characterized [5], the ESI-MS/MS fragmentation spectra are herein provided for each compound for the first time ( Figures S1-S6).
All the compounds except 4 exhibited two characteristic fragmentations: (a) the loss of 17 mass units, containing an N atom since an odd mass fragment is obtained, that has been ascribed to the loss of NH 3 (Figures S1-S3, S5 and S6); (b) the loss of 135 mass units (Figures S1, S3, S5 and S6), that becomes 134 + 59 = 193 in the case of 2 ( Figure S2).
All compounds gave a characteristic fragment that has been assigned to a positive indole-like fragment with 144 m/z (Figures S1, S2 and S4-S6) or to corresponding hydroxylsubstituted fragment with 160 m/z ( Figure S3).
The structures of the tentatively assigned lost fragments, corresponding to −135 and −193 mass units, are shown in Figure 3   Although they had been previously characterized [5], the ESI-MS/MS spectra are herein provided for each compound for the first time (Figures S All the compounds except 4 exhibited two characteristic fragmentatio of 17 mass units, containing an N atom since an odd mass fragment is obta been ascribed to the loss of NH3 (Figures S1-S3, S5 and S6); (b) the loss of 1 (Figures S1, S3, S5 and S6), that becomes 134 + 59 = 193 in the case of 2 (Figu All compounds gave a characteristic fragment that has been assigned indole-like fragment with 144 m/z (Figures S1, S2, and S4-S6) or to c hydroxyl-substituted fragment with 160 m/z ( Figure S3).
The structures of the tentatively assigned lost fragments, correspondin −193 mass units, are shown in Figure 3 (F1a and F1b, respectively); the str tentatively assigned positive fragments with 144 m/z and 160 m/z are show (F2a and F2b, respectively).  Although they had been previously characterized [5], the ESI-M spectra are herein provided for each compound for the first time (   N(1)-methyl-14,15-didehydroaspidofractinine (5) and N(1)-methyl-14,15-didehydro-12-methoxyaspidofractinine (6) exhibit the same fragmentation pattern, with fragments differing by 30 mass units, according to the presence (6) or not (5) of the -OCH 3 moiety; they are characterized by the base peak corresponding to the ion [M + H + -135] + at 158 and 188 m/z, respectively. The loss of −14 mass units corresponds to the loss of the N(1)-methyl moiety as -CH 2 gives the fragment 144 m/z for compound 5 and the fragment 174 m/z for compound 6, the last one giving the fragment 144 m/z by further loss of the methoxy moiety (−30 mass units). Moreover, 5 and 6 fragmentation patterns evidenced a characteristic ion [M + H + -120] + at 173 and 203 m/z, respectively, not assigned but whose even neutral mass value (172 and 202 Da, respectively) indicates the presence of both N atoms in the fragment (5 and 6, in Figures S5 and S6, respectively).
Compound 2, venalstonine (PubChem NSC180520), exhibited the ion [M + H + -NH 3 ] + = 320 m/z as peak base; due to the -COOCH 3 moiety, the characteristic loss of 135 discussed above appears as the loss of 193 mass units (see Figure 3, F1b). Another fragment at 260 m/z was likely due to the loss of the same group as HCOOCH 3 , as shown in Figure S2.
Compound 4, conoflorine, exhibits a fragmentation pattern slightly differing from the others. From the epoxide moiety arises a small peak for the typical fragment [M + H + -18] + = 279 m/z due to the loss of water. Also in this case, the pattern evidenced the indole-like fragment (F2a, in Figure 3) at 144 m/z; the non-indole residual fragment at 154 m/z is the base peak, confirmed by the fragment at 136 m/z corresponding to the loss of water due to the epoxide moiety (4, in Figure S4).
The strong correlation observed between the analysed alkaloid structure and the fragmentation profile provides a powerful tool for the characterization of other indole alkaloids in the future.
The purified samples of alkaloids 1-6 were used to optimize the chromatographic separation and the mass spectral parameters for the selected ion recording (SIR) mode analysis, used for their selective and sensitive unambiguous identification in the IG2019, IG2021, and RM2019 extracts.
The three different samples of V. sardoa exhibit a similar profile: in fact, alkaloids 1-6 were identified in all samples, by comparison of chromatographic and mass spectral data with the purified samples, as reported in Table 1. The typical chromatographic profile of the extracts is shown in Figure S7a, in which the total ion chromatogram (TIC) of the RM2019 extract is shown as an example. The selected m/z mass values 307, 337, 323, 297, and 293 corresponding to the alkaloids 1, 2, the isobaric 3 and 6, 4, and 5, respectively, are evidenced in the corresponding SIR chromatograms ( Figure S7b-f). On the basis of the total ions detected in each SIR channel, alkaloid 5 seemed the most abundant one ( Figure S7f, TIC = 4.00 × 10 8 ), followed by alkaloid 4 ( Figure S7e, Molecules 2023, 28, 5639 5 of 10 TIC = 2.60 × 10 8 ), while alkaloid 1 was in general the least abundant one ( Figure S7b, TIC = 10.00 × 10 7 ), in good agreement with NMR data described below.

NMR Analysis
Selected samples of V. sardoa extracts, namely RM2019, IG2019, and IG2021, were analysed by 1 H-NMR spectroscopy to quantify the identified alkaloids. The spectra showed only quantitative differences among them, but no qualitative ones, so a representative spectrum is reported in Figure S8. The spectra showed resonances attributable to sterols, fatty acids, and terpenes according to the literature data [20], as well as the resonances of the alkaloids identified by LC-MS. The resonances of the quantified alkaloids, in particular the ones of the unsaturated protons, were attributed on the basis of the literature data (PII: S0031-9422(97)00533-S) and were quantified by integration of their diagnostic aromatic resonances. In greater detail, the molecules 1, 2, and 5 were identified by their diagnostic unsubstituted aromatic ring (doublet at 6.40 ppm, triplet at 6.71 ppm, triplet at 6.83 ppm, and doublet at 7.31 ppm) and quantified by the resonance at 7.31 ppm; indole 4 was identified on the basis of its indole-like resonances (triplet at 7.29 ppm, triplet at 7.38 ppm, doublet at 7.55 ppm, and doublet at 8.14 ppm) and quantified by the resonance at 7.55 ppm. Compounds 3 and 6 were identified on the basis of the substituted 6-terms aromatic ring (doublet at 6.71 ppm, doublet at 6.81 ppm, and triplet at 7.08 ppm) and quantified by the resonance at 6.81 ppm. The integrals related to molecule signals were normalized by one of the internal standards (normalized by the number of protons) and then converted into mg/100 g of dried extract. Since the superimposition of several molecules with similar structures, the amounts of 1, 2, and 5 are reported as equivalents of molecule 1, while compounds 3 and 6 are reported in Table 2.

Cytotoxicity Assays
Cell cytotoxic activity of the extract of V. sardoa ( Figure 4) on lymphoblast cell line SUP-B15 was evaluated by MTT assay at different times (24, 48, and 72 h) with different concentrations (1 µg/mL, 5 µg/mL, 10 µg/mL, and 15 µg/mL, respectively). The results suggested that, after 48 h of the treatment, the concentration of 5 µg/mL for the IG2019 extract was able to induce a reduction in cell proliferative capacity (40-45%). Moreover, after 48 and 72 h, higher concentrations (10 and 15 µg/mL) of the IG2019 extract induced a proliferation percentage decrease (40%) ( Figure 4A). In addition, the IG2021 extract had the same effect as the IG2019 extract from 10 to 15 µg/mL at 48 and 72 h after treatments ( Figure 4B). RM2019 showed a reduction in cell viability lower than 45% ( Figure 4C) at 72 h at the concentration of 15 µg/mL. Furthermore, we evaluated the cytotoxic activity of plant extracts on normal cells such as human fibroblasts. Figure S9 shows that none of the extracts exhibited cytotoxic activity at all concentrations and times used, with the exception of IG2019, which induced cytotoxicity at higher concentration (15 µg/mL) after 72 h. Vincristine and vinblastine were used as reference drugs. The cytotoxicity assay results are reported in Figure S10.

Plant Material
The aerial parts (stem and leaves) of V. sardoa were collected in October 2019 and October 2021 in the area of Iglesias, Sardinia, Italy (geographical coordinates 41°46′16″ N, 13°21′39″ E); the botanical identification was carried out by one of us (A.M.). Representative samples of this collection are stored in the Pharmaceutical Biology laboratory (Department of Environmental Biology, Sapienza University of Rome) for further reference, under the voucher names IG2019 and IG2021, respectively. Moreover, a third sample (RM2019) was collected in late September 2019 from a living plant hosted at the Experimental Garden of Sapienza University of Rome.

Chemicals
Analytical grade chemicals were provided by Sigma-Aldrich (Milan, Italy) and used without further purification. The solvents were HPLC-grade and were provided by Carlo Erba (Milano, Italy), while deionized water was prepared daily using a Milli-Q purification system (Millipore, Vimodrone, Italy).

Plant Material
The

Chemicals
Analytical grade chemicals were provided by Sigma-Aldrich (Milan, Italy) and used without further purification. The solvents were HPLC-grade and were provided by Carlo Erba (Milano, Italy), while deionized water was prepared daily using a Milli-Q purification system (Millipore, Vimodrone, Italy).

Extraction Procedure
The extracts were obtained by a 48 h maceration of the coarsely minced aerial parts of V. sardoa in 2% aq. AcOH (plant material (g)/solvent (mL) ratio of 1:35). The aqueous solution was made alkaline with Na 2 CO 3 until pH~8-9 and extracted with dichloromethane (3×). The reunited organic phases were dried over Na 2 SO 4 and the organic solvent was evaporated by a rotary evaporator equipped with a water bath heated to 30 • C. A brown residue was obtained with a yield of 0.43, 0.57, and 0.51% w/w from IG2019, IG2021, and RM2019, respectively.

DI-ESI-MS/MS Experiments
Experiments were carried out by infusing the samples directly into the ESI source through an external syringe, with a flow rate of 5 µL/min. ES+ mass spectral data were acquired for 2 min in the appropriate mass range, with a cone voltage of 25 V, ionization source temperature of 100 • C, desolvation gas temperature of 150 • C, cone gas flow of 30 L/h, and desolvation gas flow 400 L/h. Alkaloid fragmentation patterns were obtained by selecting the precursor ion, using argon as collision gas and optimized collision energy (CE) in the range of 15-38 eV, and acquiring spectra for 2 min in the appropriate mass range. All acquisitions were carried out in duplicate.

NMR Analysis
Selected samples of V. sardoa non-polar extracts, namely RM2019, IG2019, and IG2021, were dried and resuspended in 700 µL of deuterated chloroform containing hexamethyldisiloxane at a concentration of 2 mM as both chemical shift and concentration internal standard. 1 H-NMR spectra were acquired as previously reported [21].

Cell Culture
Cell culture SUP-B15, a B lymphoblast cell line isolated from the marrow of an 8-yearold, male patient with acute lymphoblastic leukaemia, and HDF fibroblast cells (human dermal fibroblast) were both obtained from ATCC, Manassas, VA, USA. SUP-B15 cells were grown in McCoy's 5A modified medium (ATCC) while fibroblast cells were grown in Dulbecco's modified Eagle's medium (DMEM, Euroclone, Pero, Italy) both with the addition of 10% foetal bovine serum (HyClone™ Fetal Bovine Serum, USA origin, Characterized), 100 units/mL penicillin, and 100 units/mL streptomycin at 37 • C in a humidified atmosphere with 5% CO 2 .
MTT Assay SUP-B15 cells were plated at a density of 1.2 × 10 4 cells in a 96-well cell-culture-treated, U-shaped-bottom microplate (Costar) while fibroblast cells were seeded for 24 h in a 96-well plate (flat-bottom) (Corning ® , Corning, NY, USA) with a density of 1.0 × 10 4 cells/well. Both cell lines were resuspended in 90 µL of culture medium. Then, the cells were exposed (10 µL) to increased concentrations of the extracts of IG2019, IG2021, and RM2019 (1 µg/mL, 5 µg/mL, 10 µg/mL, and 15 µg/mL) in a cell culture medium for 24, 48, and 72 h. After the incubation period, 20 µL of 5 mg/mL MTT solution (Sigma, Deisenhofen, Germany) was added to each well for 2 h at 37 • C in 5% CO 2 atmosphere; cells were dissolved with 100 µL of MTT solvent (4 mM HCl, 0.1% NP40 in isopropanol). The absorbance was read on a microtiter spectrophotometric plate reader at 570 nm, in the same experiments, and standardized to 100% with respect to control cells. The results obtained were calculated as absorbance. All data were obtained in triplicate and from three independent experiments. Vincristine and vinblastine, used as reference drugs, were purchased from Merck (Milan, Italy).