Abstract

Introduction:

Rhazya stricta Decne. is considered an important medicinal plant that is rich in secondary metabolites containing anticancer alkaloids. Several indole alkaloid classes have been identified from the various parts of R. stricta, but the cytotoxic potentialities of only a few of these metabolites are known.

Methods:

In this study, an applied analytical method was used to determine the alkaloids in R. stricta from the stem (RS) and leaf (RL) and their extracts.

Results and Discussion:

This study tentatively elucidated 10 new compounds of indole alkaloids from the various parts of R. stricta. Among them, six alkaloidal glycosides had not been detected in natural resources. This investigation also examined the in vitro protective results of the four R. stricta stem (A–D) and leaf (A′–D′) extracts, each having a different group of compounds—indole alkaloids, against oxidative stress activated by H2O2/Fe2+ in human plasma in vitro. In addition, we estimated the effect of these plant extracts on DNA fragmentation in human peripheral blood mononuclear cells (PBMCs). Another aim of these in vitro experiments was to determine the result of A–D and A′–D′ on selected hemostatic parameters of human plasma, such as the activated partial thromboplastin time, prothrombin time, and thrombin time, and on the viability of PBMCs. Based on our results, we demonstrate for the first time that tested extracts of the leaves and stems of R. stricta containing different indole alkaloid compounds (especially two tested preparations from R. stricta leaf—C′ and D′) are good antioxidant in vitro models, depending on the dosage, and they may have some promising actions in vivo. For example, we observed a significant difference in the level of DNA damage induced by H2O2 in the experiment with A–D and A′–D′. Two tested extracts from R. stricta leaf (C′ and D′, at all concentrations used) were also found to protect plasma against H2O2/Fe2+—induced lipid peroxidation. In addition, preparation C′ does not induce cytotoxicity. The potential of the metabolomics of extract C′ may be correlated to the presence of rhazisidine, secamine, and their derivatives.

1 Introduction

Alkaloids are a wide range of naturally occurring metabolites that occur in medicinal plants that have one or more nitrogen atoms in their structures. Alkaloids naturally present in foods (e.g., certain indole alkaloids in cruciferous vegetables and coffee) may exert mild stimulatory or antioxidant effects, depending on their concentration and bioavailability (Liu et al., 2007; Baeshen et al., 2009; Baeshen et al., 2010; Ahmad et al., 1970; Ahmad et al., 1971; Ahmad et al., 1979; Ahmad et al., 1983; Ahmed et al., 2015; Ahmed et al., 2018; Andersen et al., 1987; Atta-ur-Rahman, 1984; Atta-ur-Rahman et al., 1987; Rehman and Rahman, 1996; Rahman et al., 1989; Marwat et al., 2012; Obaid et al., 2017; Abdul-Hameed et al., 2022; Pravin et al., 2025; Aljawdah et al., 2025; Baeshen et al., 2022; Adamski et al., 2020; Rajashekar, 2023).

Indole alkaloids are five-membered rings with one hetero-nitrogen atom and are one of the principal classes of alkaloid. Indole alkaloids are typically found in the families Rubiaceae and Apocynaceae (Liu et al., 2007). They exhibit various pharmacological activities, including antihypertensive, antimicrobial, and antitumor properties (Baeshen et al., 2009; Pravin et al., 2025; Aljawdah et al., 2025; Baeshen et al., 2022). High levels of indole alkaloids (e.g., harman and norharman) can exhibit neurotoxic effects, particularly in sensitive individuals (Liu et al., 2007; Baeshen et al., 2009; Baeshen et al., 2010; Ahmad et al., 1970; Ahmad et al., 1971; Ahmad et al., 1979; Ahmad et al., 1983; Ahmed et al., 2015; Ahmed et al., 2018; Andersen et al., 1987; Atta-ur-Rahman, 1984; Atta-ur-Rahman et al., 1987; Rehman and Rahman, 1996; Rahman et al., 1989; Marwat et al., 2012; Obaid et al., 2017; Abdul-Hameed et al., 2022; Adamski et al., 2020; Rajashekar, 2023).

The genus Rhazya comprises two species (Migahid, 1989; Mandaville, 1990; Chaudhary and Al-Jowaid, 1999; Bukhari et al., 2017; Ali et al., 2000): R. stricta Decne (syn. R. greissii Decne) and R. orientalis Decne (syn. Amsonia orientalis Decne). R. stricta, commonly known as “Harmal”, is a parched plant that belongs to the family Apocynaceae. The Rhazya genus is named after a Muslim scientist, Abu Bakr Mohammed bin Zakariya Al-Razi, known in Europe under the Latinized name of Rhazes. The plant is an erect shrub with glabrous leaves, yellow-green and broadly linear-lanceolate, which wrinkle after drying. It has a smooth central stem and dense semi-erect branches; the leaves alternate; its flowers are white in short branched cymes; its fruit has pale yellow follicles; and its seeds are short-winged (Migahid, 1989; Mandaville, 1990; Chaudhary and Al-Jowaid, 1999; Bukhari et al., 2017; Ali et al., 2000).

The medicinal plant R. stricta is known to be a rich natural source of indole alkaloids, monoterpenoid indole alkaloids, triterpenes, phenols, and glycosides (Baeshen et al., 2009; Baeshen et al., 2010; Ahmad et al., 1970; Ahmad et al., 1971; Ahmad et al., 1979; Ahmad et al., 1983; Ahmed et al., 2015; Ahmed et al., 2018; Andersen et al., 1987; Atta-ur-Rahman, 1984; Atta-ur-Rahman et al., 1987; Rehman and Rahman, 1996; Rahman et al., 1989; Marwat et al., 2012; Obaid et al., 2017; Abdul-Hameed et al., 2022). Long ago, R. stricta was used in traditional medicine to treat chronic rheumatism and fever (Rahman et al., 1989). In previous studies, R. stricta extracts and the pure alkaloidal metabolites displayed positive anti-diabetic effects (Baeshen et al., 2010; Ahmed et al., 2015; Ali et al., 2000; Ali, 1997) and antioxidant and anticancer properties (Baeshen et al., 2012; Mukhopadhyay et al., 1981; Gu and Zakarian, 2010; El-Awady et al., 2015; Shahat et al., 2016; Al-Dabbagh et al., 2018; Shaer, 2019). Other biological properties of various parts of R. stricta are less recognized, especially toward the cardiovascular system. Indole alkaloids are commonly analyzed using HPLC- or UPLC-based analytical methods, and UPLC–Q-TOF mass spectrometry achieves rapid and sensitive identification of indole alkaloids (Schnoes et al., 1962; Li et al., 2011; Nordström et al., 2006; Esquenazi et al., 2009; Demarque et al., 2016; Hamed et al., 2022; Ben Said et al., 2023; Mahida et al., 2026; Fu et al., 2025; Youssif et al., 2024; Akhgari et al., 2015a; Akhgari et al., 2015b; Baeshen et al., 2014; Baeshen et al., 2015; Baeshen et al., 2023).

Oxidative stress is a key factor in the progression of a range of human illnesses, such as cancer and cardiovascular diseases. Phytochemicals found in various plants have demonstrated significant potential in the treatment and prevention of diseases associated with oxidative stress (Milev et al., 2025; Al-Naqeb et al., 2024; Maliar et al., 2023; Diogo Gonçalves et al., 2025; Ladeira et al., 2024). So far, there are no data on the effect of R. stricta indole alkaloids on the physiology of various elements of human blood, including their interaction with human plasma and the hemostatic system. Therefore, for the first time, our study aimed to illustrate the in vitro protective results of the four R. stricta stem (A–D) and leaf (A′–D′) extracts, each containing a different structural secondary metabolite, against oxidative stress activated by H2O2/Fe2+ (donor of hydroxyl radicals) in human plasma. The Fenton reaction, driven by the interaction of Fe2+ with H2O2, generates hydroxyl radicals (•OH), which constitute one of the most potent oxidizing species in biological systems. In the initial step, Fe2+ is rapidly oxidized by H2O2, producing Fe3+, hydroxide ion, and the highly reactive •OH radical. This step is central to the reaction’s oxidative capacity as •OH readily attacks lipids, proteins, and nucleic acids. Ferric iron formed in this process is subsequently reduced back to Fe2+ by a second molecule of H2O2, yielding hydroperoxyl radicals (•OOH) and allowing the continuous cycling of iron in the Fe2+/Fe3+ redox couple. Overall, the net conversion of H2O2 results in the simultaneous formation of •OH and •OOH radicals. The reaction proceeds most efficiently under acidic conditions, where Fe2+ remains soluble and available for redox cycling. At higher pH, iron precipitation inhibits radical formation. Given the extreme oxidizing power of •OH, which surpasses most other biologically relevant oxidants, the Fenton system represents a major source of oxidative stress in both chemical assays and cellular environments (Maliar et al., 2023).

We investigated three different parameters of oxidation in human plasma: lipid peroxidation (detected by thiobarbituric acid reactive substances—TBARSs), protein carbonylation, and thiol group level. In addition, we demonstrated the effect of these plant extracts on DNA damage (by applying the comet assay) in PBMCs. Another goal of our experiments conducted in vitro was to determine the effect of the A–D and A′–D′ extracts on selected hemostatic parameters of human plasma (the stimulated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT)) and on the viability of PBMCs.

The goal of this study is to describe a rapid, sensitive ultra-performance liquid chromatography–electrospray ionization–quadrupole time-of-flight (UPLC–ESI–Q-TOF) technique for detecting and tentatively identifying alkaloid metabolites from the leaves and stems of R. stricta, which represent an important traditional natural resource for medicinal drugs.

2 Materials and methods2.1 Chemicals

Dimethylsulfoxide (DMSO), thiobarbituric acid (TBA), 4′,6-diamidino-2-phenylindole (DAPI), low-melting-point (LMP) and normal-melting-point (NMP) agarose, phosphate-buffered saline (PBS), and H2O2 were purchased from Sigma-Aldrich (St. Louis, MO, United States). All other reagents represented analytical grade and were provided by commercial suppliers.

2.2 Plant material

The plant materials were collected from Qassim Province in March 2019 at 25.8700° N, 43.5001° E in Saudi Arabia. The plant material was identified by Prof. Arafa Hamed according to Täckholm (1974) and was compared with voucher #NATKSU-108 (Department of Botany and Microbiology, King Saud University, Saudi Arabia). Our voucher plant (#13) was deposited in the Chemistry Department, College of Science, Qassim University, Al Rass, Kingdom of Saudi Arabia. The different parts of the collected materials (roots, stems, and leaves) were then separated to complete the drying process at room temperature, far from direct sunlight.

2.2.1 Plant description

The genus Rhazya comprises two species (Ali et al., 2000): R. stricta Decne (syn. Rhazya greissii Decne) and R. orientalis Decne (syn. Amsonia orientalis Decne). R. stricta, commonly known as “Harmal”, is a parched plant that belongs to the family Apocynaceae. The Rhazya genus is named after a Muslim scientist, Abu Bakr Mohammed bin Zakariya Al-Razi, known in Europe under the Latinized name of Rhazes. The plant is an erect shrub with glabrous leaves, yellow-green and broadly linear-lanceolate, which wrinkle after drying. It has a smooth central stem and dense semi-erect branches; the leaves alternate; its flowers are white in short branched cymes; its fruit has pale yellow follicles; and its seeds are short-winged (B).

2.3 Preparation of extracts A–D and A′–D′

The leaves and stems were dried for 2 weeks at room temperature (35 °C), avoiding direct sunlight. The dried leaves of R. stricta (RL-250 g), after grinding, were exhaustively extracted four times using 80% MeOH (MeOH: H2O, 80:20) by maceration at room temperature (24 h, 35 °C). The crude extract thus obtained was concentrated under reduced pressure at 600 C using a Witeg Vertical Rotary Evaporator 20–280 rpm, 500 mL, to obtain a syrupy consistency (65 g, 22.4% from the dried leaves). Part of the crude extract (10 g) was dissolved in a distilled water liquor and loaded onto a preconditioned short C18 column (6 × 10 cm, 60 lm C18, Backer) and eluted with H2O (100%, washing), 20% MeOH (MeOH: H2O, 20:80, A, 1.7 g), 40% MeOH (MeOH: H2O, 40:60, B, 2.5 g), 60% MeOH (MeOH: H2O, 60:40, D, 3.6 g), 80% MeOH (MeOH: H2O, 80:20, C, 1.5 g), and 100% MeOH (washing). The same procedures were applied for the dried stem parts (RS-250 g), which yield 77 g (30.8% from the dried stems) after removing the used solvent. Part of the RS crude extract (10 g) was fractionated in the same manner and yielded the extracts A’ (20% MeOH, 1.2 g), B’ (40% MeOH, 2.7 g), C’ (60% MeOH, 3.7 g), D’ (80% MeOH, 1.6 g), and 100% MeOH (washing). All extracts (A–D and A′ –D) were concentrated using the same method and investigated by C-18 TLC using the solvent system CH3CN:H2O (30:70 and 40:60) and sprayed with Dragendorff’s reagent, which is used for visualizing alkaloids. A–D and A′–D′ tested positive for alkaloid.

2.4 High-resolution LC-MS analyses of plant extracts

High-resolution LC-MS analyses of plant extracts were achieved according to our previous methods (41, 42, supplementary data (Liu et al., 2007)).

2.5 Preparation of stock solutions of plant extracts for bioassay

Stock solutions of R. stricta extracts A–D and A′–D′ were prepared with 50% (v/v) aq. DMSO, a universal solvent for different plant metabolomics. The final DMSO concentration in the tested samples was below 0.05% (v/v). To achieve 50% DMSO in the tested samples, 0.05% (v/v) was diluted 1,000 times during the tests.

2.6 Isolation of human plasma

Plasma was obtained from a blood bank in Łódź (Poland), and it came from regular, medication-free donors. The donors did not drink alcohol or take medicine (including antiplatelet drugs, aspirin and its derivatives, or anticoagulants) for 2 weeks before blood collection. The Bioethics Committee at the University of Łódź approved the protocol for research on human subjects (number 2/KBBN-UŁ/II/2016). The research was also conducted according to the guidelines of the Helsinki Declaration for Human Research, with the approval of the committee.

Each sample (plasma) taken for analysis came from a different donor and was an independent trial. To measure the parameters of hemostasis and auto-oxidation of biomolecules (lipids and proteins), the plasma was incubated at 37 °C for 30 min with the tested plant extracts (concentration range 0.5–50 μg/mL). To measure the oxidative stress parameters, the plasma was pre-incubated at 37 °C for 5 min with the tested plant extracts (concentration range 0.5–50 μg/mL) and then treated with a final concentration of 4.7 mM H2O2/3.8 mM Fe2SO4/2.5 mM EDTA (25 min, at 37 °C). “Control negative” refers to plasma not treated with H2O2/Fe2+; “control positive” refers to plasma treated with H2O2/Fe2+.

The protein concentration was calculated by measuring the absorbance of the tested samples at 280 nm, according to Whitaker and Granum (1980) and using the assay of Bradford (1996).

2.7 PBMCs isolation

PBMCs were isolated from leucocyte buffy coats obtained from healthy, non-smoking donors provided by the Blood Bank in Łódź, Poland, as described by Kluska et al. (2019). The leucocyte-buffy coat was diluted in a 1:1 ratio in PBS and centrifuged in a density gradient of Lymphosep (Cytogen, Zgierz, Poland) at 200 × g for 20 min at room temperature. PBMCs were then collected and washed three times by centrifugation in PBS. The pellet of the cells was resuspended in RPMI 1640 medium (Lonza, Basel, Switzerland).

All procedures were approved by the Research Ethics Committee of the University of Łódź (approval no. 12/KEBN-UŁ/I/2024–2025).

2.8 Markers of oxidative stress2.8.1 Lipid peroxidation measurement

Lipid peroxidation was quantified by measuring TBARS concentration as per Wachowicz (1984) and Bartosz (2013). After incubation, the samples were mixed with an equal volume of cold 15% (v/v) trichloroacetic acid in 0.25 M HCl and 0.37% (v/v) TBA in 0.25 M HCl and then immersed in a boiling water bath for 15 min. After cooling, the absorbance was measured at 535 nm using the SPECTROstar Nano Microplate Reader (BMG LABTECH, Germany). The TBARS concentration was calculated using the molar extinction coefficient (ε = 156,000 M-1 cm-1) and was expressed as nmol/mL of plasma.

2.8.2 Carbonyl group measurement

The carbonyl groups were determined in plasma protein according to Levine et al. (1990) and Bartosz (2013). The absorbance measurement (at 375 nm) was performed using a SPECTROstar Nano Microplate Reader (BMG LABTECH). The carbonyl group concentration was calculated using a molar extinction coefficient (ε = 22,000 M-1 cm-1) and was expressed as nmol/mg of plasma protein.

2.8.3 Thiol group measurement

After incubation, the test samples were transferred to a 96-well plate at 20 μL, followed by the addition of 20 μL of sodium dodecyl sulfate (SDS), and mixed thoroughly. Successively, 160 μL of 10 mM phosphate buffer (pH 8.0) was added to all samples and mixed thoroughly. Absorbance was measured at a wavelength λ = 412 nm (A0), and 16.6 μL Ellman’s reagent (5,5′-dithio-bis-(2-nitrobenzoic acid); DTNB) was added. The 96-well plate was incubated for 60 min (temperature 37 °C). After incubation, absorbance was measured at a wavelength λ = 412 nm (A1) using the SPECTROstar Nano Microplate Reader (BMG LABTECH) as per Ando and Steiner (1973), Ando et al. (1973), and Bartosz (2013). The absorbance difference A1-A0 was calculated. The thiol group concentration was calculated using a molar extinction coefficient (ε = 13 600 M-1 cm-1) and was expressed as nmol/mg of plasma protein.

2.8.4 DNA oxidative damage analysis

DNA oxidative damage was assessed using the alkaline comet assay, following the method described by Singh et al. (1988) as adapted by Kluska et al. (2019) and Tokarz et al. (2016). In brief, PBMCs were adjusted to a concentration of 1 × 105 cells/mL and incubated with R. stricta extracts for 2 h at 37 °C. The cells were then treated with 20 µM H2O2 for 15 min on ice.

Following treatment, the cells were centrifuged, resuspended in 0.75% LMP agarose, and spread onto microscope slides pre-coated with 0.5% NMP agarose. The slides were subsequently immersed in lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, and 1% Triton X-100; pH 10) for 1 h. DNA unwinding was performed in ice-cold alkaline buffer (300 mM NaOH, 1 mM EDTA; pH > 13) for 20 min, followed by electrophoresis in ice-cold buffer of 30 mM NaOH and 1 mM EDTA for 20 min at 0.73 V/cm (29 mA).

After electrophoresis, the slides were rinsed, stained with DAPI (2 μg/mL), and examined using a fluorescence microscope. Fluorescent imaging was conducted at ×200 magnification using an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) equipped with a ProgRes MF cool monochrome camera (JENOPTIK, Jena, Germany) and connected to a Lucia Comet Assay 7.30 image analysis system (Laboratory Imaging, Prague, Czech Republic). For each sample, 50 comets were randomly selected for analysis, and the percentage of DNA in the tail comet was quantified as an indicator of DNA damage. Two independent experiments were performed.

2.9 Cell viability

Cell metabolic activity was evaluated using the resazurin reduction assay as per O’Brien et al. (2000). PBMCs were plated in 96-well culture plates at a density of 5 × 104 cells per well and exposed to R. stricta extracts at final concentrations of 0.5, 5, and 50 μg/mL for 24 h under standard culture conditions (37 °C, 5% CO2). After treatment, 10 µL of resazurin solution (2 mg/10 mL in PBS) was added to each well, and the plates were incubated for an additional 2 h at 37 °C in 5% CO2.

Fluorescence signals were then recorded using a Synergy HT microplate reader (BioTek Instruments, United States) with excitation and emission wavelengths set at 530/25 and 590/35 nm, respectively. The impact of the alkaloid fraction on cell viability was expressed as a percentage relative to untreated control cells. Two independent experiments were performed, each in triplicate.

2.10 Parameters of hemostasis2.10.1 Measurement of prothrombin time

Human plasma was incubated at 37 °C on a block heater. After incubation, the cuvette was transferred to the measuring holes. Hence, 100 μL of Dia-PT liquid (commercial preparation) was added. The PT was determined coagulometrically using an Optic Coagulation Analyzer, model K-3002 (Kselmed, Grudziadz, Poland), as per Malinowska et al. (2012).

2.10.2 Measurement of thrombin time

Human plasma was added to a coagulometric cuvette and incubated at 37 °C on a block heater. Then, the cuvette was transferred to measuring holes, and 100 μL of thrombin (final concentration −5 U/mL) was added. The TT was determined coagulometrically using an Optic Coagulation Analyzer, model K-3002 (Kselmed, Grudziadz, Poland), as per Malinowska et al. (2012).

2.10.3 Measurement of activated partial thromboplastin time

Human plasma was added to a coagulometric cuvette. Incubation was then conducted at 37 °C on a block heater with 50 μL of Dia-PTT liquid (commercial preparation). The cuvette was transferred to the measuring holes. Then, 50 μL of 25 mM CaCl2 was added. The APTT was determined coagulometrically using an Optic Coagulation Analyzer, model K-3002 (Kselmed, Grudziadz, Poland), as per Malinowska et al. (2012).

2.11 Data analysis

Statistical analyses were conducted using Statistica software version 10 (StatSoft). Data normality was evaluated using normal probability plots, while variance homogeneity was assessed by the Brown–Forsythe test. Differences among and between experimental groups were analyzed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple comparison test. Statistical analysis was conducted using the Mann–Whitney test for samples with distributions deviating from normality in the comet assay. For clarity, only statistically significant differences between the tested preparations and the control or positive-control groups are reported.

Results are presented as the mean ± standard deviation (SD) or, for the comet assay, mean ± standard error of the mean (SEM). A p-value of less than 0.05 was considered statistically significant. To exclude outliers and uncertain data points, the Q-Dixon test was applied.

3 Results and discussion

In the current study, the structure of 11 new indole alkaloids was tentatively elucidated (Table 1), in addition to the known compounds from the various extracts of R. stricta leaves and stems that were detected (Tables 2 and 3). The analyses were achieved using the technique of electrospray ionization–quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry in positive ionization mode [M + H]+ to explore fragmentation routes.

CompoundR.tMWm/z [M + H]+Major fragment (MSn)Proposal molecular formulaExtract*Indole alkaloids from crude extract of stems (RS)ABCD12.4752753591 [M-162 + H]+, 429 [M-2X162 + H]+, 341 [M-2X162-88 + H]+, 215 [M-2X162-88–126 + H]+, 197 [M-2X162-88–126-18 + H]+C35H48N2O16+22.9470471309 [M-162 + H]+, 307 [M-164 + H]+, 263 [M-164–44 + H]+C24H27N2O8−+34.2780781601 [M-180(162 + 18)+H]+, 553 [M-228 + H]+, 391 [M-160–228-162 + H]+, 315 [M-180–48-162–76 + H]+, 229 [M-180–48-162–76-86 + H]+C37H52N2O16+45.7366367351 [M-16 + H]+, 309 [M-16–42 + H]+C21H23N2O4−+56.25368369355 [M-14 + H]+, 341 [M-2X14 + H]+, 297 [M-2X14-44 + H]+C22H28N2O3+66.33368369355 [M-14 + H]+, 311 [M-14–44 + H]+, 297 [M-2X14-44 + H]+C22H28N2O3+710.7696697349 [M-348 + H]+, 292[M-348–57+2H]+C42H40N4O6+Indole alkaloids from the crude extract of leaves (RL)A' B'C'D'82.4752753591 [M-162 + H]+, 429 [M-2X162 + H]+, 341 [M-2X162-88 + H]+, 215 [M-2X162-88–126 + H]+, 197 [M-2X162-88–126-18 + H]+C35H48N2O16+92.6546547367 [M-180 + H]+, 339 [M-180–28 + H]+, 296 [M-180–28-44 + H]+C28H38N2O9+107.8430431297 [M-133+2H]+C24H34N2O5+

Characterization of indole alkaloids first detected from R. stricta stem and leaf extracts using UPLC–ESI–MS/MS in positive ion mode [M + H]+.

+* = presence of these compounds in the extracts.

Rt (min)Molecular formulaCompound nameMWm/z [M + H]+Major fragmentExtract*ABCD4.9C21H24N2O4Rhazicine, rhazizine, or isorhazicine368369299 [M-70 + H]++5.4C21H24N2O3Akuammidine, rhazinol (analog of strictamine), geissoschizine, polyneuridine, or tetrahydroalstonine352353335 [M-18 + H]+, 317 [M-2X18 + H]++6.0C21H24N2O3Akuammidine, rhazinol (analog of strictamine), or tetrahydroalstonine352353313 [M-40 + H]+, 299 [M-40–14 + H]++6.3C21H24N2O4Rhazicine, rhazizine, or isorhazicine368369354 [M-14 + H]+, 341 [M-2X14 + H]+, 297 [M-2X14-44 + H]++6.6C21H22N2O3Leepacine isomer II350351311 [M-40 + H]+, 281 [M-40–30 + H]++6.9C21H24N2O3Akuammidine, rhazinol (analog of strictamine), and tetrahydroalstonine352353337 [M-16 + H]+, 297 [M-16–40 + H]++7.5C21H22N2O3Leepacine isomer III350351269 [M-82 + H]++7.8C27H34N2O9Strictosidine530531309 [M-222 + H]+, 283 [M-222–26 + H]+, 265 [M-222–26-18 + H]++9.9C42H56N4O4Tetrahydropresecamine680681341 [M-340 + H]+, 313 [M-340–28 + H]+, 116[M-340–28-197 + H]++11.4C40H54N4O216S, 16′-decarboxytetra-hydrosecamine622623379 [M-244 + H]+, 312 [M-244–67 + H]+, 302 [M-244–67-10 + H]++11.9C40H54N4O216S, 16‟-decarboxytetra-hydrosecamine622