Structural elucidation and evaluation of toxicity and antitumor activity of cardiac glycosides isolated from Leptadenia pyrotechnica

Abstract

Investigation of the chemical constituents of Leptadenia pyrotechnica (Forsk.) Decne (Asclepiadaceae) led to the isolation of three cardiac glycosides; 14,19-dihydroxycard-20 (22)-enolide-3-O-[β-d-glucopyranosyl-β-d-digitoxoside] C-I, 14,19-dihydroxycard-20 (22)-enolide-3-O-[β-d-glucopyranosyl-β-d-glucopyranoside] C-II and 14,19-dihydroxycard-20 (22)-enolide-3-O-β-d-digitoxoside C-III. The isolation and identification of these compounds were carried out using rotation locular counter current chromatography (RLCCC), high performance liquid chromatography (HPLC). The structures of the isolated compounds were established by fast-atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopic analysis. The acute toxicity studies of the cardiac glycosides and total alcohol extracts were examined by brine shrimp. Their lethal concentration 50 (LC₅₀) were 18.84 and 11.89 ppm, respectively. The antitumor activity potato disk assays of the cardiac glycosides and total alcohol extracts had shown good activity − 30.8% and 49.3%, respectively.

Keywords::

• Leptadenia pyrotechnica
• Asclepiadaceae
• brine shrimp
• antitumor activity
• digitoxoside
• cardiac glycosides and RLCCC

Introduction

The Asclepiadaceae family comprises many medicinal plants with a wide range of therapeutic activities (Koike et al., 1980). Therefore, many Asclepiadaceous constituents have been intensively investigated as emetics and antitumor agents (Seiber et al., 1982). The active constituents are cardenolides, polyoxypregnane derivatives, alkaloids, flavonoids, and triterpenes (Bazzaz et al., 2003; Paulo & Houghton, 2003; Atta & Mouneir, 2005; Cioffi et al., 2006; Khanna & Kannabiran, 2007).

The chosen plant for this study was Leptadenia pyrotechnica (Forssk.) Decne. This plant is widely growing in several locations in Egypt especially in the eastern desert and Sinai Peninsula. This plant was used in folk medicine to prepare antispasmodic, anti-inflammatory, antihistaminic, antibacterial, diuretic, urolithic expulsion, expectorant, gout, and rheumatism remedies (Cioffi et al., 2006; Aquino et al., 1996; Panwara & Tarafdarb, 2006). A previous study on the aerial parts of the plant led to isolation of some phenolic compounds, flavonoids, alkaloids, pregnane glycosides, fatty alcohols, sterols, terpenoids, amino and fatty acids (Cioffi et al., 2006; Noor et al., 1993; Abd El-Ghani & Amer 2003; El-Hassan et al., 2003; Panwara & Tarafdarb, 2006; Moustafa et al., 2007). The whole L. pyrotechnica plant afforded 18 new pregnane glycosides with sarcostin, 11- hydroxysarcostin, and deacetylmetaplexigenin as the aglycone moieties and acetyl, benzoyl, cinnamoyl, p-coumaroyl, and nicotinoyl ester moieties linked at C-12 and/or C-20 of the aglycone and hexopyranose, 6-deoxy-3-O-methylhexopyranose and 2,6-dideoxy-3-O-methylhexopyranose sugars linked at C-3 of their aglycone. The antiproliferative activity of all compounds was evaluated using three continuous murine and human culture cell lines, J774.A1, HEK-293, and WEHI-164. Compounds having deacetylmetaplexigenin as aglycone and a cinnamoyl ester moiety linked at C-12 were the most active constituents (Cioffi et al., 2006, Aquino et al., 1995 and 1996).

Cardenolides have been isolated from many genera of the Asclepiadaceae and several other families (Singh & Rastogi, 1970; Abisch & Reichstein, 1962; Seiber et al., 1983). Cardenolides are used to increase the tone, excitability, contractibility of the cardiac muscle and exert a diuretic action (Balbaa et al., 1976; Boulos, 1983, Braga et al., 1996; Schaller & Kreis, 2006).

A method utilizing brine shrimp (Artemia salina Leach) (Krishnaraju et al., 2006; Moustafa et al., 2007) is proposed as a simple bioassay for determining lethal concentration 50 (LC₅₀) values in μg/mL of extracts.

Crown gall is a neoplastic plant disease induced by specific strains of the Gram negative bacterium named Agrobacterium tumefaciens (Lippincott et al., 1977). This bacterium contains large tumor-inducing (Ti) plasmids which carry genetic information (T-DNA) that transforms normal plant cells into autonomous tumor cells (Chilton et al., 1980). The development of a simple antitumor pre-screen, using convenient and inexpensive plant tumor systems, could, thus, offer numerous advantages as alternatives to extensive animal testing in the search for new anti-cancer drugs (Ferrigni et al., 1982).

Crown gall tumorigenesis on disks of potato tuber (Solanum tuberosum L.) was proposed as an ideal system for investigating the transformation process (Ferrigni & McLughlin, 1984). The action of the antitumor compounds is neither via antibiosis nor through inhibition of bacterial attachment to the tumor-binding sites (Bryant et al., 1994).

This paper deals with the isolation, structural elucidation of cardiac glycosides in L. pyrotechnica and evaluation of their antitumor activity and toxicity using potato disk assay and brine shrimp, respectively.

Material and methods

Plant material

Fresh aerial parts of L. pyrotechnica (Forssk.) Decne (Asclepiadaceae) were collected in September 2004 from Wadi Khashab and Wadi Matzos, Sharm El-Sheikh to El-Tur road, southern Sinai, Egypt. The identity was established by Samia Heneidak, Department of Botany, Faculty of Science, Suez Canal University. A voucher specimen (number AMYM-1004) has been deposited in the Herbarium of the Botany Department, Faculty of Science, Suez Canal University, Ismailia, Egypt.

General methods

Melting points were determined on a Büchi 535 melting point apparatus (Büchi, Germany) and are uncorrected. FAB-MS was performed on a Mat 95 double focusing instrument operated at unit resolution. The samples of interest were mixed on the probe tip with glycerol as matrix. Xenon gas was used to generate the primary ionization beam from an Ion-Tech gun operated at 6 KV. Ion source accelerating potential was adjusted at 5 KV. Electrospray ion sources (ESI-MS) technique was equipped with a Finnigan ESI II ion source. ¹H NMR spectra were recorded in DMSO-d₆ with GE NMR (Perkin-Elmer, USA), QE-300, FT-NMR system at 300 MHz. Chemical shifts are given relative to tetramethylsilane (TMS) as an internal standard. Separation of cardiac glycosides was performed using rotation locular counter current chromatographic (RLCCC) technique, Eyela RLCC-1000 (Tokyo Rikakikai), Delivery Pump VSP-3050, UV-VIS detector UV-9000 (non-metallic separation), recorder SS-100F-MM, fraction collector DC-1200, spinning at 60-120 rpm during operation. Rotation speed of the spindle was 60–120 rpm, and flask rotation at 40 rpm; wavelength 225 nm; chart speed 0.1 mm/min. Perkin-Elmer analytical HPLC-diode array and fluorescence, model series 410 LC pump, LC 240 fluorescence detector, 235C photodiode array detector, advanced LC sample processor ISS 200, PC × 5100 post-column reaction module was used. HPLC grade solvents were used with the following conditions: column; Whatman^®, partisil 10, ODS-2, HPLC pre-packed column, Magnum 9 column 50 cm, semi-preparative. Channel parameters: channel A signal source LC235C; delay time 0 min; run time 30 min; sampling rate 2.4414 points/second injection source automatic. The eluents were A, 100% methanol, and B, 100% water. Samples were eluted according to the following gradient: 5% in A as initial conditions; 25% in A for 25 min, 50% in A for 10 min and finally, 100% in A for 5 min. The flow rate was 1 mL min⁻¹. The column was operated at room temperature and the sample volume injected was 20 μL. Absorption spectra of compounds were recorded between 225-245 nm. Solutions to be analyzed by HPLC were passed through membrane filters (0.5 μm pore size) prior to injection. Identification of compounds was made by comparing their t_R values and UV spectra with those of standards. HPLC separation was performed with a Perkin-Elmer model series 400 solvent delivery system. The instrument was equipped with Pharmacia LKB.UVICORD SII, Pharmacia LKB.Rec.102. Phenomenex^® LUNA 5u C18 column (250 × 4.6 mm internal diameter (ID)), particle size; 5 μm ± 0.3 μm was used. Solutions to be used by HPLC were passed through membrane filters (0.5 μm pore size) prior to injection. The plant samples were dried by freeze-dryer (Labconco, USA) under 133 × 10⁻³ Mbar vacuum at −50°C for 24 h. TLC was performed on Merck pre-coated silica gel 60 F₂₅₄ aluminum foil plates and reverse-phase (RP) C-18 F₂₅₄ pre-coated plates (thickness: 0.25 mm, Merck). n- Butanol-ethanol-water (4:1:5), upper layer (S1) was the best solvent elution system. Acetonitrile-water (25:75) was used for elution of RP.

The cardiac glycosides were detected on chromatograms by spraying with antimony trichloride (SbCl₃) and/or Kedde’s reagents. The colors were observed in visible and UV light (365 nm).

Extraction, isolation and characterization

The upper parts of the plant (leaves, flowers and stems) were air dried and ground all together as a fine powder. The phytochemical screening was performed in accordance with Association of Official Agricultural Chemist (AOAC) (1990).

The cardiac glycosides of L. pyrotechnica were extracted by two methods:

1) The latex was collected from the plant in the field in brown glass bottles containing methanol then kept immediately in dry ice. This was done by cutting the plant at the tip pod-stem and/or fruit-stem junctures. The solvent was evaporated under vacuum to give about 2 g latex which was kept at 4°C. 2) The aerial parts of the plant (1 kg) were shock-frozen with liquid nitrogen, lyophilized, and pulverized then percolated three times with methanol at room temperature and the extracts were concentrated under reduced pressure. The methanol extract was defatted with petroleum ether (40°–60°C) and yielded 35 g upon evaporation. The cardiac glycosides extract was isolated from the defatted methanol extract (450 g) in accordance with Abdel-Azim et al. (1996) to give a dark brown gum (7.2 g, 0.72%).

The plant latex was subjected to RLCCC system, by partitioning it with a suitable two-phase solvent system S1. The latex dissolved in the mixture of both phases, and the upper layer of the sample (3 mL, 1 g latex) was injected into the chamber. Mobile phase S1 upper layer is subsequently pumped via the sample chamber into the first of the columns.

Partial and complete acid hydrolysis of the isolated compounds was carried out according to Macek and Kiliani methods (Macek, 1972; Parrilli, et al., 1981).

Brine shrimp lethality bioassay

The cytotoxic effect of total cardiac glycosides, methanol extract and defatted methanol extract, of L. pyrotechnica were evaluated by LC₅₀ values of the brine shrimp lethality test (Krishnaraju et al., 2006; Poli et al., 2006; Ho et al., 2005; Pisutthanana et al., 2004). The eggs of brine shrimp were obtained from San Francisco Bay Brand, Newark, CA. The tested samples were dissolved in methanol, and three graded doses, 10, 100, and 1000 ppm, respectively, were used for 5 mL seawater containing 10 brine shrimp nauplii in each group. The number of survivors was counted in each well after 6 h. Counting of the chronic LC₅₀ was begun 24 h from starting the test. LC₅₀ was determined by probit analysis described by Meyer et al. (1982). Control disks were prepared using only methanol. Five replicates were prepared for each dose level. The negative control solution was simply the same saline solution used to prepare the stock test sample solution. Potassium dichromate was used as standard toxicant and dissolved in artificial seawater, to obtain concentrations of 1000, 100, and 10 ppm. Reed-Muench method was used to treat the data derived from a test series (Krishnaraju et al., 2006). The dose that killed 50% of the animals could be derived by graphic procedure, plotting the number of the accumulated survivors and the number of accumulated deaths on the same axes versus log dose. The two curves would cross at the log dose, where the number of survivors was equal to the number of deaths. This method was used to estimate the median tolerance. The second method plotted dosage against percentage mortality and the dosage at 50% mortality was obtained by intersection. This method was used to estimate the standard error (Nguyen et al., 1998; Yang et al., 2001).

Antitumor screening of L. pyrotechnica: Potato disk assay

The potato disk bioassay for the plant extracts was carried out in accordance to (Ferrigni & McLughlin, 1984; Ferrigni et al., 1982; Anderson et al., 1988, 1992; Bryant et al., 1994). Tumors were initiated on potato disks (usually Pontiac red or red russet variations). DMSO was used as a solvent. Fresh, disease-free potato tubers (preferably red) were obtained from local markets and were kept under refrigeration until used. About 8 mg of each cardiac glycoside, total methanol and defatted methanol extracts were dissolved in 2 mL DMSO. These solutions were filtered through Millipore filters (0.22 μm) into sterile tubes. From these solutions 0.5 mL was added to 1.5 mL sterile water and 2 mL of a broth culture of A. tumefaciens strain B₆ (48-h culture containing 5x10⁹ cell/mL). Controls were made in the following way: 0.5 mL DMSO was filtered through Millipore filter (0.22 μm) into 1.5 mL sterile distilled water and added to tubes containing 2 mL of the same A. tumefaciens strain B₆. A standard solution of Camptothecin was made as follows: 8 mg Camptothecin was dissolved in 2 ml DMSO. From this solution 0.5 mL was added to 1.5 mL sterile water and 2 ml of the same broth culture of A. tumefaciens strain B₆. A blank solution was made in the following way: 0.5 mL DMSO was added to 1.5 mL sterile water. One drop (0.05 mL) from these tubes was used to inoculate each potato disk, spreading it over the disk surface. Twelve days after inoculation at room temperature, the tumors were counted with the aid of a dissecting scope, after staining with Lugol`s solution lodine (I), potassium iodide (KI) and compared with controls. The tumor cells lack starch. The control with 12.5% dimethyl solfoxide (DMSO) yielded 30 tumors/disk. The results were expressed as + or − percentages versus the number of tumors on the control disks; inhibition was expressed as a negative percentage and stimulation was expressed as a positive percentage. Significant activity was indicated when two or more independent assays gave consistent negative values of 20% or greater inhibition.

A. tumefaciens strain B₆ was maintained on solid slants under refrigeration. Subcultures were grown in 0.8% nutrient broth (Difco, Detroit, Michigan, USA) supplemented with 0.5% sucrose and 0.1% yeast extract.

Results and discussion

The results obtained showed that the cardiac glycosides formed colored complexes, varied in color and fluorescence (blue, red-violet or brown color). Seven cardiac glycosides, three major constituents with R_f-values, 0.52, 0.65, and 0.74 and four as minors with R_f-values 0.62, 0.8, 0.85, and 0.9 were detected in latex. Two major spots in the chloroform extract (R_f-values, 0.65, and 0.74), beside others as minor spots were also detected. Four fractions were isolated, fraction A (0.13 g, R_f; 0.9, 0.85, 0.8, 0.74, 0.65, 0.56, and 0.52; S1), fraction B (0.45 g, R_f; 0.74, 0.65, 0.56, and 0.52, S1), fraction C (0.11 g, R_f; 0.74, 0.65, 0.56 and 0.52, S1) and fraction D (0.15 g, R_f; 0.52; S1) were detected using antimony tri-chloride solution reagent, Figure 1. Almost all cardiac glycosides constituents were detected in fraction A as minor spots.

Figure 1.  TLC chromatogram of the collective fractions isolated from RLCCC; 1: total latex, 2-6: Fractions A, B, C, C2 and D, respectively.

These fractions were integrated using a densitometer scanner, which revealed that fractions B and D contain the major characteristic spots of cardenolides, as shown in Figure 2. These fractions were detected by analytical HPLC-diode array and their UV spectra were recorded online (Figure 3A, 3B, 4, 5A, 5B).

Figure 2.  Densitometer scanning of collective fractions isolated from RLCCC.

Figure 3.  (A) HPLC diode-array of cardiac glycosides constituents of fraction A and (B) Its UV-detection at 245 and 225 nm wavelengths.

Figure 4.  HPLC chromatogram of the fraction B components.

Figure 5.  (A) HPLC diode-array of cardiac glycosides constituents of fraction B and (B) Their UV-absorption spectra at 245 and 225 nm.

Fraction B contains four major peaks, possessing retention times t_R; 8.9 (C-III), 10.5 (C-I), 12.5 (C-II) and 19 (C-IV) others as minor constituents. Fraction A contains six major peaks, possessing retention times t_R; 8, 8.9, 10.5, 12.5 and 19 others as minor constituents, while fraction D contains one major peak, possessing retention time t_R; 19 other peaks as minors.

The four major peaks, i.e. C-I to C-IV, were isolated by preparative HPLC as single peaks at the same retention times as mentioned above (Figure 6).

Figure 6.  HPLC analysis of the isolated cardenolides.

14,19-dihydroxycard-20(22)-enolide-3-O-(β-d-glu copyranosyl-β-d-digitoxoside) C-I was a white amorphous powder, 26.8% from the total cardiac glycosides, R_f; 0.74, S_i, t_R; 10.5. NIFAB-MS spectra of C-I exhibited a quasi molecular ion at m/z 681, and fragment ions at 551 and 389, assigned to [M-H]⁻, [M-H-162]⁻ and [M-H-162-130]⁻ respectively. This indicated the presence of two glycosyl moieties. Also, ESI-MS spectral data gave the molecular ion of aglycone at m/z 391, which ascribed to [M+H-162-130]⁺ and in accordance with the molecular formula C₂₃H₃₅O₅ of trihydroxy substitution pattern for 3,14,19-trihydroxycard-20 (22)-enolide (Dhawan & Singh, 1976; Jolad et al., 1986; Noor et al., 1993). This was also evidenced with PIFAB-MS, exhibiting readily interpretable fragmentation peaks at m/z 683, 553 and 391 and were ascribed to [M+H]⁺, [M+H-162]⁺ and [M+H-162-130]⁺. The presence of trihydroxyl groups in aglycone residue was clearly marked by their specified peaks at m/z 373 [391-H₂O]⁺, 355 [373-H₂O]⁺ and 337 [355-H₂O]⁺.

In ¹H-NMR spectrum, (300 MHz, DMSO-d₆): δ (ppm) 1.15 (1Hα, m, H-1), 2.67 (1Hβ, m, H-1), 4.28 (1H, m, H-2), 4.50 (1H, m, H-3), one singlet signal at 0.82 (-CH₃, 3H, s, H-1) corresponding to methyl group at H-18 was observed. The other signals were also observed at δ 5.02-5.29 (2H, m, H-21), and δ 6.68 (1H, br. s, H-22). These signals are characteristic of protons constituting the γ-lactone ring and H-17 at δ 2.77(1H, m) (Majinda et al., 1997). The nature and stereochemistry of glycosyl moieties were determined by anomeric proton resonances at δ 5.21 (1H, dd, H-1′), and δ 4.84 (1H, d, J=7.5, H-1″), assigned to retain β-linkage and to be a D-sugar. The presence of a methyl signal at δ 1.56 (3H, br. s, CH₃- sugar) suggested it to be a 6-deoxysugar. In addition to one set of protons 3.31-3.73 (sugar protons, m) which was characteristic for sugar moiety. The signal locations of primary carbinol methylene protons were observed at δ 3.93 and 4.06 (-CH₂, each 1H, br. d, H-19) (Majinda et al., 1997; Abdel-Azim et al., 1996).

C-I sugar moiety was identified as glucose and digitoxose (2, 6-dideoxy-β-d-ribo-hexose) by partial and complete acid hydrolysis. PIFAB-MS spectra of C-I aglycone, exhibited a quasi molecular ion at m/z 391 [M+H]⁺ gave the molecular ion of the aglycone in accordance with the molecular formula C₂₃H₃₅O₅ of trihydroxy substitution pattern for 3, 14, 19-trihydroxycard- 20(22)-enolide. This aglycone was previously described as Coroglaucigenin and Carpogenol (Shoeba et al., 2004; Noor et al., 1993; Jolad et al., 1986; Dhawan & Singh, 1976; Abisch & Reichstein, 1962; Jäggi et al., 1967; Golab et al., 1960).

14,19-Dihydroxycard-20(22)-enolide-3-O-(β-d- glu copyranosyl-β-d-glucopyranoside) C-II was a white amorphous powder, 28.3% from the total cardiac glycosides, R_f; 0.65, S1, t_R; 12.5. NIFAB-MS spectra exhibited a quasi-molecular ion at m/z 713, and fragment ions at 551 and 389, were assigned to [M-H]⁻, [M-H-162]⁻ and [M-H-162-162]⁻ and suggested the presence of two glycosyl moieties. ESI-MS and PIFAB-MS spectral data gave the molecular ion of aglycone as mentioned before in C-I.

¹H-NMR spectrum (300 MHz, DMSO-d₆) showed the characteristic signals of aglycone and the glucosyl moieties determined by anomeric proton resonances as previously described in C-I. δ (ppm) 1.20 (1Hα, m, H-1), 2.57 (1Hβ, m, H-1), 4.19 (1H, m, H-3), 2.67 (1H, m, H-17), 1.02 (3H, s, H-18), 5.01-5.22 (2H, m, H-21), 6.10 (1H, br. s, H-22), 4.48 (1H, dd, H-1`), 4.84 (1H, d, J=7.5Hz, H-1``), 3.13-3.75(m, sugar protons), 3.89 and 4.06 (-CH₂, each 1H, br. d, H-19). The sugar moiety was identified as glucose. PIFAB-MS spectra of aglycone m/z 391. This compound was isolated before from Coronilla scorpioides and known as Coronillobiosidol (Kopp et al., 1996).

14,19-Dihydroxycard-20(22)-enolide-3-O-β-d-dig italoside C-III was a white amorphous powder, 12.0% from total cardiac glycosides. R_f; 0.8, S1, t_R; 8.9. PIFAB-MS spectra exhibited quasi-molecular ions at m/z 551 and 391, assigned to [M+H]⁺ and [M+H-160]⁺ and suggested the presence of one glycosyl moiety, deoxyhexose. This was evidenced with the ESI-MS spectral data, which gave the molecular ion of aglycone at m/z 391 [M+H-160]⁺. C-I D-digitalose moiety and aglycone were identified as previously described in C-I.

Four compounds C-IV to C-VII with t_R; 19, 8, 9.5, and 3.8, respectively, were isolated from all the fractions A-D. These compounds gave white amorphous powder upon crystallization from methanol-water and possessed the chromatographic properties of cardenolides (R_f-values, color, and response towards antimony trichloride spray reagent). They represented 21%, 4.1%, 3.5%, and 1.6%, respectively, from the total cardiac glycosides.

The results obtained for all tested samples from the brine shrimp toxicity test, are represented in Table 1. The data of mortality rates point out that with 1000 ppm concentration, latex (cardiac glycosides) and methanol extracts exhibited 100% mortality, while the defatted methanol extract represented 98.1%. Moreover, with 100 ppm concentration, the extracts latex, methanol, and defatted methanol, exhibited high mortality and represented 94.12%, 98.83%, and 79.41%, respectively. On the other hand, with 10 ppm concentration, latex represented 33.33%, but the other extracts were represented 45.1% and 25% respectively.

Table 1.  Mortality of brine shrimp at various concentrations of the different extracts of L. pyrotechnica.

Plant extract	Dose(ppm)	Dead	Alive	Accumulated dead	Accumulated alive	Ratio dead:total	Mortality(%)	LD50^*`ppm
Latex (cardiac glycosides)	1000	50	0	114	0	114/114	100	18.84
	100	46	4	64	4	64/68	94.12
	10	18	32	18	36	18/54	33.33
Methanol extract	1000	50	0	122	0	122/122	100	11.89
	100	49	1	72	1	72/73	98.63
	10	23	27	23	28	23/51	45.1
Defatted methanol extract	1000	48	2	102	2	102/104	98.1	28.19
	100	38	12	54	14	54/68	79.41
	10	16	34	16	48	16/64	25

*Where transformation which does data was insufficient for probit analysis, LC₅₀`s were estimated using logit not.

The acute median LC₅₀ of latex is low (18.84 ppm) and exhibited high toxicity compared with the other detected extracts. Moreover, the 95% confidence limits of the extracts were calculated as shown in Table 1. The results obtained revealed that the estimated LC₅₀ and its 95% confidence limits for the latex, methanol and methanol after defatted extracts were 18.84 (11.22-31.61), 11.89 (11.23-21.05) and 28.19 (16.27-48.81) ppm, respectively.

The results obtained from the potato disk assay showed significant antitumor activity for methanol extract (−49.3%) and total cardiac glycosides (−30.8%) compared with Camptothecin standard which represents 100%. Figure 7 showed the inhibition tumor development by applying different extracts of L. pyrotechnica. The first picture (I) showed the maximum inhibition of tumors while the degree of inhibition decreased from picture (II) to (VI) respectively.

Figure 7.  Tumor inhibition development by cardiac glycosides on potato disks. The first picture I shows the maximum inhibition of tumors, while the degree of inhibition decreased from pictures II to VI, respectively.

Conclusion

In this work, three cardiac glycosides were isolated from the target plant and identified as 14,19-dihydroxycard-20 (22)-enolide-3-O-[β-d-glucopyranosyl-β-d-digitoxoside] C-I, 14,19-dihydroxycard-20 (22)-enolide-3-O-[β-d-glucopyranosyl-β-d-glucopyranoside] C-II and 14,19-dihydroxycard-20 (22)-enolide-3-O-β-d-digitoxoside C-III. The LC₅₀ of the cardiac glycosides and total alcohol extract were 18.84 and 11.89 ppm respectively. The antitumor activity potato disk assays of the cardiac glycosides and total alcohol extracts showed good activity. The antitumor activity of L. pyrotechnica could be attributed to its cardenolides and the other methanol constituents, while the cardenolides of this plant exhibited high toxicity. Therefore, applying these active cardenolides as antitumor agent must be used in a limited range.

Acknowledgements

AMYM is grateful to the Egyptian Ministry of higher Education, and the Environmental Chemistry and Toxicology Laboratory, Texas Southern University, Houston, for the Fellowship it has provided her with to undertake this work.

Declaration of interest: This work was supported by RCMI grant number R003045-17A and NASA/URC grant number NCC 9.165. The authors alone are responsible for the content and writing of the paper.