Phytochemical, cytotoxicity and antioxidant investigation of Cassia alata leaves growing in Egypt

Mohammed I. Ali¹, Ahmed M. Aboul-Enein², Samy M. Mohamed¹, Faten M. Abou elella², Magdy M. D. Mohammed³, Ahmed R. Hamed⁴

Introduction

Cassia alata (L.) Roxb., (syn. Senna alata L) family Fabaceae. It is widely distributed in the tropical countries including Egypt [1]. Chemical constituents of C. alata leaves include emodin [2], chrysoeriol, kaempferol, quercetin, 5,7,4'-trihydroflavanone, kaempferol-3-O-β-D- glucopyranoside, kaempferol-3-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside, fatty acids, hydrotetratriacontane, n-dotriacontanol, n-triacontanol [3], aloe-emodin, rhein methyl ester, aloe-emodin-O-β- glucoside, kaempferol [4]. Kaempferol [5,6], Kaempferol3-O-gentiobioside [7], 5, 7-dihydroxy-3', 4'- dimethoxy flavone and 2, 5, 7, 4' tetrahydroxy Isoflavone [8,9], propelargonidins, is a condensed tannins [10] and volatile oil [11].

C. alata leaf extract has been reported to have various pharmacological activities including antibacterial [12,13], cytotoxicity [14,15], anti-inflammatory [16], antidiabetic [17], antihepatotoxic and hepatoprotective effects [18,19], antiseptic [20], antiviral [21] and exhibited strong DPPH Natural plant compounds are highly varied in structure. However, there is an increase attention on extracts and biologically active compounds isolated from plant species used in herbal medicine, due to the fewer side effects. Medicinal plants are playing an important role as anticancer agents and it is significant that many of currently used anticancer agents are derived from natural source such as plants [23].

The present study is focused in isolation and chemical identification of unsaponifiable matter, fatty acid and anthraquinones, in addition to the antioxoidant and cytotoxicity activities of chloroform fraction of Cassia

Experimental

Material and Methods

Chemicals

Folin–Ciocalteu reagent (Sigma Chemical Co., St. Louis, Mo., USA). All solvents and chemicals were HPLC grade, Fisher Scientific, USA. Silica gel 60 mesh (Merck, column chromatography. TLC silica gel F or 254 polyamide aluminum sheets 20 x 20 cm and HPTLC Silica gel F aluminum sheets 20 x 20 cm (Merck, 254 Germany).

Dulbecco’s modified Eagle’s medium (DMEM), TrypsinVersene (EDTA) Mix, Dulbecco’s Phosphate-Buffered Saline (DPBS), L-Glutamine and penicillin/streptomycin stock from Lonza Verviers SPRL, Belgium. Fetal Bovine Serum (FBS; SeraLab, UK), Doxorubicin (trade name: Adriamycin; Pfizer company, Australia), and DPPH (Sigma, Germany). MTT dye (3-(4,5-Dimethylthiazol-2- yl)-2,5-Diphenyltetrazolium Bromide) and Di-methyl sulfoxide (DMSO) from SERVA Electrophoresis GmbH, Germany.

Apparatus and Instruments

Nuclear magnetic resonance (NMR) spectra were measured on Bruker AV-400 spectrometer operating at a frequency of 400 MHz using DMSO as solvent at room temperature with tetramethylsilane (TMS) as an internal standard. Electron ionization (EI) mass spectra (EI-MS) were carried out on a THERMO Scientific Corp.; USA), mass spectrometer 70 eV. FLUOstar Optima microplate reader (BMG labtech, Germany) and inverted microscope (Olympus CK2, Japan).

Gas Chromatography/Mass Spectrometry (GC/ MS) analysis of unsaponifiable matter

GC-MS analysis of unsaponifiable matter was carried out using gas chromatography-mass spectrometry instrument stands with the following specifications Instrument: a TRACE GC Ultra Gas Chromatographs (THERMO Scientific Corp.; USA), coupled with a thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer). The GC-MS system was equipped with a TG-5MS column (30 m x 0.25 mm i.d.; 0.25 μm film thickness). Analyses were carried out using helium as carrier gas at a flow rate of 1.0 ml/min and a split ratio of ° 1:10 using the following temperature program: 50 C for ° ° 3 min; rising at 5 C /min to 300 C and held for 20 min.

° The injector and detector were held at 280 C. Diluted samples (1:10 hexane, v/v) of 0.2 μl of the mixtures were always injected. Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 40- 450. Most of the compounds were identified using two different analytical methods: mass spectra (authentic chemicals, Wiley spectral library collection and NSIT library) and by comparison with previous references and data.

Gas chromatographic-mass spectrometry (GC-MS) analysis for fatty acid methyl esters

GC-MS analysis of the fatty acid methyl esters was carried out using gas chromatography-mass spectrometry instrument stands with the following specifications. Instrument: a TRACE GC Ultra Gas Chromatographs (THERMO Scientific Corp.; USA), coupled with a THERMO mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer). The GC-MS system was equipped with a PR-5MS column (30 m x 0.25 mm i.d.; 0.25 m film thickness). Analyses were carried out using helium as the carrier gas at a flow rate of 1.0 ml/min at a split ratio of 1:10 and the following temperature program:

° o ° 50 C for 3 min; rising at 4.0 C/min to 260 C and held ° ° for 6 min; rising at 6 C /min to 300 C and held for 1 ° min. The injector and detector were held at 200 C. Diluted samples (1:10 hexane, v/v) of 0.2 μl of the mixtures were always injected. Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 40-450. Most of the compounds were identified using two different analytical methods: mass spectra (authentic chemicals, Wiley spectral library collection and NSIT library) and by comparison with previous data.

Plant materials

Cassia alata (L.) Roxb., leaves were collected from Faculty of Agriculture Farm, Cairo University and were kindly identified by, Orman Botanical garden, Giza and Dr. Mona Marzok, Taxonomist in the Herbarium of National Research Centre (NRC), Cairo, Egypt. A voucher specimen was deposited at the Herbarium of the NRC, Cairo, Egypt.

Extraction

Fresh leaves were dried at room temperature in shad and grinded to fine powder. Air-dried, powdered (2 kg) were extracted by methanol at room temperature and o concentrated under reduced pressure at 40 C. The residue of methanolic extract (22.57 %) was suspended in water with assistance of ultrasonic water bath and partitioned successively with hexane (3.6%), chloroform (3.33%), ethyl acetate (0.74%), n-butanol (4.63%) and aqueoussoluble fractions (10.27%). All extracts were ° concentrated till dryness in rotor vapor at 40 C. The ° obtained extracts were stored at -20 C until used.

Isolation and identification of saponifiable and unsaponifiable matter of n-hexane extract

About 2g of n-hexane fraction were saponified with alcoholic KOH 10% (ethanol 95%) and the residue was fractionated into unsaponifiable and saponifiable matters according to the method of Farag et al. [24], as well as, the fatty acids were liberated by acidification of the saponifiable matter, extracted with ether, dried over anhydrous Na²So⁴ and concentrated under reduced pressure. The fatty acid methyl esters were prepared according to the method adopting by Kinsella [25]. Fatty acid methyl esters and unsaponifiable matter were analyzed by GC/MS and were identified on the basis of fragmentation pattern of mass spectra data and a library database [Wiley (Wiley Institute, Los Angeles, CA), NIST (National Institute of Technology, Los Angeles, CA)] and by comparison with previous data. Quantitative determination was carried out on the basis of peak area measurements of the GC/MS chromatograms.

Isolation and characterization of anthraquinones

Chloroform crude fraction was subjected to silica gel (mesh 60) column chromatography (CC) and was successively eluted gradient with a mixture of solvents of increasing polarity made up n-hexane: ethyl acetate (100%, up to 25:75 v/v), then CHCl³: ethyl acetate (100%, up to 70:30 v/v) yielding 98 fractions (20 ml) and the fractions were monitored by TLC silica gel F²⁵⁴ sheets (Merck) using different solvent systems as follows: toluene: ethyl acetate (8:2 v/v) and n-hexane: ethyl acetate (70:30 v/v), CHCl³: ethyl acetate (9:1 v/v). Visualization was carried out using p-anisaldehyde - ° sulphuric acid reagent heated at 110 C and alcoholic KOH (10%). Similar fractions of the same 𝑅𝑓 (TLC) in different solvent systems were combined and pooled together, to give 18 major fractions.

F1 to F5 corresponding to the n-hexane (100%) and F6 to F8 corresponding hexane: ethyl acetate (95:5 v/v) were fatty acids and lipids. F9 to F11 corresponding the n-hexane: ethyl acetate (90:10), F12 corresponding to the n-hexane: ethyl acetate (75:25 v/v). F13 corresponding the n-hexane: ethyl acetate (50:50), F14 corresponding to the n-hexane: ethyl acetate (25:75 v/v), F15 corresponding to the CHCl³: ethyl acetate (90:10 v/v), F16 corresponding the CHCl³: ethyl acetate (80:20 v/v), F17 and F18 corresponding to the CHCl³: ethyl acetate (70:30).

F9 to F18 were checked for the presence of anthraquinones using HPTLC silica gel F²⁵⁴ and TLC polyamide and the presence of yellow spots which upon spraying with 10% ethanolic KOH (Bornträger reaction) showed red spots in visible and red fluorescence spots in UV-365 nm indicated the presence of anthraquinones [26].

This was finally F9, combined of F10 and F11, F12 and F13 were separated by HPTLC silica gel F²⁵⁴ and TLC polyamide using CHCl³: ethyl acetate (9:1 v/v) as the mobile phase. The bands were visualized and detected under UV light at 254 and 365 nm then scraped off from TLC plates, eluted from the silica with absolute methanol, and filtered. The isolated compound(s) appeared as yellow spots which upon spraying with 10% ethanolic KOH (Bornträger reaction) showed red spots in visible and red fluorescence spots in UV-365 nm. The purity of the isolated compounds was confirmed by TLC using various solvent systems. Three different pure anthraquinone compounds were obtained; each pure compound was applied to a Sephadex LH-20 column reversed-phase SPE cartridge C18 column previously activated by elution of methanol.

Cell culture

Three different types of human cancer cells: triple negative breast cancer cells (MDA-MB-231), hepatocellular carcinoma cells (HepG2) and colon adenocarcinoma cells (Caco2) were purchased from vacsera, Egypt and maintained as monolayer culture in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% L-glutamine 200 mM and 1% penicillin/streptomycin stock. Monolayer was passage at 70-90% confluence using trypsin-EDTA solution. All cell incubations were maintained at humidified CO² incubator with 5% CO² at 37°C.

Cytotoxicity activity (MTT assay)

The cytotoxicity in the present study is based essentially on previous report of Mosmann [27] with some modification. Briefly, HepG2, MDA-MB-231 (8000 cells/well) or Caco2 (5000 cells/well) cells were seeded onto 96-well plates in a total volume of 200 μl and left overnight to form a semi-confluent monolayer. Cell monolayers were treated in octuplates with vehicle (DMSO, 0.1% v/v) or test sample (100 μg/ml) for an exposure time of 48 h. Doxorubicin hydrochloride was used as cytotoxic reference drug. At the end of exposure, 30 μl/well of MTT solution in DPBS (5 mg/ml) were added to all wells and left to incubate for 90 min. The formation of formazan crystals was visually confirmed using phase contract inverted microscope (Olympus CK2, Japan). DMSO (100 μl/well) was added to dissolve the formazan crystals with shaking for 10 min after which the absorbance was read at 540 nm against no cell blanks on a FLUOstar Optima microplate reader (BMG labtech, Germany). The percentage of cell death was calculated using the following formula:

𝐴𝑡 −𝐴𝑏 % cell death= 100 − [ × 100] 𝐴𝑐 −𝐴𝑏 Whereas, At: Absorbance of treated cells, Ab: Absorbance of blank (medium only without cells) and Ac: Absorbance of control.

Dose-response experiment was performed on sample producing ≥ 50% loss of cell viability using five serial 2- fold dilutions (100, 50, 25, 12.5 and 6.25 μg/ml). IC 50 values were calculated using the dose response curve fit to non-linear regression correlation using Graph Pad Prism® V6.0 software.

Morphological assessment

Assessment of morphological changes of cells following treatments was performed using phase contrast inverted microscope (Olympus CK2, Japan) and photomicrographs were taken with digital eyepiece camera (Total magnification is 150×).

Antioxidant activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay

The method used in the present study is based essentially on previous reports of Braca et al. and Nara et al. [28,29] with some modification. Briefly, chloroform fraction was prepared in DMSO as 10× stock solution (1 mg/ml). Stock solution (20 μl/well) was dispensed in triplicates onto 96-well plates (flatbottomed, Greiner bio one, Belgium). The assay was started with the addition of DPPH reagent (0.004% wt/v in methanol, 180μl/well) to give a final concentration of the sample as 100 μg/ml. Appropriate blank was prepared using the solvent only in addition to the same amount of DPPH reagent to get rid of any inherent solvent activity. Negative control was also run in parallel to correct for any non-DPPH absorbance by colored fraction at the test wavelength. The plate was immediately shaken for 30 seconds and incubated in the dark for 30 min at room temperature. The remaining DPPH was measured at 540 nm on a FLUOstar Optima microplate reader (BMG labtech, Germany). Quercetin dihydrate was used as a positive control. DPPH radical scavenging activity was calculated using the equation below.

Ab − As % DPPH Scavenging Activity = 100 × Ab Whereas, Ab: Absorbance of blank and As: Absorbance of sample. Absorbance of blank and sample are the averages of triplicates determinations of the corrected readings of blank and sample at 540 nm, respectively.

Results and Discussion

Fatty acids composition of Cassia alata leaves

Fatty acids composition of leaves is presented in Table 1. The major fatty acids were palmitic (37.02%), linolenic (24.27%), linoleic (15.22%) and stearic (14.18%) acid and composed 90.69% of the total fatty acids. The fatty acid palmitic represented the predominate one. The major unsaturated fatty acids consist mainly of linolenic, linoleic followed by oleic acid and linolenic acid represented the most abundant unsaturated one. The total fatty acids are characterized by much higher proportion of saturated fatty acids (54.76%) followed by unsaturated fatty which represented 45.24% of the total fatty acids. Oleic (5.04%) and myristic acid (2.65%) were present in minor level while the other identified fatty acids methyl esters were less than 1%.

2-Hexadecenoic acid, methyl ester 42.40 0.71 Octadecanoic acid, methyl ester 44.91 14.18 (Stearic acid) C18:0 9-Octadecenoic acid (Oleic acid) C18:1 45.38 5.04 9,12-Octadecadienoic acid methyl ester 46.69 15.22 (Linoleic acid) C18:2 9,12,15-Octadecatrienoic acid, methyl 48.42 24.27 ester, (α Linolenic acid) 18:3 Mono unsaturated fatty acids 5.75 Poly unsaturated fatty acids 39.49 Unsaturated fatty acids 45.24 Saturated fatty acids 54.76 RT: retention time by min

Unsaponifiable matter composition of Cassia alata leaves

The data of the GC/MS analysis of the unsaponifiable matter revealed the presence of 23 compounds from which 14 major compounds accounted (99.02%) were identified whereas the minor compounds represented (0.98%) and couldn’t be identified. Results in table 2 shows that phytol C²⁰H⁴⁰O (3,7,11,15- tetramethylhexadec-2-en-1-ol) acyclic diterpene alcohol represented the major constituents (74.59%) and β- sitosterol C²⁹H⁵⁰O (17.45%) was the second major one. Among the phytosterols namely stigmasterol C²⁹H⁴⁸O was identified in minor level and constituted 4.13%. So phytosterol accounted 21.58% of the total unsaponifiable fraction. On the other hand, fatty acid, volatile monoterpene and hydrocarbones were identified in traces levels. Phytol was found to be major constituent of unsaponifiable matter of Cassia alata leaves. In this concern, several studies addressed on its biological activity [30,31].

Anthraquinones

TLC purification of F9 resulted in the isolation of compound I (22 mg) in pure form. Purification based on TLC result to give compound II (15 mg) from combined F10 and F11. Compound III was obtained in pure form from combined F12 and F13 (10 mg). Identification of the isolated pure compound was based on spectrometric 1 analyses HNMR, EI-MS and by comparison with previous data (Figure 1).

Uk = unknown, RT: retention time by min

Compound I was obtained from F9 as orange-yellow 1 amorphous powder. H-NMR spectrum revealed the presence of a sharp singlet signal appeared at δH 2.42 (3H, s, CH³), two sets of double of doublets appeared at δH 6.54 and 7.10 (each, 1H, J = 2.4 Hz) corresponding to H-7 and H-5 respectively, moreover, at δ 7.18 and 7.52 H (each, 1H, J = 1.2 Hz) corresponding to H-2 and H-4 respectively. EI-MS: m/z 269,3 m/z 241.2 [M − 28], 225. The identity of the isolate was achieved by analysis of 1 HNMR and mass spectrum, and comparison data with literature [32–34] and data confirmed the compound as emodin C H O (4,5,7-trihydroxy-2-methyl 15 10 5 anthraquinone).

Emodin has previously reported as anticancer [35] and was isolated from C. alata leaves [2,36], it has been found in a wide variety of plant species belonging to the genus Cassia [37,38].

Compound II was obtained from F10 and F11 as orange-yellow amorphous powder, the compound gives positive Borntrager's test which indicates its presence of a methylene group at δ 4.63 (2H, br s, CH - H 2 11) which broadened due to long range coupling with aromatic protons [39]. Furthermore, two sets of broad signals appeared at δH 7.29 (1H, br s, H-2) and δH 7.69 (1H, br s, H-4) corresponding to the aromatic protons of ring-B. Moreover, two sets of doublets appeared at δH 7.37 (1H, br d, J = 8.4 Hz, H-7) and at δH 7.71 (1H, br d, J = 8.4 Hz, H-5) corresponding to the ortho-coupled aromatic protons (Ring-A) respectively, in addition to a triplet signal appeared at δ 7.80 (1H, t, J = 8.4 Hz) H corresponding to H-6 of (ring-A) due to the orthocoupling with both of H-5 & H-7, ESI-MS: m/z 269.98, m/z 240.2 [M − 28]. Hence, and according to the above data the compound can be identified as aloe-emodin and through comparison with NMR and MS spectra of published data [33,40,41].

Generally aloe-emodin has previously reported as anticancer [42–44]. It has been reported to exhibit an anticancer activity on neuroectodermal tumors, lung squamous cell carcinoma, and hepatoma cells [42,45–47] and was isolated from C. alata leaves [36,48,49] . It has been found in a wide variety of plant species belonging to the genus [44].

Compound III was obtained from F12 and F13 as orange-yellow amorphous powder and the compound gives positive Borntrager's test which indicates its anthraquinone nature. Rhein (1,8- dihydroxyanthraquinone-3-carboxylic acid) was structurally identified as one compound out using MS and 1 H NMR spectra as follows: EI-MS: m/z 284, 267, 256, 239, 228, 211, 183, 155, 142, 126. It showed the molecular ion at m/z 284, which was corresponding to the 1 molecular formula C H O of rhein. The H NMR 15 8 6 spectrum revealed five aromatic protons of which two 1 were broad singlets due to meta coupling. H NMR (400 MHz, DMSO): 11.9 (1H,brs, C1–OH), 11.5 (1H,brs, C8– OH), 8.13 (1H,brs, C2–H), 7.40 (1H, d, J = 8.5 Hz, C5– H), 7.73 (1H, m, C6–H), 7.75 (1H,br s, C4–H), 7.81(1H, - d, J = 8.0 Hz, C7–H). EI-MS m/z 284.31 (M-1) , rhein is characterized by decarboxylation that is molecular ions resulting in [M-COOH] m/z 239. The ions at m/z 253 and 267 were also observed in the mass spectrum of rhein. The structure identification of rhein was performed by 1 HNMR, MS analysis and with comparison published data [33,50,51].

The anticancer activity of rhein has previously reported [51–53] and was isolated from C. alata leaves [49,54,55].

Cytotoxicity activity (MTT assay)

The evaluation of the anti-proliferative activity of the chloroform fraction of C. alata leaves against the proliferation of three different cancer cell lines at a final concentration of 100 μg/ml was illustrated in Figure 2. The pre-screen revealed that the chloroform fraction has high anti-proliferative effect on HepG2 cells only with 100% loss of cell viability relative to solvent control. It exhibited weak anti-proliferative effect on Caco2 and MDA-MB-231 cells with 8.2% and 11.6% loss of cell viability relative to solvent control, respectively. The assay was further investigated against HepG2 cells only in a dose-response experiment for IC determination, 50 whereas, IC values of chloroform fraction against Caco2 50 and MDA-MB-231 cells were considered to be >100

chloroform fraction on HepG2 cells to reveal the IC .

50 The IC of chloroform fraction was 37.4 μg/ml and 50 doxorubicin was tested as positive control and gave IC 50 of 1.4 μg/ml (Table 3). This effect has stimulated us to ongoing work to isolate and identify the active ingredients of the fraction. This led to the isolation of valuable compounds in the light of their ability to inhibit HepG2 cell proliferation. Three anthraquinones were isolated and identified from the fraction, these anthraquinones composition may be responsible for the cytotoxic activity against HepG2 cancer cells. The cytotoxic effects (e.g., cell death) may be attributed to the arrest of the cell cycle and the induction of apoptosis in cancer cells or the fraction could effectively inhibit the uptake of glucose in tumor cells, caused changes in membrane-associated functions and led to cell death.

50

Several other studies have dealt with the cytotoxic activity evaluation of C. alata leaves extracts. Hexane fraction of C. alata leaves exhibited cytotoxic activity against parental A549 (lung cancer cells) and OV2008 (ovarian cancer cells) cell lines [56,57], also the hexane fraction and f6l (a mixture of polyunsaturated fatty acid esters) exerted a cytotoxic effect on MCF-7 (breast carcinoma cells), T24 (bladder carcinoma cells) and Col 2 (colorectal carcinoma cells) cell lines in a dose dependent manner, but were not effective against A549 (non-small cell lung adenocarcinoma) and SK-BR-3 (breast carcinoma cells) cell lines [14]. On the other hand, petroleum ether extract possessed anticancer activity against HCT-15 (colon carcinoma) and Hep2 (Human cervix carcinoma) cell lines [15]. From other species, petroleum ether extract of C. roxburghii Linn. leaves exhibited cytotoxic activity against HCT-116 (colon carcinoma cells) and MCF-7 (breast carcinoma cells) cell lines while against HepG-2 cell line showed no activity [44].

The petroleum ether extract of C. fistula seeds significantly induced antitumor activity against HeLa cell line [58]. The n-hexane extract of Senna gardneri and the ethyl ether extracts of Senna splendida showed cytotoxic effect against human colon and human glioblastoma cell lines [59]. Six bioactive compounds from Cassia italica, showed anticancer properties using Ehrlich ascites carcinoma cell line and Hepatoma cell (HepG2) cell line. The identified compounds showed variable antioxidant activities [60]. Three anthraquinones compounds (emodin, aloe-emodin and Chrysophanol) isolated from C. garrettiana heart wood showed appreciable antitumor activity against HT-29 (human colon adenocarcinoma), HeLa (human cervical adenocarcinoma), KB (oral cavity cancer) and MCF-7 cell lines [61].

Microscopic examination of chloroform fraction of C. alata leaves against HepG2 cell line

Morphological assessment of control/treated cells was performed using phase contrast microscopy equipped with digital camera after 48 h exposure time. Morphological signs of toxicity included cell rounding, shrinking and loss of contact with neighboring cells and the substratum of the wells (cell floating). These morphological features have led to the loss of monolayer integrity in comparison to the control (0.1% DMSO) as

magnification is 150X

Antioxidant activity (DPPH radical scavenging assay)

The pre-screen of antioxidant activity revealed that chloroform fraction showed weak antioxidant activity with 7.8% DPPH scavenging activity. These results revealed that may be the absence of antioxidant compounds. Previous studies have dealt with antioxidant activity of cassia alata leaves [22,62,63], in addition indole alkaloid of cassia alata leaves exhibited strong antioxidant potential [64].

Conclusion

Chloroform fraction of Cassia alata L leaves were evaluated for its potential antitumor properties in vitro. The fraction showed remarkable cytotoxicity against HepG2 cells, this observation was confirmed by morphological investigation. However, the fraction exhibited weak anti-proliferative effect on Caco2 and MDA-MB-231 cells and exhibited weak antioxidant activity. The anticancer activity against HepG2 cells is possibly due to anthraquinones content. Further bioassayguided fractionation of the cytotoxic fraction led to the isolation of three anthraquinones (rhein, emodin and aloeemodin) all known to have useful bioactivities including anticancer activities. On the other hand, the fatty acids and the unsaponifiable matter were fractionated and identified by GC/MS. These findings show the importance of screening Cassia alata as medicinal plant for various cancers cell lines and the responsible active compounds. The cytotoxicity profile might represent a new promising extract with potential for development as an anticancer drug against HepG2 cells with low or no toxicity to non-cancer cells. Further work is certainly needed to develop and evaluate the anthraquinones compounds as anticancer.

Conflict of Interests

The authors declare that there is no conflict of interest.

Acknowledgements

The authors would like to acknowledge the financial support from the project: The use of traditional medicinal plants in the treatment of viral skin herpes simplex disorders ID: 449 financed by Science and Technology Development Fund (STDF), Academy of Scientific Research and Technology, Ministry of Higher Education, Cairo, Egypt.

References

References

1. Parsons WT and Cuthbertson EG: Noxious weeds of Australia. Melbourne: In data Publication. 1992.
2. Prasenjit M, Tanaya G, Sumanta G, Basudeb B and Kumar MP: Isolation and characterization of a compound from the leaves of Cassia alata Linn. ECronicon Chemistry 2016; 2(2):138–144.
3. Liu A, Xu L, Zou Z and Yang S: Studies on chemical constituents from leaves of Cassia alata. Zhongguo Zhong Yao Za Zhi 2009; 34(7):861–
5. Duong NTT, Chinh HT, Din TS, Phong TLH, Phuong PN, Quynh PQ, Truc NTT and Quang TT: Contribution to the study on chemical constituents from the leaves of Cassia alata l., (Caesalpiniaceae). Science and Technology Development 2013; 16(T2):26–31.
6. Rahman MS, Hasan AM, Ali M y. and Ali M u.: A flavone from the leaves of Cassia alata. Bangladesh Journal of Scientific and Industrial
7. Rhazri K, Hamri S, Lebri M, Onguéné R, Ouchetto H, Hafid A, Izikki M and Khouili M: Isolation of 3,5,7,4’-Tetrahydroxyflavone (Kaempferol) from the methanol extract of the leaves of Cassia alata and in-vitro evaluation of its potential inhibitory effect on the human pulmonary arterial hypertension (PAH). Der Pharma Chemica 2015; 7(7):241–245.
8. Moriyama H, Iizuka T, Nagai M and Murata Y: HPLC quantification of kaempferol-3-O-gentiobioside in Cassia alata. Fitoterapia 2003; 74:425–
10. Rahman MS, Yeasmin MS, Begum MN and Rahman MS: Studies on the isolation of 2, 5, 7, 4′ - tetrahydroxy isoflavone from the leaves of Cassia alata. Asian Journal of Phytomedicine and Clinical Research 2015; 3(2):64–70.
11. Rahman MS, Yeasmin MS, Das K and Rahman MS: Studies on the isolation of 5, 7-dihydroxy-3´, 4´-dimethoxy flavone from the leaves of Cassia alata. American Chemical Science Journal 2015; 8(4):1–8.
12. Ramsay A and Mueller-Harvey I: Senna alata leaves are a good source of propelargonidins. Natural Product Research 2016; 30(13):1548–1551.
13. Igwe OU and Onwu FK: Leaf essential oil of Senna alata linn from South East Nigeria and its antimicrobial activity. International Journal of Research in Pharmacy and Chemistry 2015; 5(1):27–33.
14. Muthuselvam D: Screening for antibacterial and antifungal activity from leaf extract of Cassia alata L., Cassia fistula L. and Cassia tora L. Asian Journal of Pharmaceutical Science & Technology 2016; 6(1):17–18.
15. Tatsimo SJN, Tamokou J, Tsague VT, Lamshoft M, Sarkar P, Bag PK and Spiteller M: Antibacterial-guided isolation of constituents from Senna alata leaves with a particular reference against Multi-Drug-Resistant Vibrio cholerae and Shigella flexneri. International Journal of Biological and Chemical Sciences 2017; 11(1):46–53.
16. Olarte EI, Herrera AA, Villaseñor IM and Jacinto SD: In vitro antitumor properties of an isolate from leaves of Cassia alata L. Asian Pacific journal of cancer prevention 2013; 14(5):3191–3196.
17. Raji P, Sreenidhi J, Sugithra M, Renugadevi K and Samrot A V.: Phytochemical screening and bioactivity study of Cassia alata leaves. Biosciences Biotechnology Research Asia 2015; 12(Spl. Edn. 2):291–
19. Lewis A and Levy A: Anti-inflammatory activities of Cassia alata leaf extract in complete freund’s adjuvant arthritis in rats. West Indian Medical Journal 2011; 60(6):615–621.
20. Laishram P, Behari MP, Heisanam P and Choudhury MD: Effect of aqueous extract of Cassia alata Linn. on oral glucose tolerance test in normal and STZ induced diabetic mice. European Journal of Medicinal Plants 2016; 15(1):1–7.
21. Neharkar VS and Gaikwad KG: Hepatoprotective activity of Cassia alata (Linn.) leaves against paracetamol-induced hepatic injury in rats. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2011; 2(1):783–788.
22. Patrick-iwuanyanwu KC, Wegwu MO and Makhmoor T: Hepatoprotective effect of crude methanolic extract and fractions of Ring worm plant Senna alata ( L . Roxb ) leaves from Nigeria against carbon tetrachloride –induced hepatic damage in rats. European Journal of Experimental Biology 2011; 1(1):128–138.
23. Esimone CO, Nworu CS, Ekong US and Okereke BC: Evaluation of the antiseptic properties of Cassia alata-based herbal soap. The Internet Journal of Alternative Medicine [Internet] 2007; 6(1). Available from: https://ispub.com/IJAM/6/1/5856
24. Shaheen M, Mostafa S and El-esnawy N: In Vitro and in Vivo Antirotaviral Activity of C. alata Extracts. Journal of Research in Applied Sciences 2015; 2(3):63–71.
25. Chatterjee S, Chatterjee S, Dey KK and Dutta S: Study of antioxidant activity and immune stimulating potency of the ethnomedicinal plant, Cassia alata (L.) Roxb. Medicinal & Aromatic Plants 2013; 2(4):131.
26. Kumar RS, Rajkapoor B and Perumal P: In vitro and in vivo anticancer activity of Indigofera cassioides Rottl. Ex. DC. Asian Pacific Journal of Tropical Medicine 2011; 4(5):379–385.
27. Farag RS, Hallabo SAS, Hewedi FM and Basyony AE: Chemical evaluation of rapeseed. Fette, Seifen, Anstrichmittel 1986; 88(10):391–
29. Kinsella JE: Metabolic patterns of the fatty acids of Periplanteta americana (L.) during its embryonic development. Canadian Journal of Biochemistry 1966; 44(2):247–258.
30. Wagner H and Bladt S: Plant Drug Analysis: A Thin Layer Chromatography Atlas. Berlin; 1996.
31. Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 1983; 65:55–63.
32. Braca A, Tommasi N De, Bari L Di, Pizza C, Politi M and Morelli I: Antioxidant principles from Bauhinia tarapotensis. Journal of Natural Products 2001; 64(7):892–895.
33. Nara K, Miyoshi T, Honma T and Koga H: Antioxidative activity of bound-form phenolics in potato peel. Bioscience, biotechnology, and biochemistry 2006; 70(6):1489–1491.
34. Hultqvist M, Olofsson P, Gelderman KA, Holmberg J and Holmdahl R: A new arthritis therapy with oxidative burst inducers. PLoS Medicine 2006; 3(9):1625–1636.
35. Ogunlesi M, Okiei W, Ofor E and Osibote AE: Analysis of the essential oil from the dried leaves of Euphorbia hirta Linn (Euphorbiaceae), a potential medication for asthma. African Journal of Biotechnology 2009; 8(24):7042–7050.
36. Guo S, Feng B, Zhu R, Ma J and Wang W: Preparative isolation of three anthraquinones from Rumex japonicus by high-speed counter-current chromatography. Molecules 2011; 16(2):1201–1210.
37. Liu R, Li A and Sun A: Preparative isolation and purification of hydroxyanthraquinones and cinnamic acid from the Chinese medicinal herb Rheum officinale Baill. by high-speed counter-current chromatography. Journal of Chromatography A 2004; 1052(1–2):217–
39. Zhang X, Thuong PT, Jin W, Su ND, Sok DE, Bae K and Kang SS: Antioxidant activity of anthraquinones and flavonoids from flower of Reynoutria sachalinensis. Archives of Pharmacal Research 2005; 28(1):22–27.
40. Hsu SC and Chung JG: Anticancer potential of emodin. BioMedicine 2012;2(3):108–116.
41. Narayanan S and Jadhav AP: Simultaneous estimation of aloe emodin and emodin from Rheum emodi, Cassia alata and Aloes by HPTLC. Indian Journal of Pharmaceutical Sciences 2015; 77(6):783–787.
42. Kelly TR, Ma Z and Xu W: Revision of the structure of alatinone to emodin. Phytochemistry 1994; 36(1):253–254.
43. Chukwujekwu JC, Coombes PH, Mulholland DA and Staden J van: Emodin, an antibacterial anthraquinone from the roots of Cassia occidentalis. South African Journal of Botany 2006;72(2):295–297.
44. Berhanu E and Dagne E: Aloe-emodin acetate, an anthraquinone derivative from leaves of Kniphofia foliosa. Planta medica 1984; 50(6):523–524.
45. Wan J, Liu G, Xu X, Xie C, Ding W and Xu J: Simplex optimization for aloe-emodin separation and purification using aqueous double-phase extraction system. Journal of Liquid Chromatography & Related Technologies 2014; 37(5):653–663.
46. Dong JW, Cai L, Fang YS, Duan WH, Li ZJ and Ding ZT: Simultaneous, simple and rapid determination of five bioactive free anthraquinones in Radix et Rhizoma Rhei by quantitative 1H NMR. Journal of the Brazilian Chemical Society 2016; 27(11):2120–2126.
47. Kuo PL, Lin TC and Lin CC: The antiproliferative activity of aloeemodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines. Life sciences 2002; 71(16):1879–1892.
48. Chen SH, Lin KY, Chang CC, Fang CL and Lin CP: Aloe-emodininduced apoptosis in human gastric carcinoma cells. Food and chemical toxicology 2007; 45(11):2296–2303.
49. Mohammed MMD, El-Souda SS, El-Hallouty SM and Kobayashi N: Antiviral and cytotoxic activities of anthraquinones isolated from Cassia roxburghii Linn. leaves. Herba Polonica 2013; 59(4):33–44.
50. Pecere T, Gazzola MV, Mucignat C, Parolin C, Vecchia FD, Cavaggioni A, Basso G, Diaspro A, Salvato B, Carli M and Palù G: Aloe-emodin is a new type of anticancer agent with selective activity against neuroectodermal tumors. Cancer Research 2000; 60(11):2800–2804.
51. Pecere T, Sarinella F, Salata C, Gatto B, Bet A, Vecchia FD, Diaspro A, Carli M, Palumbo M and Palù G: Involvement of p53 in specific antineuroectodermal tumor activity of aloe-emodin. International Journal of Cancer 2003; 106(6):836–847.
52. Lee HZ, Hsu SL, Liu MC and Wu CH: Effects and mechanisms of aloeemodin on cell death in human lung squamous cell carcinoma. European Journal of Pharmacology 2001; 431(3):287–295.
53. Hazni H, Ahmad N, Hitotsuyanagi Y, Takeya K and Choo C-Y: Phytochemical constituents from Cassia alata with inhibition against methicillin-resistant Staphylococcus aureus (MRSA). Planta Medica 2008; 74(15):1802–1805.
54. Panichayupakaranant P, Sakunpak A and Sakunphueak A: Quantitative HPLC determination and extraction of anthraquinones in Senna alata leaves. Journal of Chromatographic Science 2009; 47(3):197–200.
55. Wei Y, Zhang T and Ito Y: Preparative separation of rhein from Chinese traditional herb by repeated high-speed counter-current chromatography. Journal of Chromatography A 2003; 1017(1–2):125–130.
56. Duraipandiyan V, Baskar AA, Ignacimuthu S, Muthukumar C and AlHarbi NA: Anticancer activity of rhein isolated from Cassia fistula L. flower. Asian Pacific Journal of Tropical Disease 2012; 2(SUPPL.1):S517–S523.
57. Huang Q, Lu G, Shen HM, Chung MCM and Ong CN: Anti-cancer properties of anthraquinones from rhubarb. Medicinal Research Reviews 2007; 27(5):609–630.
58. Aviello G, Rowland I, Gill CI, Acquaviva AM, Capasso F, McCann M, Capasso R, Izzo AA and Borrelli F: Anti-proliferative effect of rhein, an anthraquinone isolated from Cassia species, on Caco-2 human adenocarcinoma cells. Journal of Cellular and Molecular Medicine 2010; 14(7):2006–2014.
59. Saito ST, Trentin DDS, Macedo AJ, Pungartnik C, Gosmann G, Silveira JDD, Guecheva TN, Henriques JAP and Brendel M: Bioguided fractionation shows Cassia alata extract to inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa growth and biofilm formation. Evidence-Based Complementary and Alternative Medicine 2012; 2012(Article ID 867103):13 pages.
60. Singh B, Nadkarni JR, Vishwakarma RA, Bharate SB, Nivsarkar M and Anandjiwala S: The hydroalcoholic extract of Cassia alata (Linn.) leaves and its major compound rhein exhibits antiallergic activity via mast cell stabilization and lipoxygenase inhibition. Journal of Ethnopharmacology 2012; 141(1):469–473.
61. Levy A and Lewis A: Cassia alata leaf extract induces cytotoxicity in A549 lung cancer cells via a mechanism that is caspase 8 dependent. West Indian Medical Journal 2011; 60(6):608–614.
62. Levy AS and Carley S: Cytotoxic activity of hexane extracts of Psidium guajava L (Myrtaceae) and Cassia alata L (Caesalpineaceae) in Kasumi-1 and OV2008 Cancer Cell Lines. Tropical Journal of Pharmaceutical research 2012; 11(2):201–207.
63. Awan SJ, Tariq K, Nasir GA, Batool W, Sheraz MA, Ahmed S, Maqbool T, Ali U and Ansari F: Cassia fistula seed extract enhances apoptosis in HeLa cell line via activating Annexin V and p53. Science Letters 2017; 5(1):93–104.
64. Silva JGA, Silva AA, Coutinho ID, Pessoa CO, Cavalheiro AJ and Silva MG V.: Chemical profile and cytotoxic activity of leaf extracts from Senna spp. from northeast of Brazil. Journal of the Brazilian Chemical Society 2016; 27(10):1872–1880.
65. Aboul-Enein AM, Abu EL-Ela F, Shalaby E and EL-Shemy H: Potent anticancer and antioxidant activities of active ingredients separated from Solanum nigrum and Cassia italica extracts. Journal of Arid Land Studies 2014; 24(1):145–152.
66. Yuenyongsawad S, Bunluepuech K, Wattanapiromsakul C and Tewtrakul S: Anti-cancer activity of compounds from Cassia garrettiana heartwood. Songklanakarin Journal of Science and Technology 2014; 36(2):189–194.
67. Sagnia B, Fedeli D, Casetti R, Montesano C, Falcioni G and Colizzi V: Antioxidant and anti-inflammatory activities of extracts from Cassia alata, Eleusine indica, Eremomastax speciosa, Carica papaya and Polyscias fulva medicinal plants collected in Cameroon. PLoS ONE 2014; 9(8):e103999.
68. Sarkar B, Khodre S, Patel P and Mandaniya M: HPLC Analysis and Antioxidant Potential of Plant Extract of Cassia Alata. Asian Journal of Pharmaceutical Science & Technology 2014; 4(1):4–7.
69. Olarte EI, Herrera AA, Villaseñor IM and Jacinto SD: Antioxidant Activity of a New Indole Alkaloid from Cassia alata L. The Philippine Agricultural Scientist 2010; 93(3):250–254.