Z. Naturforsch. 2015; 70(1-2)c: 31–37

Farouk R. Melek, Fawzia A. Aly, Iman A.A. Kassem*, Mona A.M. Abo-Zeid, Ayman A. Farghaly and Zeinab M. Hassan Three further triterpenoid saponins from caspica and protective effect of the total saponin fraction on cyclophosphamide-induced genotoxicity in mice

Abstract: Three triterpenoidal saponins were isolated 1 Introduction from the saponin fraction derived from a Gleditsia caspica Desf. methanolic extract. The isolated saponins were The genus Gleditsia (family ) comprises 14 identified as gleditsiosides B, C, and Q based on spectral species of [1]. Gleditsia caspica (Caspian data. The saponin-containing fraction was evaluated in locust), a that grows up to 12 m, is cultivated in public vivo for genotoxic and antigenotoxic activities. The frac- gardens in Egypt mainly for ornamental purposes due tion caused no DNA damage in Swiss albino male mice to its graceful habit, elegant form, and delicate fern-like treated with a dose of 45 mg/kg body weight for 24 h, foliage. Gleditsia species have been widely used in folk although it significantly inhibited the number of chromo- medicine. The thorns of G. sinensis have been used for the somal aberrations induced by cyclophosphamide (CP) in treatment of carbuncles, scabies, and suppurate skin dis- bone marrow and germ cells when applied before or after eases, whereas the mature pods and anomalous fruits are CP administration. The inhibitory indices in chromosomal mainly used for treating apoplexy, headache, productive aberrations were 59% and 41% for bone marrow and 48% cough, and asthma. The dried fruits of G. japonica Miq. and 43% for germ cells, respectively. In addition, the have long been known in oriental medicine as diuretic saponin fraction was found to reduce the viability of the and expectorant [2]. Saponins, the main constituents of human tumor cell line MCF-7 in a dose-dependent manner Gleditsia fruits, were previously reported from fruits of with an extrapolated IC value in the range of 220 μg/mL. 50 different Gleditsia species [3–10]. From G. caspica, we recently reported the isolation and characterisation of 11 Keywords: antigenotoxicity; Gleditsia caspica; triterpe- new triterpenoidal and acylated triterpenoidal saponins noidal saponins. named caspicaosides A–K [11, 12], as well as the known Gleditsia saponins C′ and E′ and gleditsioside I [13]. DOI 10.1515/znc-2014-4132 As a part of our continuous interest in bioactive sapo- Received July 24, 2014; revised September 27, 2014; accepted nins from cultivated in Egypt [12, 14–19], we report ­February 10, 2015 here the isolation and identification of three further sapo- nins from the saponin fraction of G. caspica fruits (SFGC). Also, the genotoxic and antigenotoxic activities of SFGC are presented.

*Corresponding author: Iman A.A. Kassem, National Research Centre, Chemistry of Natural Compounds Department, Dokki 12622, 2 Materials and methods Giza, Egypt, E-mail: [email protected] Farouk R. Melek and Zeinab M. Hassan: National Research Centre, Chemistry of Natural Compounds Department, Dokki, Giza, Egypt 2.1 General Fawzia A. Aly and Ayman A. Farghaly: National Research Centre, Genetics and Cytology Department, Dokki, Giza, Egypt 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on an Jeol Mona A.M. Abo-Zeid: National Research Centre, Genetics and α-400 FT-NMR spectrometer (Tokyo, Japan), and chemical shifts are Cytology Department, Dokki, Giza, Egypt; and Cancer Biology given as δ values with tetramethylsilane (TMS) as internal standard at

Laboratory, Center of Excellence for Advanced Sciences, National 35 °C in pyridine-d5. High-performance liquid chromatography (HPLC) Research Centre, Dokki, Giza, Egypt was performed on a Jasco system 800 instrument (Tokyo, Japan). 32 Melek et al.: Saponins of Gleditsia caspica and their antigenotoxic activity

2.2 Chemicals (1H, dd, J = 10.5, 2.0 Hz, H-8a), 5.53 (1H, dd, J = 17.0, 2.0 Hz, H-8b), 6.11 (1H, dd, J = 17.0, 10.5 Hz, H-7), 7.20 (1H, t, J = 8.0 Hz, H-3). – 13C NMR (C H N, 100 MHz): [6]. Cyclophosphamide (CP) and all other material used in cell culture 5 5 were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used in extraction and isolation of saponins were pur- 2.6 Gleditsioside B (2) chased from ADWIC (Cairo, Egypt).

1 Amorphous powder (20 mg). – H NMR (C5H5N, 400 MHz): aglycone: 2.3 material δ 0.87, 0.91, 0.97, 0.99, 1.08, 1.33, 1.36 (each 3H, s), 5.47 (1H, br t, J = 3.0 Hz, H-12); sugar units: δ 1.76 (3H, d, J = 6.1 Hz, Rha Me-6), 4.97 (1H, d, J = 7.0 Hz, Xyl H-1), 5.06 (1H, d, J = 7.0 Hz, Xyl′ H-1), 5.14 (1H, d, J = 5.2 Hz, Fruits of G. caspica were collected from El-Orman public garden, Ara H-1), 5.17 (1H, d, J = 7.8, Xyl″ H-1), 6.13 (1H, d, J = 9.0 Hz, Glc′ H-1), Giza, Egypt, in November 2012. A voucher specimen was deposited in 6.35 (1H, d, J = 1.3 Hz, Rha H-1); monoterpene unit (MT): δ 1.44 (3H, s, the herbarium of the National Research Centre (CAIRC), Giza, Egypt. Me-10), 5.14 (1H, dd, J = 10.8, 1.8 Hz, H-8a), 5.53 (1H, dd, J = 17.3, 1.8 Hz, H-8b), 6.10 (1H, dd, J = 17.3, 10.8 Hz, H-7), 7.23 (1H, t, J = 7.9 Hz, H-3). – 13C NMR (C H N, 100 MHz): [5]. 2.4 Preparation of the saponin fraction of G. caspica 5 5 (SFGC) and isolation of saponins 2.7 Gleditsioside C (3) The air-dried fruits of G. caspica (2.0 kg) were defatted with n-hexane, then extracted twice with CHCl , followed by MeOH until exhaustion. 1 3 Amorphous powder (10 mg). – H NMR (C5H5N, 400 MHz): aglycone δ The combined MeOH extract was evaporated under reduced pres- 0.92, 0.94, 0.99, 1.08, 1.11, 1.31, 1.84 (each 3H, s), 5.19 (1H, br s, H-16), sure to dryness. The residue (66 g) was dissolved in the least pos- 5.64 (1H, br t, J = 3.0 Hz, H-12); sugar units: δ 1.62 (3H, d, J = 6.5 Hz, Rha sible amount of MeOH, and the solution was diluted with the 10-fold Me-6), 4.92 (1H, d, J = 7.5 Hz, Glc H-1), 4.99 (1H, d, J = 6.9 Hz, Xyl H-1), amount of acetone to precipitate 18 g of a crude saponin mixture. 5.10 (1H, d, J = 7.5 Hz, Xyl′ H-1), 5.11 (1H, d, J = 7.0 Hz, Xyl″ H-1), 5.12 (1H,

The mixture was dissolved in H2O (0.2%), and the aqueous solution d, J = 5.0 Hz, Ara H-1), 5.15 (1H, d, J = 10.5 Hz, Gal H-1), 5.93 (1H, d, J = 9.0 passed through a chromatographic column packed with 500 g Diaion Hz, Glc′ H-1), 6.43 (1H, d, J = 1.3 Hz, Rha H-1); monoterpene unit (MT): HP-20 polymer gel (Mitsubishi, Tokyo, Japan). After washing the col- δ 1.45 (3H, s, Me-10), 5.15 (1H, dd, J = 10.8, 1.8 Hz, H-8a), 5.53 (1H, dd, umn with distilled water for several times, elution was carried out J = 17.3, 1.8 Hz, H-8b), 6.10 (1H, dd, J = 17.3, 10.8 Hz, H-7), 7.22 (1H, t, with 25%, 50%, 60%, 75% aqueous MeOH and finally with 100% 13 J = 7.8 Hz, H-3). – C NMR (C5H5N, 100 MHz): [5]. MeOH. The collected fractions were examined by silica gel thin layer chromatography (TLC) (Merck, Darmstadt, Germany) using the sol- 2.8 Animals vent systems CHCl3/MeOH/H2O (60:30:5, v/v/v) and n-BuOH/EtOH/

NH4OH (7:2:5) and visualized by spraying with 20% sulfuric acid in MeOH followed by heating at 110 °C. Based on TLC analysis, simi- Laboratory-bred strain Swiss albino male mice, 10–12 weeks old lar fractions were combined. Fractions eluted with 75% and 100% with an average weight of 25±2.5 g were obtained from the National MeOH were found similar and contained saponin constituents. The Research Centre, Giza, Egypt. Animals were housed in groups (five two fractions were combined, and part of the combined fraction animals/group) and maintained under standard conditions of tem- (SFGC) (3 g) was kept in a refrigerator until used for the biological perature, humidity, and light. The animals were given standard food study. The remainder (2.5 g) was applied onto a chromatographic and water ad libitum. column packed with 120 g PSQ 100B silica gel (Fuji Silysia, Nagoya,

Japan) and eluted with CHCl3/MeOH/H2O with increasing polarity (70:27:3–58:35:7). A total of 50 fractions, 50 mL each, were collected. 2.9 In vitro study Similar fractions were combined after TLC analysis to yield 20 sub- fractions, A–T. Subfractions A (605 mg) and B (410 mg) were sub- 2.9.1 Cell culture: The breast cancer cell line (MCF-7) (ATCC, Rock- jected to repeated HPLC (column, TSK gel ODS-80TS, 5 mm × 60 cm; ville, MD, USA) was routinely cultured in RPMI-1640 media sup- solvent, 30%–45% CH3CN in H2O, linear gradient; flow rate, 45 mL/ plemented with 10% fetal bovine serum (FBS), 2 mM glutamine, min; detection, UV at 205 nm). Part of fraction A (150 mg) yielded containing 100 U/mL penicillin G sodium, 100 U/mL streptomycin gleditsioside Q (1; 22 mg). Part of fraction B (180 mg) afforded gledit- sulfate, and 250 ng/mL amphotericin B. Cells were maintained in sioside B ( ; 20 mg) and gleditsioside C ( ; 10 mg). 2 3 humidified air containing 5% CO2 at 37 °C. SFGC was dissolved in deionized distilled water before being added to cultured cells.

2.5 Gleditsioside Q (1) 2.9.2 MTT Assay of cell viability: The proliferation of the MCF-7 cells was estimated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 1 Amorphous powder (22 mg). – H NMR (C5H5N, 400 MHz): aglycone: 2H-tetrazolium bromide (MTT) assay [20]. Cells were cultured in δ = 0.92, 0.95, 1.00, 1.09, 1.10, 1.32, 1.85 (each 3H, s), 5.46 (1H, br t, J = 3.0 6-wells plates for 24 h in medium (5·105 cells/well), then SFGC was Hz, H-12), 5.21 (1H, br s, H-16); sugar units: δ = 1.71 (3H, d, J = 5.8 Hz, added at concentrations from 12.5 to 100 μg/mL in triplicate. The Rha Me-6), 4.88 (1H, d, J = 7.5 Hz, Glc H-1), 4.98 (1H, d, J = 6.8 Hz, Xyl plates were incubated for 24 h at 37 °C in humidified air containing

H-1), 5.13 (1H, d, J = 7.5 Hz, Xyl′ H-1), 5.13 (1H, d, J = 5.0 Hz, Ara H-1), 5.19 5% CO2, before being submitted to the MTT assay. The absorbance (1H, d, J = 7.9 Hz, Xyl″ H-1), 6.11 (1H, d, J = 7.9 Hz, Glc′ H-1), 6.34 (1H, d, was measured with an ELISA reader (BioRad, Munich, Germany) at J = 1.5 Hz, Rha H-1); monoterpene unit (MT): δ = 1.44 (3H, s, Me-10), 5.15 570 nm. The relative cell viability was determined by the amount of Melek et al.: Saponins of Gleditsia caspica and their antigenotoxic activity 33

MTT converted to the insoluble formazan precipitate. The data were 3.2 In vitro study expressed as the mean percentage of viable cells as compared to the respective control untreated cultures. 3.2.1 Cell viability

2.10 In vivo study Cells of the breast cancer cell line (MCF-7) were incubated with increasing concentrations of SFGC for 24 h. The MTT 2.10.1 Animals and treatment: Animals were divided into nine assay revealed that cell viability decreased in a dose- groups of five animals each. Group I received distilled water and was dependent manner but statistically significant only with used as negative control. Group II, the positive control, was admin- doses of 50 and 100 μg/mL. By linear extrapolation, the istered CP [20 mg/kg body weight (b.w.)] intraperitonealy (i.p.) in μ two instalments within a 24-h interval. Group III was treated orally IC50 value would be expected in the range of 220 g/mL. with 45 mg/kg b.w. SFGC, for 24 h. Groups IV through VI were treated orally with 4.5, 9.0, and 45 mg/kg b.w. SFGC 24 h before CP adminis- tration, and groups VII through IX were treated orally with 4.5, 9.0, 3.3 In vivo study and 45 mg/kg b.w. SFGC 24 h after CP administration. Animals were sacrificed 24 h after the last treatment. For preparation of somatic and germ cells, animals from the different groups were i.p. injected 3.3.1 Chromosomal aberrations in somatic cells with colchicine (10 mg/kg b.w.) 2–3 h before sacrifice. Table 1 shows the number and percentage of the chromo- 2.10.2 Chromosomal aberrations in somatic cells Chromosome somal aberrations in control animals and animals treated preparations from bone marrow (somatic cells) were carried out with SFGC. The percentage of aberrant cells in animals according to the method of Yosida and Amano [21]. One hundred treated with SFGC alone was not significantly differ- well-spread metaphases were analysed per mouse. Metaphases with ent from that in the control group. But SFGC was found gaps, chromosome or chromatid breakage, fragmentation, dele- to reduce the number of chromosomal aberrations when tions, Robertsonian translocation, as well as numerical aberrations (polyploidy) under 100-fold magnification with a light microscope administered 24 h either before or after administration of (­Olympus, Saitama, Japan), were recorded. CP. Upon treatment with a dose of 45 mg/kg b.w. of SFGC, the reduction of chromosomal abnormalities, excluding 2.10.3 Chromosomal abnormalities in germ cells Chromosome gaps, was 59% when added before and 41% when added preparations from spermatocytes (germ cells) were made according after CP administration, respectively. The reduction was to the technique of Evans et al. [22]. One hundred well-spread diaki- nase-metaphase I cells were analysed per animal for chromosomal highly significant (p < 0.01) in comparison with CP alone. aberrations. Metaphases with univalents, fragments, and chromo- some translocations were recorded. Evaluation of the activity of SFGC to reduce chromosomal aber- 3.3.2 Chromosomal abnormalities in germ cells rations induced by CP was carried out according to the formula of Madrigal-Bujaidar et al. [23] as follows: There were no significant differences between the animals inhibitory index(II)= treated with SFGC alone and the control animals (Table 2). [1 −− (SFGC and CP control) /(CP −⋅ Control] 100. The mean percentage of diakinesis-metaphase I cells were (21.60±0.70) % and (18.20±0.44) % (p < 0.01) with 20 mg/kg 2.11 Statistical analysis b.w. of CP administered after 24 h and 48 h, respectively. This percentage was decreased after treatment with SFGC. The significance of the results from the data of the negative control Upon treatment with a dose of 45 mg/kg b.w. of SFGC, the and between SFGC with CP compared to CP alone was calculated using the t-test for chromosomal aberrations. Results in case of SFGC maximum reductions were 48% and 43% when added 24 h before CP were compared with CP after 24 h (first instalment), before and after CP administration, respectively. Table 2 while results in case of SFGC 24 h after CP was compared with CP after illustrates the protective effect of SFGC on the different 48 h (second instalment). types of aberrations, such as XY-univalents and/or auto­ somal univalents, fragments, and chain (IV). 3 Results 4 Discussion 3.1 Phytochemistry In the present work, the crude saponin fraction from the The NMR data of the isolated compounds are recorded in Gleditsia caspica fruit extract was subjected to Diaion the materials and methods section. HP-20 polymer and silica gel column chromatography, 34 Melek et al.: Saponins of Gleditsia caspica and their antigenotoxic activity

Table 1: The effect of SFGC on cyclophosphamide-induced chromosomal aberrations in mouse bone marrow cells in vivo.

Treatments, Total abnormal metaphases Number of different types of metaphases Inhibitory index mg/kg b.w. (excluding gaps) Number Mean±SE, % G. Frag. and/or Br. Del. C.F. M.A. Polyp.

Including Excluding gaps gaps

I. Control 24 4.80±0.38 2.80±0.40 10 8 6 0 0 0 – II. CP (20) 117a 23.40±0.42c 18.80±0.60c 23 55 8 5 20 6 – 104b 20.80±0.54c 16.60±0.50c 21 55 6 4 15 3 – III. SFGC (45) 23 4.60±0.50 2.40±0.62 11 8 4 0 0 0 – IV. SFGC (4.5) Before CP 82 16.40±0.30c,d 13.00±0.62c,d 17 51 5 2 7 0 30 V. SFGC (9.0) Before CP 68 13.60±0.50c,d 11.40±0.50c,d 11 52 3 0 2 0 46 VI. SFGC (45) Before CP 57 11.40±0.58c,d 9.40±0.70c,d 10 45 2 0 0 0 59 VII. SFGC (4.5) After CP 92 18.40±0.42c 14.60±0.44c 19 56 5 1 10 1 14 VIII. SFGC (9.0) After CP 80 16.00±0.56c,d 12.00±0.68c,d 20 51 4 0 5 0 33 IX. SFGC (45) After CP 63 12.60±0.40c,d 11.00±0.42c,d 8 47 6 0 1 1 41

Total number of examined metaphases, 500 (five animals/group). G., gap; Frag., fragments; Br., breaks; Del., deletions; C.F., centric fusions; M.A., multiple aberrations; Polyp., polyploidy. aSamples taken after 24 h. bSamples taken after 48 h. cSignificant compared to vehicle control (p < 0.01); dSignificant compared to CP treatment (p < 0.01) (t-test). respectively, followed by repeated HPLC to afford three Antigenotoxicity of SFGC was established in bone marrow triterpenoidal saponins (Fig.1). The isolated saponins and spermatocyte cells in CP treated mice. were characterised based on their spectral data (materials SFGC at a dose of 45 mg/kg b.w. had no apparent and methods section) as gleditsiosides B, C [5] and Q [6]. genotoxic effect, as the proportion of aberrant cells was

Table 2: The effect of SFGC on cyclophosphamide-induced chromosomal abnormalities in mouse spermatocyte cells in vivo.

Treatment, Total abnormal metaphases Number of different types of metaphases Inhibitory mg/kg b.w. index Number Mean±SE, % XY-uni. Auto. uni. XY-uni.+Auto. uni. Frag. Chain (IV)

I. Control 15 3.00±0.48 9 6 0 0 0 – II. CP (20) 108a 21.60±0.70c 73 21 4 2 8 – 91b 18.20±0.44c 58 24 3 1 5 – III. SFGC (45) 22 4.40±0.40 13 9 0 0 0 – IV. SFGC (4.5) Before CP 75 15.00±0.40c,d 54 21 0 0 0 35 V. SFGC (9.0) Before CP 67 13.40±0.48c,d 55 10 1 1 0 44 VI. SFGC (45) Before CP 54 10.80±0.64c,d 45 9 0 0 0 48 VII. SFGC (4.5) After CP 83 16. 60±0.45c 60 18 1 0 4 11 VIII. SFGC (9.0) After CP 74 14.80±0.57c,d 52 18 1 1 2 22 IX. SFGC (45) After CP 58 11.60±0.40c,d 48 10 0 0 0 43

Total number of examined metaphases, 500 (five animals/group). XY-uni., XY- univalent; Auto. uni., autosomal univalent; XY-uni.+Auto. uni., XY-univalent+autosomal univalent; Frag., fragment. aSamples taken after 24 h. bSamples taken after 48 h. cSignificant compared to vehicle control (p < 0.01). dsignificant compared to CP treatment (p < 0.01) (t-test). Melek et al.: Saponins of Gleditsia caspica and their antigenotoxic activity 35

20 19 21

12 18 13 17 22 11 25 26 O 1 H Ara 9 14 28 OH 16 2 10 8 5 O O 15 O Glc 3 5 1 1 4 27 R 5 O 7 1 2 3 HO O 6 3 HO R R R O 5 O HO 1 24 23 3 OH HO MT O 1 OH CH2OH H HO 1 3 OH 6 8 O Glc′ Xyl HO 2 5 O 2 H CH2OH H 5 3 HO 2 HO ′1 3 OH CH2OH Gal R 3 5 O Rha H C Xyl′ 3 O 1 5 O O Xyl′′ 3 Q3: 5 O HO HO 3 OR Please HO O 1 3 OH ­indicate HO OH 1 3 where Figure 1 Figure 1: Chemical structures of the isolated gleditsiosides B,C, and Q. should be mentioned Glc, β-D-glucopyranosyl; Ara, α-L-arabinopyranosyl; Xyl, β-D-xylopyranosyl; Rha, α-L-rhamnopyranosyl; Gal, β-D- galactopyranosyl; in text MT, monoterpene unit. not significantly different from that of the negative control concentrate from a smoker (SU). All the saponins were in both somatic and germ cells, whereas conversely, it found to be nontoxic and nonmutagenic up to doses of displayed significant antigenotoxic activity against CP- 400 μg. Four saponins from Calendula arvensis and three induced mutagenesis in bone marrow cells. saponins from Hedera helix counteracted the mutagenic Cyclophosphamide is a bi-functional alkylating agent. activity of BaP (1 μg) and SU (5 μL) as a function of their It is used to treat a wide range of cancers, in addition to concentration. A modified liquid incubation technique of its use as an immunosuppressive agent. However, it is a the Salmonella/microsomal assay (Ames test) was used known carcinogen in humans and transforms into sec- in this study [30]. The triterpenoid saponin ginsenoside ondary metabolites. Its cytotoxic effects result from these Rb1 from Panax ginseng also provided significant pro- reactive metabolites that alkylate DNA, RNA, and proteins tection against DNA damage and apoptosis induced by [24]. Cyclophosphamide can cause DNA single strand CP [31]. Soy saponins were proven to be antimutagens breaks, as well as DNA-DNA and DNA-protein cross-links in Salmonella typhimurium TA98 against mutagenic het- in post-implantation rat embryos and in testicular cells. erocyclic amines and arylamines [32]. The maesasaponin CP treatment can induce structural chromosomal aberra- mixture B, consisting of six homologous oleanane-type tions and sister-chromatid exchanges in embryos, Chinese triterpenoid saponins isolated from Maesa lanceolata, hamster cells, human chorionic villi, and germ cells at was reported to exhibit moderate antimutagenic activity various stages of spermatogenesis [25, 26]. DNA breaks [33]. The antimutagenic mechanism of saponins may be induced by CP are important markers of genotoxicity [27]. related to the promotion of DNA repair [34]. Our findings are in agreement with previous reports The antigenotoxic property of SFGC provides addi- demonstrating the antimutagenic activity of saponins. tional health supplemental value to the other claimed Amara-Mokrane et al. [28] suggested a desmutagenic therapeutic properties of Gleditsia plants. Saponins from activity of the triterpenoid saponin α-hederin isolated G. sinensis fruits have been suggested for the therapy of from Hedera helix against doxorubicin at all doses tested. rheumatoid arthritis [35]. The cytotoxic activity of G. sin- This was later confirmed by Villani et al. [29] who dis- ensis fruit extract (GSE) against breast cancer [36] and cussed their mechanism of action. Furthermore, 13 nasopharyngeal carcinoma [37] has also been reported. saponins from Hedera helix, Calendula officinalis, and Moreover, GSE was suggested as a potential chemother- Calendula arvensis were assayed for their mutagenic apeutic drug to treat patients with acute and chronic and antimutagenic activity using the known promuta- myelogenous leukemia [38], and it was also proposed to gen benzo-[a]-pyrene (BaP) and a mutagenic urine be of use as an angiogenic inhibitor in both solid tumor 36 Melek et al.: Saponins of Gleditsia caspica and their antigenotoxic activity

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