Chemical composition and therapeutic potential of three Cycas species in brain damage and pancreatitis provoked by γ-radiation exposure in rats

Metabolomic profiling of the crude extracts of the three Cycas plants leaves; Cycas armstrongii Miq., Cycas circinalis L., and Cycas revoluta Thunb. showed the presence of diverse secondary metabolites. A detailed phytochemical study of C. armstrongii fractions led to isolation of 15 known components of different classes, but this is the first time to report them for this species. These components were chemically identified as (naringenin, dihydroamentoflavone, 2,3dihydrohinokiflavone, amentoflavone, 2,3-dihydrobilobetin, isoginkgetin, prunin, naringin, vanillic acid, p-coumaric acid, β-sitosterol, stigmasterol, β-sitosterol glucoside, 3,7,9,11-tetramethyl heptadecanoic acid, and N-(3ʹ-one-5ʹ-methyl)-hexyl-alanine). Moreover, the radioprotective potential of the three Cycas plant species was also investigated, and the ionizing radiation was performed by the exposure of rats whole-body to 8 Gy. The extracts of the three Cycas species were administered at a dose of 200 mg/kg each using an intra-gastric tube. Results indicated that Cycas spp. extracts significantly ameliorated radiation-induced brain and pancreatic damage as well as showing protection against radiation-induced oxidative stress. The results were also proved by histopathological study. ARTICLE HISTORY Received 21 November 2019 Accepted 26 December 2019


Introduction
Cycas is known to be the only genus of family Cycadaceae and represented in Egypt, by nine species; C. armstrongii Miq., C. revoluta Thunb., C. circinalis L., C. litoralis K.D. Hill, C. thouarsii R.Br., C. media R.Br, C. tansachana K.D. Hill, C. rumphii Miq., and C. pectinata Griff. In a survey for benzoic and cinnamic acids and their derivatives in the Cycadaceae, the presence of caffeic, p-hydroxybenzoic, vanillic, p-coumaric, protocatechuic, and ferulic acids in C. revoluta and C. circinalis leaves was reported (Wallace, 1972).
Plants provide a great challenge in metabolomics due to the high chemical and physical diversity of their metabolites (Alonso & Stepanova, 2015). Therefore, no single analytical method can determine all plant metabolites simultaneously. Liquid chromatography-mass spectrometry (LC-HRESIMS) is considered as an advantageous analytical technique for metabolic profiling by detecting a broad range of chemical compounds at the same time without the tedious isolation procedures.
Radiation damage is mediated by free radicals which react with body tissues to generate lipid peroxidation, DNA lesions, and enzyme inactivation. Wholebody exposures to any form of radiation are known to alter its general physiology of the animal (Sharma, Parmar, Sharma, Verma, & Goyal, 2011). Total body irradiation (TBI) has been used in the clinical treatment of many malignancies to produce sufficient immunosuppression and prevent allograft rejections (Ravichandran et al., 2013).
The lack of chemical and biological reports on C. armstrongii Miq. encouraged us to perform a phytochemical study of this species to explore its active constituents and main therapeutic purposes. In this study, we performed comparative analytical and biological studies of three Cycas species growing in Egypt, since no chemical or biological reports on Cycas armstrongii Miq. had been reported so far.
In addition, their potential as therapeutic agents in radiation-induced brain and pancreatic damage was also explored.

Instruments and reagents
Nuclear Magnetic Resonance analysis was performed on Bruker Avance III 400 MHz for 1 HNMR and 100 MHz for 13 CNMR (Bruker AG, Switzerland) with BBFO Smart Probe. Thin-layer chromatography was performed on precoated Si gel 60 F 254 plates (Fluka-Sigma-Aldrich Chemicals-Germany), vacuum liquid chromatography (VLC) and column chromatography were done using Si gel H and Si gel 60 (E. Merck, Darmstadt, Germany), respectively. Sephadex LH20 for CC (Amersham Pharmacia Biotech B, Uppsala, Sweden).

Metabolomic analysis
The metabolomic analysis of crude ethanolic extracts of three Cycas species under investigation was performed using the previous published analytical technique of LC-HRESIMS (Abdelhafez et al., 2018).
2.3. Ameliorative role of Cycas species extract on γ radiation toxicity in brain and pancreas of albino rats 2.3.1. Animals. In this study, 48 adult female albino rats were used, their weight range was from (180-200 g). The animals were obtained from the National Center for Radiation Research and Technology (NCRRT), Cairo, Egypt. Animals were kept on a standard diet for 1 week until the experiment had been started. Ionizing radiation was performed by whole-body exposure of rats to 8 Gy. The tested extracts of Cycas species were administered by intragastric tube in the dose of 200 mg/kg body weight for each. Animals were divided into eight groups; Normal (group 1), C. circinalis extract (group 2), C. armstrongii extract (group 3), C. revoluta extract (group 4), Radiation group (Group 5), Radiation plus C. circinalis extract (group 6), Radiation plus C. armstrongii extract (group 7) Radiation plus C. revoluta extract (group 8).
2.3.2. Radiation process. Irradiation processing was performed at (NCRRT), using a Canadian Gamma Cell-40 ( 137 Cs). Animals were subjected to γ-radiation; 2 Gy installments every week at a dose rate of 0.5 Gy/min up to 8 Gy (total dose).
2.3.3. Samples collection. Animals were fasted overnight prior to sacrificing. Six animals from each group were randomly sacrificed by cervical dislocation 7 days post irradiation. Blood samples were collected by heart puncture from the anaesthetized rats. Serum samples were prepared by centrifugation at 3000 rpm and brain samples were collected and prepared following normal laboratory procedures, for the measurement of the biochemical parameters as well as histological examination. The abdomen was dissected to remove the pancreas. Brains and pancreases were then divided into two equal portions, the first portion was homogenized in ice-cold phosphate-buffered saline (PBS) to form 10% homogenate for biochemical assays while the other portion was kept for the histopathological examination.

Hematological parameters
Platelet count and total leucocytic count were determined using (Sysmex XE 2100 and XT 2000i operator manual, USA) a previously reported method (Bain, 2001).

Biochemical assays
Serum hepatic enzymes: alanine transaminase (ALT) and aspartate transaminase (AST) activities, glucose, urea, creatinine, amylase, and lipase were estimated. Brain and pancreas homogenates were used to estimate malondialdehyde (MDA) levels as one of the main end products of lipid peroxidation, GPx methods using available commercial kits (Biodiagnostics, Cairo, Egypt), and TAC levels by using Randox total antioxidant status kit (UK). Serum insulin, amyloid A, IL-18 assayed using a commercially available ELISA kit (MyBiosource, USA) according to the manufacturer's protocol. Also, MCP-1 in brain assayed by ELISA kit.

MicroRNA216a
The TaqMan® MicroRNA Assays were performed using the using Applied Biosystems real-time PCR instrument protocols mentioned in Brattelid et al. (2011).

Real-time PCR analysis
Polytron homogenizer and TriPure isolation reagent (Roche Diagnostics, Basel, Switzerland) was used to homogenize the tissue samples at 10,000 rpm, then the total RNA was extracted and isolated according to the manufacturer's protocols (Dasgupta, Das, Izumi, Venkatesan, & Barat, 2004). Livak and Schmittgen method was applied to measure the level of mRNA (Livak & Schmittgen, 2001). GAPDH (R&D Systems Inc., USA) was used as a housekeeping gene. qPCR was performed using the specific primers (Table 1).

Histopathological examination
Liver tissue specimens were fixed and stained with hematoxylin and eosin (H&E) reagent, then examined with a light microscope (Bancroft & Stevens, 1996).

Plant material
Cycas species were purchased from Zoheria garden, Giza, Egypt in October 2014. Dr Abd-Elhaleem Mohamed (Department of Plant Taxonomy, Agricultural Research Center, Egypt) confirmed the identity of the plants. Voucher specimens of C. armstrongii Miq., C. revoluta Thunb., and C. circinalis L., species no. BuPD; 41-43, respectively, were deposited at the department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University.

Preparation of extracts
The aerial parts of three Cycas species under investigation: C. armestrongii (4.25 kg), C. revoluta (4.5 kg), and C. circinalis (4 kg), were air-dried, powdered and saved in tightly closed amber colored glass containers at room temperature. One kilogram of dried powder of each species was extracted by 70% ethanol at room temperature by maceration. The solvent was, in each case, removed by vacuum distillation to give residues of total alcoholic extracts used for analytical and metabolomic analysis. Aliquots of these extracts were also used for biological screening.
The hexane extract of the leaflets of Cycas armstrongii Miq. was saponified according to Ismail (2013). An aliquot of the unsaponifiable matter of the leaflets of Cycas armstrongii Miq. (2.5 g) was fractionated on Si gel VLC column (12.5 × 7 cm, 150 g). Hexane/EtOAc mixtures with increasing polarity were used as eluent in 5% stepwise increments till 100%. Fractions (30 ml, each) were pooled and similar fractions were combined to give two main fractions (A and B). Fraction A; (0-30% EtOAc in hexane, 610 mg). The residue was further purified by rechromatography on a Si gel column (50 × 2.2 cm, 30 g). Gradient elution was started with hexane followed by increasing amounts of ethyl acetate. Subfraction A 1 upon evaporation of the solvent yielded compound 54 (13mg) as a white powder while subfraction A 2 was subjected to further purification and recrystallization giving compound 55 (17 mg) as white needle crystals. Fraction B; 40-100% EtOAc in hexane, 420 mg), the residue obtained upon evaporation of the eluent was further purified by rechromatography on Si gel column (15 cm×1 cm, 10 g). Gradient elution was performed out using CHCl 3 with increasing amounts of MeOH (5% increments) yielding compound 51 (8 mg) as a white powder and mixture of two compounds 51 and 52 (25 mg).

Statistical analysis
Biochemical data were reported as means ± SE. The results were submitted to one-way ANOVA, and means were compared between groups by Duncan's multiple range tests and least-significant difference test. Statistically significant results were achieved when P-value<0.05 (Graph Pad Prism 5).

Metabolomic analysis
Chemical profiling of the three Cycas species using LC-HRESIMS resulted in the identification of 40 phenolic metabolites (1-40) which belonged to different chemical classes including flavonoids and phenolic acids. The identification of these metabolites was performed by employing macros and algorithms that coupled MZmine with online and in-house databases (METLIN and DNP databases for plant natural products) (Table 2, Figure 1).

Liver and kidney functions
To show the toxicity of the extracts, liver and kidney functions were tested. Compared to the control group, γ-radiation induced a significant increase in serum activities of ALT and AST by 363% and 341%, respectively (p < 0.001). Blood urea and serum creatinine concentrations turned out to be markedly increased by 63% and 441%, respectively, in γ-irradiated rats as compared to control group. Three extracts, Ext 1 (C. circinalis), Ext 2 (C. armstrongii) and Ext 3 (C. revoluta) treatment induced a significant reduction in serum activities of ALT (56%, 61.8% and 61% respectively) and AST (56.6%, 56.3%, and 57.7%, respectively) as compared to γradiation group. Furthermore, a significant decrease in the concentration of blood urea returning to normal values and serum concentrations of creatinine (46%, 60%, and 51%, respectively) as compared to radiation group (Table 3). Data were expressed as mean±SEM. Ext 1 (C. circinalis), Ext 2 (C. armstrongii) and Ext 3 (C. revoluta).

Glucose and insulin levels
Compared to the control group, γ-radiation induced a significant increase in serum glucose by 67.6% (p< 0.001) while γ-radiation caused a significant reduction in serum insulin level by 47.2%. Cycas treatment (3 extracts) produced a significant decrease in serum glucose (22.5%, 29%, and 29%, respectively) and elevation of insulin (53%, 49%, and 80% respectively) as compared to γradiation group (Figure 3).

White blood count and platelet count
γ-Radiation induced a significant decrease in WBCs by 77.5% (p< 0.001). Platelets count was exhibited by the same trend where γ-radiation caused a significant reduction in platelet count by 77%. Cycas treatment (3 extracts) induced a significant increase in WBCs by (101%, 142%, and 157%, respectively) and non-significant elevation of platelet count (31%, 26.8%, and 31%, respectively) as compared to γ-radiation group (Figure 4).

Oxidative stress parameters
Compared to the control group, γ-radiation showed a significant elevation in MDA in tissues of brain and pancreas by 58.2% and 67.26%, respectively (p < 0.001) while γ-radiation induced a significant   Ext 2 (Cycas armstrongii) and Ext 3 (Cycas revoluta). * Irradiation only, # Significantly different from irradiation.
decrease in Gpx and TAC in tissues of brain and pancreas by 58.5% and 65.5% for brain, respectively (p < 0.001) and 49% and62% for pancreas, respectively. Cycas treatment (3 extracts) induced a significant reduction in MDA of brain by 62.6%, 46.7%, 56.7%, respectively and MDA for pancreas 49%, 50%, 58%, respectively as compared to the γ-radiation group. Furthermore, a significant increase in GPx and TAC were shown. The increase of GPx in brain was 137%, 138%, 131%, respectively. The increase of TAC in brain was 137%, 165%, 169%, respectively, as compared to the γ-radiation group.

Histopathological examination
To further characterize the brain and pancreatic damage induced by γ-radiation, histopathological examination of the two tissues was performed. No histopathological alteration was observed on the structure of the neurons in the cerebral cortex, subiculum and fascia dentate, striatum and cerebellum both normal and three extract groups (Figure 8(a-e)) and (Figure 9(a-c)). In contrast, histological examination of brain tissue from γ-irradiated animals revealed nuclear pyknosis and degeneration in the neuronal cells of the cerebral cortex (Figure 8(f)), subiculum of the hippocampus (Figure 8(g)) and striatum (Figure 8(h)). Treatment by Ext1 showed that the neurons in the subiculum of the hippocampus were intact (Figure 9(d)) while the neurons of fascia dentate exhibited nuclear pyknosis and degeneration (Figure 9(e)). Furthermore, in the R+ Ex2 group, nuclear pyknosis and degeneration were observed in fewer neurons in the subiculum of the hippocampus (Figure 9(f)), while the fascia dentate showed more (Figure 9(g)). The examination of the R+ Ex2 group revealed that few neurons in subiculum of the hippocampus appeared nuclear pyknosis and degeneration (Figure 9(h)). On the other hand, the histology of pancreas of control and extracts alone groups, there was no histopathological alteration of the islands of Langerhans cells as endocrine portion and the acini with the ducts system as exocrine one was recorded in (Figure10(a,d,e,f)). In the radiation group, there was an atrophy in the islands of Langerhans cells (Figure10(b)) associated with congestion in the stromal blood vessels (Figure10(c)). In the R+ Ext1 group, there was atrophy in some of the islands of Langerhans while others showed moderate size (Figure10(g)). In the R+ Ext 2 group, no histopathological alteration was observed (Figure10(h)). In the R+ Ext 3 group, there was no histopathological alteration as recorded in (Figure 10(i)). The variability in chemical components observed in Cycas species is correlated to the variability in response upon evaluation of their biological activities. The correlation between Cycas species and their biological activities might be attributed to a combinatorial synergistic effect of these components. Cycas armstrongii Miq. was selected for phytochemical study based on its prevalence effects in most of the assessed biological activities. The alcoholic extract of C. circinalis L. showed the best antioxidant activity followed by C. armstrongii Miq., then C. revoluta Thunb. as a result of the ease of donation of hydrogen radicals, which is related to the radical scavenging ability of antioxidants as a reason of flavonoids and phenolic contents that could be responsible for the antioxidant capacity of medicinal plants (Zalabani et al., 2012).
Phenolic compounds were reported to exert various physiological activities as antioxidant effect (Shahwar et al., 2012) and improve a variety of conditions such as microbial, protozoa, and malaria infection; vanillic acid reported to have a moderate inhibition effect against Pseudomonas aeruginosa (Chatterjee et al., 2015). Naringin was recently considered as antimalarial agent (Rudrapal & Chetia, 2017), while amentoflavone and its derivatives have previously been assessed as antileishmanial agents (Rizk et al., 2014). Regarding the antifungal activity, the isolated sterols and diterpene alcohol were reported as antifungal agents (Minhas, Rehaman, Yasin, Awan, & Hussain, 2013;Omoruyi, Afolayan, & Bradley, 2014). Few reports were recorded on the phytochemical studies of different Cycas species (Laishram et al., 2014) and no records on radiation injury. The improvement of the studied parameters in treating rats could be attributed to their active constituents and their antioxidative and anti-inflammatory effects. The extracts attenuated the oxidative stress in brain and pancreas. Serum SAA and IL-18 declined significantly than radiation exposure alone. Kumar and kumar (2015) observed that C. circinalis does not produce any toxic effect in male albino rats with no adverse histopathological presentations when different organs including brain, liver, and kidney were examined. Their findings of the sub-acute toxicity tests suggest that C. circinalis with oral administration is considered nontoxic at therapeutic doses for an extended period showing potency for enhancing the immune system and liver protection, kidney, and cardiovascular system. The sub-acute toxicity study proved that when the ethanolic extract of C. circinalis given orally until 1000 mg/kg body weight proved its safety and exhibited no toxicity.
Histopathological examination confirmed the biochemical evaluation and showed ameliorating effects in brain tissue. The histology of pancreas showed better histopathological findings than brain in which extracts render pancreatic tissues as normal control.

Conclusion
Although Cycas plants were used for decades as edible food and drug, the reported secondary metabolites of these plants are few and the phytochemical and biological profiles have been scarcely studied. Accordingly, this work was designed to explore the role of Cycas species in ameliorating the deleterious effects of ionizing radiation via attenuation of oxidative stress, improving blood count, alleviating inflammatory mediators in serum, brain, and pancreatic tissues after exposure of high dose of ionizing radiation. The obtained data proved the potential role of these extracts in radiation treatment and provided a proposal for Cycas therapeutic applications.

Disclosure statement
No potential conflict of interest was reported by the authors.