Anticancer and Neuroprotective Activities of Ethyl Acetate Fractions from Morus macroura Miq. Plant Organs with Ultraperformance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry Profiling.

Column chromatography afforded the isolation of seven secondary metabolites (1-(2,4,6-trihydroxy phenyl)-ethanone-4-O-β-d-glucopyranoside, naringenin-7-O-β-d-glucopyranoside, kaempferol-3-O-α-l-rhamnoside, kaempferol-3-O-β-d-glucopyranoside, quercetin-3-O-β-d-glucopyranoside, quercetin-3-O-β-d-galactopyranoside, rutin) from the ethyl acetate (ET) fractions of Morus macroura Miq. stems (S), leaves (L), and fruits (F). Their identification based on ultraviolet (UV), electron ionization (EI), electrospray ionization-mass spectrometry (ESI-MS), and 1D and 2D NMR data. In addition, profiling of ET fractions using ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) resulted in the identification of 82 compounds belonging to different classes, mainly polyphenolic constituents. Chemical profiling as well as molecular docking directed us to biological evaluation. Interestingly, the ET-L fraction exhibited a robust cytotoxic activity against HepG-2, MCF-7, and HELA cell lines. Also, it displayed a neuromodulatory activity against cisplatin neurotoxicity in rats by ameliorating the neurobehavioral dysfunction visualized in the open field and Y-maze test and modulating the neurochemical parameters such as brain amino acid levels (glutamate, aspartate, serine, and histidine), oxidative stress markers (GSH, MDA, and 8-hydroxy-2'-deoxyguanosine), and purinergic cell energy (adenosine triphosphate (ATP) and adenosine monophosphate (AMP)). In conclusion, the isolated compounds (kaempferol-3-O-β-glucoside and quercetin-3-O-β-glucoside) from the ET-L fraction could serve as potent anticancer agents due to their strong antioxidant, in vitro cytotoxicity, and in vivo neuroprotective activity.

Introduction

The genus Morus family Moraceae includes 40 genera and 1000 species of monoecious rarely dioecious plants distributed in tropical and subtropical regions.[1] This genus is famous for the biosynthesis of bioactive metabolites such as Diels–Alder type adducts, phenolic acids, flavonoid aglycone, prenylated flavonoids, flavonoid glycosides, anthocyanins, 2-arylbenzofurans, alkaloids, stilbenes, steroids, and triterpenoids.[2,3] Moreover, plants belonging to this genus had exhibited many significant biological activities such as antidiabetic, anticancer, antioxidant, anti-inflammatory, antimicrobial, antihyperlipidemic, and antihypertensive activities.[4] An up to date survey revealed the presence of a few reports about the isolation and identification of bioactive metabolites from M. macroura Miq, vice Diels–Alder type adducts such as guangsangons F, G, H, I, J, mulberrofuran J, and kuwanon J,[5] stilbene dimer as andalasin A,[6] triterpenoids such as α-amyrin acetate, 3-β-hydroxylup-20(29)-en-28-oic acid, 3-β-hydroxylup-12-en-28-oic acid, and butyrospermol acetate[7] and arylbenzofuran derivatives including moracins.[8]

Cancer is the most predominant cause of high mortality globally after cardiovascular diseases.[9] The incidence is usually increasing in middle and low-income countries.[10] There are many cancer treatment varieties, such as surgical interventions, radiotherapy, and/or chemotherapy. Cisplatin (cis-diamminedichloroplatinum II), one of the platinum chemotherapeutic drugs, is used heavily to treat many cancers.[11] Despite its potent antitumor efficacy, it has many side effects that negatively affect cancer patients’ health and quality of life.[12] Neurotoxicity is one of these side effects that occurred due to the ability of cisplatin to cross the blood-brain barrier (BBB) and induce weakness in the mature neurons of the brain.[13] It is manifested by many symptoms like locomotion incoordination, ototoxicity, and encephalopathy.[14] The prevalence of neurotoxicity increases with increasing the cumulative cisplatin dose above 300 mg/m2 or 500 mg/m2.[15]

Recent strategies recommend using pain killers or natural antioxidants along with cisplatin medication to reduce its side effects.[16] A considerable amount of studies has been published on the neuroprotectant effect of some herbal plants against cisplatin-induced neurotoxicity in rats like Ginkgo biloba,[17]Azadirachta indica,[18] and Polygonum minus.[19] However, no study was conducted on the M. macroura Miq and their role in protecting the brain against cisplatin neurotoxicity. Therefore, in our current study, we aimed to investigate the neuroprotectant activity of the ET-L fraction from M. macroura Miq against cisplatin-induced neurotoxicity in rats using behavioral and neurochemical assays. Besides, phytochemical investigations of the ET-L, ET-S, and ET-F fractions is done through UPLC-ESI-MS/MS profiling and other techniques used for secondary metabolites isolation.

Experimental Section

Plant Material

Fresh plant was gathered from a private nursery in Belbis in May 2014 and was kindly identified by Prof. Dr. Abdelhalem Abdelmogali, taxonomy researcher, Ministry of Agriculture, Dokki-Cairo, Egypt. A voucher specimen (MM100) was preserved in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, University of Zagazig.

Extraction, Fractionation, And Metabolites Isolation

Dried powdered stems (3.5 kg), leaves (2.5 kg), and fruits (1.0 kg) of M. macroura Miq were separately macerated with 80% aqueous ethanol at room temperature. The total ethanolic extracts of different plant organs were concentrated using a Buchi rotary evaporator (236, 704, and 86 g, respectively) and subsequently partitioned against light petroleum followed by dichloromethane and ethyl acetate (ET). Aliquots of the ET soluble fractions of different organs (5 g of the stem, 10 g of leaves, and 2.5 g of fruit) were separately subjected to silica gel column chromatography packed with methylene chloride, and the polarity of the mobile phase was gradually increased using methanol. The isolated pure compounds (Scheme S1) were subjected to an ESI-MS spectral analysis triple quadruple instrument (XEVO TQD), and acetonitrile–H2O (1:5) was used as a matrix. In addition, 1H NMR and 13C NMR spectral analyses were recorded on a Bruker instrument (Switzerland) at 400 and 100 MHz, respectively, using CD3OD as the solvent.

UPLC-ESI-MS-MS Analysis

ET fractions were subjected to UPLC-ESI-MS/MS analysis using negative and positive ion acquisition modes on a triple quadruple instrument (XEVO TQD, Waters Corporation, Milford, MA) mass spectrometer; column: ACQUITY UPLC-BEH C18 1.7 μm × 2.1 mm × 50 mm column. The samples were injected automatically using a Waters ACQUITY FTN autosampler. The mobile phase was filtered using a 0.2 μm filter membrane disc and degassed by sonication before injection. Its flow rate was 0.2 mL/min using a gradient mobile phase (methanol and water acidified with 0.1% formic acid that applied from 10% to 30% in 5 min, then from 30% to 70% in 10 min, then from 70% to 90% in 5 min, then holds the gradient for 3 min, then from 90% to 10% in 3 min). The instrument was controlled by Masslynx 4.1 software.

In VitroEvaluation

Cytotoxic Activity

The tested samples (ET-L, ET-S, and ET-F) were investigated at different concentrations (500, 250, 125, 62.5, 31.25, 15.6, 7.8, and 3.9 μg/mL) against breast carcinoma (MCF-7), cervical carcinoma (HELA), and liver carcinoma (HepG-2) cell lines that were obtained from the Faculty of Pharmacy, Pharmacology Department, Al-Azhar University and maintained in Dulbeccoʼs modified Eagleʼs medium (DMEM). Also, quercetin-3-O-β-d-glucopyranoside and kaempferol-3-O-β-d-glucopyranoside, isolated from ET-L, were investigated against the HELA cell line seeded in 96-well plates (10 000 cells per well in 100 μL of growth medium). According to published protocols, cells were fixed and stained for 10 days.[20] Optical density readings were carried out at 490 nm using a microplate reader (SunRise, TECAN, Inc., USA) to determine the percentage of viability.

Antioxidant Activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) was used to evaluate the antioxidant activity of ET-S, ET-L, and ET-F fractions at different concentrations according to Rice-Evans, Halliwell, Lunt, and Halliwell.[21] Additionally, methanol solution (40 mL) of tested samples were added to 3 mL of methanolic solution of freshly prepared DPPH radicals (0.004% w/v) according to Yen and Duh.[22] The absorbance of the tested samples, control, and ascorbic acid as a reference standard were measured at λmax 517 nm. The percentage of DPPH radical scavenging was calculated depending upon the following formula:, where Ac is the absorbance of methanol solution of DPPH and At is the absorbance of the extracted sample with DPPH.

Molecular Docking

Cyclin-Dependent Kinase 9 (CDK9) was selected for docking. The protein structural file of the human CDK9/cyclinT1 in complex with Flavopiridol (PDB accession code 3BLR) was downloaded from the protein data bank repository (www.rcsb.org). The protein structure was prepared for docking by adjusting the bond orders, adding missing hydrogen atoms, filling in missing side chains and loops with prime, and deleting artificial water molecules. Although it does not affect the docking simulation, we kept cyclinT1. The hydrogen bond network was assigned by sampling water orientations and adjusting the protonation states of the amino acids. Restrained energy minimization was carried out using OPLS3e to remove atomic clashes. ATP atomic coordinates were selected to define the receptor grid for docking. To impart some degree of flexibility during the docking process, we modified the van der Waals radius scaling factor to 0.85 with a partial charge cutoff of 0.25. This will allow the ligand to use more space for docking. We allowed the rotation of receptor hydroxyl and thiol groups to simulate the actual ligand fitting. The 3D structures of the compounds were downloaded from PubChem (pubchem.ncbi.nlm.nih.gov). All acceptable protonation and tautomerization states were generated at pH 7.4 using LigPrep. After ligand preparation, we run the docking simulation using Glide SP (Standard precision). The ligand was treated as flexible, and the receptor was considered rigid with softened potentials.

In Vivo Evaluation

Animals

Twenty-eight male Wistar rats weighing between 100–120 g were purchased from a commercial animal house (Giza, Egypt). They were maintained at standard environmental conditions and fed with commercial rodent ration and water ad libitum. All the experimental procedures agreed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). They were approved by the Veterinary Institutional Animal Care and Use Committee (VET-IACUC: Approval Number, Vet CU28/04/2021/269) of the Faculty of Veterinary Medicine, Cairo University.

Experimental Design

Animals were randomly divided into four groups of seven animals each, as follows: Group I: control, rats were administrated 2 mL/kg distilled water daily. Group II: cisplatin (Mylan, The Netherlands) at a single dose 10 mg/kg body weight, i.p. Group III: ET-L200, 200 mg/kg body weight ET-L fraction gavaged daily for 15 days. Group IV: cisplatin + ET-L200, 200 mg/kg body weight ET-L fraction gavaged daily for 15 days + cisplatin 10 mg/kg body weight, i.p. as a single dose. Cisplatin administration was performed on the tenth day of the experiment, 1 h after administration of ET-L200. The dosage of cisplatin was selected according to previous studies,[23] while the dose of the ET-L fraction was selected according to a previous study.[24]

Behavioral Testing

At a time 24 h after the last dose of ET-L200, rats were submitted to open field testing and the Y-maze to assess the locomotor behavior and working memory. The behavioral tests were performed as previously mentioned.[25,26] The measuring parameters for the open field test were the number of crossing squares and rearing activity which were recorded for 3 min. However, the number of arm entries and spontaneous alternation percentage (SAP%) were evaluated for 5 min as working memory indicators in the Y-maze test. After the last behavioral test, all rats were euthanized by cervical dislocation, and the brains were excised gently and preserved at −80 °C for further neurochemical analyses.

Neurochemical Analyses

HPLC is a convenient method used for the detection of several neurochemicals, where the level of glutamate in the brain was detected according to Heinrikson et al.,[27] while brain monoamines levels were measured according to Pagel et al.[28] Oxidative stress markers (reduced glutathione (GSH), malondialdehyde (MDA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG)) were also measured according to Jayatilleke et al., Karatepe et al., and Lodovici et al.,[29−31] respectively. Concerning brain cell energy levels, adenosine triphosphate (ATP) and adenosine monophosphate (AMP) were determined according to Teerlink et al.[32]

Statistical Analysis

The in vitro studies were performed in triplicates. The in vivo study was analyzed using SPSS 24.0 software. ANOVA followed by Bonferroni post hoc analysis for multiple comparisons and Pearson correlation for neurochemical parameters were used for statistical analysis. Data were expressed means ± SEM, and significance was set at P ≤ 0.05. Graphs were drawn using SPSS and Graph Pad Prism version 6.00.

Results

Phytochemical Results

Column chromatography packed with different stationary phases in combination with semipreparative HPLC of the ET soluble fractions afforded the isolation and identification of seven metabolites, where compounds (1, 2, 3) isolated from ET-S, (4, 5, 6) from ET-L and (7) ET-F (Figures and 2, Supplementary text sections ST1–ST7, Figure S1–S27, and Scheme S1).

Figure 1

UPLC-ESI-MS/MS chromatograms (negative mode) of ET-L (A), ET-S (B), and ET-F (C) fractions of M. macroura, ET-L: ethyl acetate fractions of leaves; ET-S: ethyl acetate fractions of stems; ET-F: ethyl acetate fractions of fruits.

Figure 2

UPLC-ESI-MS/MS chromatograms (positive mode) of ET-L (A), ET-S (B), and ET-F (C) fractions of M. macroura, ET-L: ethyl acetate fractions of leaves; ET-S: ethyl acetate fractions of stems; ET-F: ethyl acetate fractions of fruits.

Characterization of Secondary Metabolites Analyzed by UPLC -ESI-MS/MS

Phenolic and nonphenolic compounds were identified in the ET-L, ET-S, and ET-F fractions of M. macroura Miq by UPLC-ESI-MS/MS. The distribution of these compounds and their percentages are gathered in Table and described in Supplementary text section ST8.

Table 1

Compounds Identified in the Ethylacetate Fractions of Leaves (ET-L), Stems (ET-S), and Fruits (ET-F) of M. macroura Miq by Using UHPLC-ESI/MS/MS in Positive and/or Negative Ionization Modes

peak no.	RT	RRT	MS 1–/+	MS2	leaves	stems	fruits	tentative assignment	refs
1	0.82	0.032	353/–	191(100%)	0.59	10.47	14.98	chlorogenic acid	(33)
				179 (15%)
2	2.1	0.084	153/–	109		0.74		protocatecheic acid	(34)
3	2.43	0.097	339/–	177 (100%)			0.82	aesculin	(35)
4	3.63	0.145	137/–	93 (100%)		0.57		hydroxybenzoic acid	(36)
5	3.86	0.155	353/–	191 (100%)			2.76	neochlorogenic acid
				179 (32%)
6	4.84	0.194	353/–	173 (100%)			1.7	cryptochlorogenic acid	(33)
				179 (90%)
7	4.84	0.194	137/–	93 (100%)		0.57		hydroxybenzoic acid isomer	(36)
8	6.27	0.251	339/–	177, 175, 161, 135, 109	0.39		0.77	morachalcone A	(37)
9	6.27	0.251	431/–	269 (100%)		2.71	0.31	apigenin-7-O-β-glucoside	(38)
10	6.73	0.269	163/–	119 (100%)		6.66		coumaric acid	(36)
11	6.92	0.277	463/–	270.7	0.85	0.61	0.31	naringenin derivatives	(39)
12	7.26	0.291	447/–	285, 256.9, 242.5, 198.8	0.36	0.61	1.22	luteolin-7-O- glucoside	(38)
13	7.46	0.299	–/355	193	0.29			scopolin	(40)
14	7.62	0.305	431/–	341, 311, 269		0.61		vitexin (apigenin-8-C-glucoside)	(39)
15	7.92	0.317	463/–	301, 257			0.88	morin-O-β-glucoside	(41)
16	8.05	0.323	667/–	6, 49, 407		1.02		3-hydroxy kuwanol E	(42)
17	8.1	0.325	–/449	287		0.35		cyanidin-3-O-glucoside	(43)
18	8.25	0.331	463/–	301 (100%)	50.85	10.91	√	aquercetin-3-O-β-d-glucoside (isoquercitrin)	(35)
19	8.3	0.333	609/–	463, 301			17.63	arutin	(44)
20	8.32	0.333	–/949	717, 465, 303 (100%) from MS 1	6.61			delphinidin derivative	(45)
21	8.38	0.336	463/–	301	√	4.93		aquercetin-3-O-β-galactoside (hyperoside)	(46)
22	8.53	0.342	431/–	285, 284, 255, 227		0.2		kaempferol-3-O-β- rhamnoside (afzelin)	(47)
23	8.71	0.349	447/–	269	0.71	0.95	1.22	apigenin-7-O-glucuronide	(48)
24	8.96	0.359	431/–	269		11.38		apigenin-7-O- glucoside isomer	(38)
25	9.24	0.37	447/–	285	20.63	0.95	0.88	akaempferol-3-O-β-glucoside (astragalin)	(35)
26	9.24	0.37	447/–	343, 301	0.71	0.95	0.88	quercetin-8-C-rhamnoside	(33)
27	10.95	0.439	–/611	303, 328	18.93	7.53	4.54	hesperidin	(49)
28	10.95	0.439	–/610	317.5, 256.8, 151.2	0.46		0.99	petunidin dirhamnoside	(50)
29	11.14	0.446	447/–	301, 255	0.71	1.95	0.88	quercetin-3-O-β- rhamnoside (quercitrin)	(33)
30	11.18	0.448	–/397	351		1.35		unknown
31	11.34	0.454	301/–	139 (100%), 256	1.3	0.43	0.77	ellagic acid	(35)
32	11.34	0.454	355/–	193.3 (100%)			0.67	ferulic acid-O-glucoside	(35)
33	11.37	0.456	431/–	285, 284, 255, 227		1.43	0.3	kaempferol-3-O-β- rhamnoside isomer (afzelin)	(33)
34	11.49	0.46	285/–		6.33			kaempherol	(51)
35	12.1	0.485	301/–	179, 151, 121	1.3	0.43	0.77	quercetin	(35)
36	12.85	0.515	271/–	271, 58			1.5	moracin J
37	12.17	0.488	301/–	256, 151, 107	1.42	4.92		morin
38	13.39	0.537	–/535	255			0.48	unknown
39	14.06	0.563	467/–	305		√		gallocatechin glycoside	(35)
40	14.15	0.567	467/–	305		√		epigallocatechin glycoside
41	14.39	0.577	329/–	287 [M – H – COCH3]	0.91	0.77		aphloracetophenone-4-O-glucoside	(52)
42	14.68	0.588	575/–			√		unknown
43	16.04	0.643	467/–			√		unknown
44	17.23	0.691	–/343				1.2	unknown
45	17.77	0.712	–/579			0.48	0.91	unknown
46	17.83	0.715	–/355	203	0.86			epoxybergamottin	(53)
47	17.83	0.715	–/355	299, 69	0.29			albanin A	(54)
48	18.38	0.737	293/–	220.8 (100%)		0.01		hydroxy octadecatrienoic acid	(55)
49	18.87	0.756	591/–	287	√	0.33		cyanidin cinnamoyl glucuronide	(56)
50	19.77	0.792	317/–		0.86	1.73	1.05	unknown
51	20.29	0.813	271/–	150.7 (100%)			√	naringenin	(57)
52	20.86	0.836	–/369				√	unknown
53	21.08	0.845	475/–	299		0.05		chrysoeriol-uronic acid	(38)
54	21.1	0.846	–/355	177	0.29		1.29	sanggenon F	(37)
55	21.4	0.858	433/–	271		2.14		anaringenin-7-O-β-glucoside	(58)
56	21.49	0.861	–/381	318, 163, 71, 69	√			brasicasterol	(59)
57	21.74	0.871	475/–	410.9, 299.0	√			diosmetin glucuronide	(60)
58	21.99	0.881	475/–	255			0.01	palmitic acid ester	(61)
59	22.03	0.883	–/427	363.5			√	lupeol	(62)
60	22.06	0.884	423/–				4.5	unknown
61	22.51	0.902	279/–			1.02		unknown
62	22.73	0.911	–/683	331	6.65	3.9		malvidin diglucoronide	(63)
63	23.02	0.922	451/–	249.8, 390.5, 406.7, 435.1		1.73		melissic acid	(64)
64	23.06	0.924	–/683	597, 435, 287	5.4		√	cyanidin derivative	(65)
65	23.24	0.931	255/–		√	3.86	√	unknown
66	23.47	0.941	281/–		√		1.71	unknown
67	23.55	0.944	–/391	279, 149 (100%)			10.77	bis(2-ethyl hexyl) phthalate	(66)
68	23.6	0.946	–/611	302, 328, 229, 201	4.46	7.53	4.54	peonidin rutinoside	(67)
69	23.67	0.949	–/663	301	√			peonidin derivative	(68)
70	23.72	0.951	–/593	287, 434, 595	0.43	5.18	0.81	cyanidin rutinoside	(69)
71	23.83	0.955	349/–		0.68		0.8	unknown
72	23.86	0.956	395/–	281			2.31	oleic acid ester	(70)
73	24.14	0.967	–/607	286	6.4		1.75	australisine A	(71)
74	24.44	0.979	283/–	239 [M – H – COO]			√	octadecanoic acid	(72)
75	24.45	0.981	339/–	162.9		0.33	1.09	coumaric acid glucuronide	(38)
76	24.65	0.988	–/535	317, 153	6.99	15.18	0.61	isorhamnetin-3-O-acetyl glucuronide	(73)
77	24.65	0.988	–/536	317, 153	0.99	15.18	0.61	petunidin 3-O-acetyl glucuronide	(74)
78	24.78	0.993	–/463	301		4.37		peonidin glucoside	(75)
79	24.95	1	339/	163 (100%)	0.71	0.78	13.61	euchrenone a7	(76)
82	25.53	1.023	–/683	597 (loss of 86), 435 (loss of glucose), 287.7	10.15	12.42	4.39	cyanidin-3-O-malonyl glucoside derivative	(77)
83	25.84	1.035	–/535	287	6.99	15.18	17.51	cyanidin-3-O-malonyl glucoside	(77)
90	27.68	1.109	–/398	314	6.4	9.35	1.31	24-methylene-ergosta-5-en-3β-ol	(75)

Compounds isolated as pure compounds from M. macroura Miq are listed in bold. √ denotes that compounds are minor.

Biological Results

In Vitro Cytotoxic Activity 2

Different concentrations of ET fractions were evaluated against different tumor cells MCF, HELA, and HepG-2. The ET-L was the most active fraction against HepG-2 (13.2 μg/mL) and MCF-7 (20.5 μg/mL). Moreover, ET-S and ET-F showed moderate activity against HepG-2 (23.2 μg/mL and 25.8 μg/mL) and MCF-7 (51.0 μg/mL and 43.7 μg/mL), respectively (Figure S2). Additionally, quercetin-3-O-β-d-glucopyranoside and kaempferol-3-O-β-d-glucopyranoside exhibited strong and moderate activity against the HELA cell line with IC50 = 19.8 μg/mL and 61.4 μg/mL, respectively compared to doxorubicin (Figures –5).

Figure 3

Effect of ethyl acetate fractions of leaves (ET-L), stems (ET-S) and fruits (ET-F) of M. macroura Miq. on the viability % MCF-7 (A), HELA (B), and HepG-2 (C) cell lines.

Figure 5

IC50 (μg/mL) of quercetin-3-O-glucoside, kaempferol-3-O-glucoside, and doxorubicin as standards against the HELA cell line.

Effect of quercetin-3-O-glucoside and kaempferol-3-O-glucoside on the viability % HELA cell line.

In Vitro Antioxidant Activity

The DPPH scavenging percentage of ET fractions and ascorbic acid and SC50 values (the concentration required to scavenge DPPH by 50%) are shown in Figure S27. The ET-L fraction showed the highest antioxidant activities as indicated by their high SC50 followed by ET-F and ET-S fraction (SC50 5.53 ± 0.04, 10.6 ± 0.02, and 44.2 ± 0.11, respectively) compared to ascorbic acid (SC50 5.3 ± 0.04).

Molecular Docking

We selected CDK9 to investigate the expected mechanism of kaempferol-3-O-β-glucoside and quercetin-3-O-β-glucoside as anticancer agents. The results showed that kaempferol-3-O-β-glucoside and quercetin-3-O-β-glucoside fit well within the binding site of CDK9 with binding energy values of −8.4 kcal/mol for quercetin-3-O-β-glucoside and −7.5 kcal/mol for kaempferol-3-O-β-glucoside compared to −9.1 kcal/mol for the reference compound (flavopiridol). The compounds showed a hydrogen bond to the hinge amino acid residues Cys 107 and Asp 110 and electrostatic interactions to the Mg2+ ion. These interactions are conserved in the native ligand’s complexes. The conserved Lys residue (Lys 49) showed strong ion–cation interactions with the aromatic rings of these flavonoids. In general, the oxygen containing functionalities orient themselves in the proper position to face the hinge region allowing for forming the required hydrogen bonds (Figures and 7).

Figure 6

Interaction profile of quercetin-3-O-glucoside. (Left) 2D interaction map showing the interacting amino acids and the type of interaction. (Right) 3D figure of the protein–ligand complex showing the protein as a cartoon and the ligand as sticks. The Mg2+ is shown as a sphere.

Figure 7

Interaction profile of kaempferol-3-O-glucoside. (Left) 2D interaction map showing the interacting amino acids and the type of interaction. (Right) 3D figure of the protein–ligand complex showing the protein as a cartoon and the ligand as sticks. The Mg2+ is shown as asphere.

In Vivo Evaluation

Open Field Test

As shown in Figure A,B, the locomotor behavior as visualized by the number of crossing squares and rearing activity was markedly decreased in the cisplatin intoxicated rats compared to the control group. On the other hand, the rats treated with cisplatin + ET-L200 showed a significant increase in the locomotion compared to the cisplatin intoxicated rats. There was no significant difference between control rats and ET-L200 treated rats.

Figure 8

Effect of cisplatin and/or ET-L fraction of M. macroura on the locomotion and cognitive behavior of rats. (A) Open field test: number of crossings, (B) open field test: rearing frequency, (C) Y-maze: number of arm entries, and (D) Y-maze: spontaneous alternation percentage. Data are expressed as mean ± SEM, one-way ANOVA, followed by a post hoc test and Bonferroni test for seven rats in each group. *Significant from the control group, @significant from the cisplatin group, &significant from the ET-L200 group. p < 0.05.

Y-Maze

As demonstrated in Figure C,D, the cisplatin intoxicated rats showed a marked decrease in the number of Y-maze arms entries and spontaneous alternation percentage compared to control rats. Rats treated with cisplatin + ET-L200 showed a substantial increase in the arm entry frequency and spontaneous alternation percentage compared to cisplatin intoxicated rats.

Brain Amino Acid Level

As shown in Figure A–D, cisplatin intoxicated rats exhibited a significant increase in most brain amino acid levels (glutamate, aspartate, serine, and histidine) in comparison to the control and ET-L200 treated rats. On the other hand, cisplatin + ET-L200 treated rats displayed a marked decrease in the amino acids level (glutamate and histidine) in comparison to control ET-L200 treated rats. Interestingly, ET-L200 administration decreased aspartate and serine levels near the control in cisplatin + ET-L200 treated rats.

Figure 9

Effect of cisplatin and/or ET-L fraction of M. macroura on the brain amino acid levels of rats: (A) glutamate, (B) aspartate, (C) serine, and (D) histidine. Data are expressed as mean ± SEM, one-way ANOVA, followed by a post hoc test and Bonferroni test for seven rats in each group. *Significant from the control group, @significant from the cisplatin group, &significant from the ET-L200 group. p < 0.05.

Brain Oxidative Stress Markers

As depicted in Figure A–C, cisplatin intoxicated rats displayed a substantial increase in the MDA and 8OHdG levels and a marked decrease in the GSH level compared to the control and ET-L200 treated rats. In contrast, cisplatin + ET-L200 treated rats showed a mid amelioration of oxidative stress markers and recovered near the control group.

Figure 10

Effect of cisplatin and/or ET-L fraction of M. macroura on the oxidative stress markers of rats: (A) reduced glutathione, (B) malondaldhyde, and (C) 8-hydroxy-2′-deoxyguanosine. Data are expressed as mean ± SEM, one-way ANOVA, followed by a post hoc test and Bonferroni test for seven rats in each group. *Significant from the control group, @significant from the cisplatin group, &significant from the ET-L200 group. p < 0.05.

Brain Cell Energy Level

Cisplatin intoxicated rats displayed a marked exhaust of ATP and accelerated the degradation resembling the AMP content compared to the control and ET-L200 treated rats (Figure A,B). The cisplatin + ET-L200 treated rats displayed a mild recovery for ATP and a complete recovery for the AMP content compared to the control and ET-L200 treated rats.

Figure 11

Effect of cisplatin and/or ET-L fraction of M. macroura on the brain cell energy level of rats: (A) adenosine triphosphate and (B) adenosine monophosphate. Data are expressed as mean ± SEM, one-way ANOVA, followed by a post hoc test and Bonferroni test for seven rats in each group. *Significant from the control group, @significant from the cisplatin group, and &significant from the ET-L200 group. p < 0.05.

Correlation Matrix Parameters

Figure and Table showed a positive correlation for Glu against ASP, Ser, His, MDA, 8OHdG, and AMP and negative against GSH and ATP. Also, GSH presents a positive correlation against ATP and negative from Glu, ASP, Ser, HIS, MDA, 8OHdG, and AMP.

Figure 12

Scatter correlation matrixes against GLU and GSH and subsequent parameters.

Table 2

Correlation Matrix of Some Biochemical Parametersa

	Glu	GSH	ASP	Ser	His	MDA	8OHdG	ATP	AMP
Glu	ND	–0.388	0.089	0.04	0.507	0.562	0.51	–0.435	0.383
		0.061	0.678	0.853	0.011	0.004	0.011	0.034	0.065
GSH	–0.388	ND	–0.653	–0.448	–0.739	–0.624	–0.839	0.975	–0.633
	0.061		0.001	0.028	0	0.001	0	0	0.001
ASP	0.089	–0.653	ND	0.439	0.317	0.382	0.598	–0.656	0.591
	0.678	0.001		0.032	0.131	0.065	0.002	0	0.002
Ser	0.04	–0.448	0.439	ND	0.504	0.484	0.513	–0.494	0.24
	0.853	0.028	0.032		0.012	0.017	0.01	0.014	0.26
His	0.507	–0.739	0.317	0.504	ND	0.647	0.725	–0.805	0.601
	0.011	0	0.131	0.012		0.001	0	0	0.002
MDA	0.562	–0.624	0.382	0.484	0.647	ND	0.659	–0.667	0.341
	0.004	0.001	0.065	0.017	0.001		0	0	0.103
8OHdG	0.51	–0.839	0.598	0.513	0.725	0.659	ND	–0.836	0.568
	0.011	0	0.002	0.01	0	0		0	0.004
ATP	–0.435	0.975	–0.656	–0.494	–0.805	–0.667	–0.836	ND	–0.672
	0.034	0	0	0.014	0	0	0		0
AMP	0.383	–0.633	0.591	0.24	0.601	0.341	0.568	–0.672	ND
	0.065	0.001	0.002	0.26	0.002	0.103	0.004	0

0 to 0.3 (0 to −0.3) negligible correlation; 0.3–0.5 (−0.3 to −0.5) low positive (negative) correlation; 0.5–0.7 (−0.3 to −0.7) moderate positive (negative) correlation; 0.7–0.9 (−0.7 to −0.9) high positive (negative) correlation; 0.9–1 (−0.9 to −1) very high positive (negative) correlation.

Discussion

Traditional column chromatography and hyphenated HPLC techniques were applied for profiling the ET-L, ET-S, and ET-F, where eight secondary metabolites were isolated by column chromatography and purified by preparative TLC as well as crystallization. Additionally, UPLC-ESI-MS/MS profiling afforded the identification of altogether 82 compounds, where about half of the identified compounds are present for the first time in the family Moraceae, such as phenolic acid glycosides (ferulic acid-O-glucoside, phloracetophenone-4-O-glucoside, and coumaric acid glucuronide), flavonoid glycosides (apigenin-O-glucoside, its isomer, kaempferol-3-O-rhamnoside, its isomer, luteolin-7-O-glucoside apigenin glucuronide, hesperidin, morin-3-O-glucoside, chrysoeriol-uronic acid, naringenin-7-O-glucoside, diosmetin glucuronide, isorhamnetin 3-O-acetyl glucuronide), tannin compounds (gallocatechin glycoside, epigallocatechin glycoside). Moreover, aesculin and epoxybergamottin are first reported in Morus macroura Miq. Also, anthocyanin derivatives such as compounds 20, 28, 49, 62, 64, 69, 77, 78, 82, and 83 as well as melissic acids, steroids, and triterpenoids (cholesterol, brasicasteol, and 24-methylene ergosta-5-en-3β-ol) are first reported in Morus macroura Miq.

Furthermore, all the phenolic acids, compounds 3-hydroxy kuwanol E, moracin J, bis (2-ethylhexyl) phthalate, all flavonoid aglycones and glycosides, prenylated flavonoids, coumarins, all anthocyanins, fatty acids and their derivatives, all steroids and triterpenoids and their derivatives were identified for the first time in Morus macroura Miq. On top of these metabolites, quercetin-O-glucoside and kaempferol-O-glucoside, in a ratio of 50.85% and 20.63%, respectively, are the most abundant compounds in the ET-L fraction. In conclusion, M. macroura Miq. leaves exhibit higher contents of polyphenolics, and these findings are in agreement with that previously reported.[78]

In vitro cytotoxic and antioxidant activity of the ET-L, ET-S, and ET-F fractions were evaluated. The cytotoxic activity evaluation was based on IC50 values, according to Srisawat et al.[79] The ET-L fraction displayed the highest activity against HepG-2 and MCF-7 tumor cell lines. Also, they exhibited the most increased antioxidant activity compared to ascorbic acid. These findings can be attributed to their high content of polyphenolics, especially quercetin-3-O-β-d-glucopyranoside and kaempferol-3-O-β-d-glucopyranoside.[78] They also showed potent cytotoxic activity against the HELA tumor cell line compared to doxorubicin.

In general, plant polyphenolics as free radical scavengers have attracted tremendous interest as possible natural therapeutics against free radical-mediated diseases such as atherosclerosis, cancer, asthma, diabetes, dementia, and inflammatory joint and degenerative eye diseases.[78,80] Therefore, based on the strong cytotoxic and antioxidant activity of the ET-L fraction, in vivo cisplatin-induced neurotoxicity was conducted on rats to further confirm the ET-L fraction cytotoxic and antioxidant activities against cisplatin.

Our in vivo results showed that single intraperitoneal administration of cisplatin (10 mg/kg) causes evident neurobehavioral and neurochemical changes. The neurobehavioral changes, including anxiety-like behavior, and the general activity is reduced in cisplatin intoxicated rats in the open field test. The open field test is generally used to evaluate the anxiety-like behavior and the locomotor activity of rodents. These results agreed with previous studies.[23] Furthermore, cisplatin causes cognitive impairment, a condition known as chemobrain, as evidenced by a reduction in arm entries and spontaneous alternation percentages in the Y-maze. The Y-maze test is used to evaluate the working memory in rodents. These data agreed with those recorded in earlier studies.[81,82] However, the administration of the ET-L fraction was able to ameliorate most of the detrimental effects caused by cisplatin as evidenced by an increase in the general motor activity in the open field test and an increase in the arm entries and spontaneous alternating percentages in the Y-maze. Previous studies supported these findings that discussed the role of quercetin and kaempferol in ameliorating the behavioral dysfunction associated with lipopolysaccharide-induced neuroinflammation[83] and stressful events.[84]

A possible explanation for these behavioral dysfunction might be due to the disturbance in the glutamatergic system in the cisplatin intoxicated rats. Glutamate is one of the most excitatory neurotransmitters responsible for the common cognitive functions in the cerebral cortex.[85] It is worth noting that the secretion of a lot of glutamates leads to excitatory toxicity.[86] Glutamate oversecretion leads to the influx of calcium ions into the cell, leading to the activation of many enzymes, such as phospholipase, nuclease, and proteases such as calbin. These enzymes then destroy cell structures such as the cytoskeleton, the cell membrane, and DNA.[87] Downstream of glutamate overexpression leads to a negative correlation for GSH and ATP in which glutamate decrease GSH and purinergic cell energy remarked by ATP content. The glutamate-induced cytotoxicity is likely mediated through inhibition of cystine uptake through cystine/glutamate transporter inhibition leading to a depletion of cellular glutathione synthesis.[88,89] Glutathione played the main factor in the defense against oxidative stress; therefore, prolonged depletion of intracellular glutathione may lead to cell degeneration.

On the other hand, the administration of the ET-L fraction enhances the ROS scavenging properties accompanied by significantly lowering the lipid peroxidation status. This antioxidant property could be attributed to the presence of kaempferol and quercetin metabolites in the ET-L fraction.[90,91] Moreover, kaempferol ameliorates cell energy depletion, which may be the homeostasis’s main causes via increasing mitochondrial ATP and decreasing turnover.[92] Also, it can indirectly reduce Ca2+ and regulate various Ca2+ subordinate enzymes (phospholipids, proteases, and nucleases), which leads to diminishing oxidative pressure and cell death.[93,94]

Additionally, cisplatin intoxicated rats exhibited elevated histidine and serine levels in the brain. Histidine is a precursor of brain histamine, responsible for maintaining the brain functions when releasing in an adequate amount. In contrast, histamine over secretion is associated with many disorders such as anxiety and appetite regulation.[95] In contrast, histidine showed amelioration level after the administration of the ET-L fraction which may depend on the anti-inflammatory and antihistaminic effects of quercetin.[96] In addition, serine is a nonessential amino acid in the body necessary for tumor cell survival.[97] The antitumor role of cisplatin exists in the reduction of phosphoglycerate dehydrogenase (PHGDH) and converts approximately 3-phosphoglyceric acid produced at glycolysis to 3-phosphatedehydropyruvate, which is a precursor of serine.[98] This conversion increases serine accumulation and increases the inhibitory amino acids. However, the administration of ET-L fraction may decrease the serine level. This may be attributed to its quercetin content as confirmed by ref (99) who found that quercetin can reduce the serine level via the Drp1 phosphorylation inhibited at serine 616 in cultured endothelial cells.

Conclusion

The high contents of phenolic compounds in the extract support their potent antioxidant and in vitro cytotoxic activity. The expected mechanisms of action of kaempferol-3-O-β-glucoside and quercetin-3-O-β-glucoside as anticancer agents is CDK9 inhibition. Moreover, The ET-L fraction modulated the neurobehavioral and neurochemical deficits associated with cisplatin in rats. As it increases the locomotor and cognitive behavior visualized by an increase in the number of crossing squares and rearing activity in the open field test and the number of arm entries and spontaneous alternation percentage in the Y-maze test. Moreover, it moderates the brain amino acid levels (glutamate, aspartate, serotonin, and histamine), oxidative stress markers (GSH, MDA, and 8-hydroxy-2′-deoxyguanosine), and purinergic cell energy (ATP and AMP). All of these results render the ET-L fraction a promising candidate in the treatment of various types of cancer or even as a supplementary agent in cisplatin-treated patients.