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Evaluating ancient Egyptian prescriptions today: anti-inflammatory activity of Ziziphus spina-christi.

ABSTRACT

Background: Ziziphus spina-christi (L.) Desf. (Christ's Thorn Jujube) is a wild tree today found in Jordan, Israel, Egypt, and some parts of Africa, which was already in use as a medicinal plant in Ancient Egypt. In ancient Egyptian prescriptions, it was used in remedies against swellings, pain, and heat, and thus should have anti-inflammatory effects. Nowadays, Z. spina-christi, is used in Egypt (by Bedouins, and Nubians), the Arabian Peninsula, Jordan, Iraq, and Morocco against a wide range of illnesses, most of them associated with inflammation. Pharmacological research undertaken to date suggests that it possesses anti-inflammatory, hypoglycemic, hypotensive and anti-microbial effects.

The transcription factor NF-[kappa]B (nuclear factor kappa-light-chain-enhancer of activated B cells) is critical in inflammation, proliferation and involved in various types of cancer. Identification of new anti-inflammatory compounds might be an effective strategy to target inflammatory disorders and cancer. Therefore, extracts from Z. spina-christi are investigated in terms of their anti-inflammatory effects.

Our intention is to evaluate the effects of Z. spina-christi described in ancient Egyptian papyri, and to show whether the effects can be proven with modern pharmacological methods. Furthermore, we determine the active ingredients in crude extracts for their inhibitory activity toward NF-[kappa] pathway. Materials and methods: To determine the active ingredients of Z. spina-christi, we fractionated the extracts for bioassays and identified the active compounds. Epigallocatechin, gallocatechin, spinosin, 6"' feruloyl-spinosin and 6"' sinapoylspinosin and crude extracts of seed, leaf, root or stem were analyzed for their effect on NF-[kappa]B DNA binding by electromobility shift assay (EMSA) and nuclear translocation of NF-[kappa]-p65 by Western blot analysis. The binding mode of the compounds to NF-[kappa]B pathway proteins was compared with the known inhibitor, MG-132, by in silico molecular docking calculations. [Log.sub.10][IC.sub.50] values of gallocatechin and epigallocatechin as two main compounds of the plant were correlated to the microarray-based mRNA expression of 79 inflammation-related genes in cell lines of the National Cancer Institute (NCI, USA) as determined. The expression of 17 genes significantly correlated to the [log.sub.10][IC.sub.50] values for gallocatechin or epigallocatechin.

Results: Nuclear p65 protein level decreased upon treatment with each extract and compound. Root and seed extracts inhibited NF-xB-DNA binding as shown by EMSA. The compounds showed comparable binding energies and similar docking poses as MG-132 on the target proteins.

Conclusion: Z. spina-christi might possess anti-inflammatory activity as assumed by ancient Egyptian prescriptions. Five compounds contributed to this bioactivity, i.e. epigallocatechin, gallocatechin, spinosin, 6"' feruloylspinosin and 6"' sinapoylspinosin as shown in vitro and in silico.

Keywords:

Ancient Egyptian medicinal papyri

Ethnopharmacology

Inflammation

Microarray

Molecular docking

Rhamnaceae

Introduction

Egyptological background

From ancient Egyptian times about 40 papyri, papyri fragments, and ostraca survive, dating from around 1900 BC to 300 BC giving us nearly 2000 prescriptions with ingredients to treat ancient Egyptian illnesses (Pommerening 2012). One of the most interestingly scientific tasks located between the disciplines of Egyptology and pharmacology is to evaluate, whether the ancient prescriptions could supply us with valuable information for therapeutic potentials of plants and other drugs today. This should always be done by considering the original concepts and contexts (Pommerening 2005; 2006), and with awareness of the cultural changes and differences in the understanding of illnesses and their treatment (Pommerening 2010a) as well as the consideration of relevant dosages (Pommerening 2006, 2010b; Gertsch 2009).

Ziziphus spina-christi (L.) Desf. (Christ's Thorn Jujube, Rhamnaceae) is one of the original constituents of the wild flora of Ancient Egypt with its various parts being used in medicine, diet, and rituals (Nicholson and Shaw 2000). As Z. spina-christi did not undergo appropriate pharmacological research as of yet, the plant seems to offer a good example for interdisciplinary investigation, starting with its ancient Egyptian usage, and progressing to in vitro and in silico evidence concerning its compounds from a modern point of view.

Under the ancient Egyptian name nebes (pl. 1, blue frame for hieroglyphic writing), Z. spina-christi occurs as an ingredient of 33 ancient Egyptian prescriptions in the papyri Ramesseum V, Edwin Smith (Sm), Ebers (Eb), Hearst (H), Berlin 3038 (Bln), and Brooklyn 47.218.48 + 85 (Brk) (Table 1).

Besides that, nebes and a kind of bread made of nebes are some of the main items in the food offering rituals for the dead written down in tombs. It occurs among the typical cereals, fruits, and drinks of the ancient Egyptians (pi. 1, blue frame), and, thus, seems to have played an important role in the diet of the living as well. The tree was a typical part of the indigenous ancient Egyptian wild flora, and was also planted in ancient Egyptian gardens (Pommerening 2015; Nicholson and Shaw 2000). Z spina-christi still occurs widely in both wild and cultivated forms in Africa, and is used for its ability to provide shade, timber and edible fruits (Boulos 2000). Today, Nubians and Bedouins in Egypt use the wood, fruits, and leaves for their medicinal properties (Dafni et al. 2005; Moursi 1992). There is a lack of precise field studies examining which plants are used today in Egyptian folk medicine.

In most of the ancient Egyptian prescriptions (examples: Table 2; complete lists: Table 3 and 4), the leaves have been used. Sometimes however, bread made from Ziziphus fruits, or the pulp of the fruit, the fruit, or the 'wood' was prescribed. Some recipes do not specify the part of the plant to be used. Only two prescriptions recommend Ziziphus as a single drug (Eb 536; H 134, Table 2). Those prescriptions in particular demonstrate which properties Ziziphus was credited with by the ancient Egyptians. In Eb 536, the heading of the prescription reads "Healing all things from which a man suffers, namely any Setscha". Setscha is normally associated with wounds and swellings, and seems to indicate a swollen part of the body after injury. Bread of Ziziphus fruits has to be boiled in water, and the suffering body part has to be bandaged with the drug at a pleasant warm temperature.

The prescription H 134, by contrast, is not very specific: bread of Ziziphus fruits with water is used to drive out illness from all body parts by bandaging the ill part with the drug. This prescription is included in a group of prescriptions against swellings. If we consider the composed recipes including Ziziphus, most of them appear in a context of visible swellings, which are externally cured (Table 3). Some of these prescriptions are specifically concerned with cooling (Eb 616, H 95, H 173b, H 226, H 238, Ram V, Nr, XIII, Sm Fall 41/1). The other compositions appear in the context of internally cured pain (Table 4). In total, there seems to be a correlation of Ziziphus with swellings, pain and heat, which are the typical signs of inflammation. For internal applications in particular, the exact measures of the ingredients were noted, leaves or breads mostly around 40ccm had to be used (1/8 dja), sometimes only 20ccm (1/16 dja), or lOccm (1/32 dja) (Table 5, Pommerening 2010b). Even though the explanatory models of pharmacology vary considerably across different times and cultures, an empirical selection of useful remedies was possible on the basis of the evident effects of the drug. As Z spina-christi is used throughout history, and today in the Arabian peninsula, Jordan, Iraq, and Morocco against all kinds of illnesses, including inflammations (Dafni et al. 2005), it can be assumed that Z spina-christi may contain valuable anti-inflammatory compounds. No distinction appears to be made between the use of the fruit, seed, leaves, branches, or bark.

Pharmacological background

Inflammation is a protective response of the organism to eliminate injurious stimuli and to initiate the healing process. NF-[kappa]B plays a central role in inflammation as transcription factor, since it regulates the expression of various pro-inflammatory and proliferative genes such as IL-1 (interleukin-1), TNF-[alpha] (tumor necrosis factor-[alpha]), IFN (interferon) and COX-2 (cydooxygenase-2) upon response to carcinogens, growth factors and inflammatory stimuli (Gasparini and Feldmann 2012; Luqman and Pezzuto 2010). Over-expression of NF-[kappa]B in various types of cancer is referred as an important factor for upregulated cell proliferation and cancer progression (Dhanalakshmi et al. 2002; Ghosh and Hayden 2008).

Z. spina-christi protects against carbon tetrachloride-induced liver fibrosis and severe inflammation. Carbon tetrachloride induces oxidative stress and is widely used to study liver fibrosis in rats. The anti-oxidative and anti-inflammatory effects of Z spina-christi on liver fibrosis depend on increased superoxide dismutase (SOD), catalase (CAT) and decreased myeloperoxidase (MPO) activity (Amin and Mahmoud-Ghoneim 2009). Z spina-christi diminished TGF-[[beta]1, which is a target gene of NF-[kappa]B and a potent inductor of fibrosis (Garcia et al. 2002). Its leaf extracts improved glucose utilization in diabetic rats via increased insulin secretion, which is possibly due to their saponin and polyphenol content. Moreover, hyperglycemia is diminished through decreased meal-derived glucose absorption, decreased hepatic glucose-6-phosphatase and increased glucose-6-phosphate dehydrogenase activities together with [alpha]-amylase inhibition (Michel et al. 2011). Fruit extracts of Z. spina-christi protected against aflatoxin B1-induced oxidative stress and hepatocarcinogenesis in Sprague-Dawley rats possibly due to the decreased DNA damage by activating the phase II enzymes glutathione S-transferase (GST) and GSH peroxidase (GSH-Px) (Abdel-Wahhab et al. 2007). Fruit extracts also inhibited early stages of colon carcinogenesis by preventing oxidative stress and inducing apoptosis and prevent aberrant cryptic foci development in azoxymethane-treated Sprague-Dawley rats (Guizani et al. 2013). Leaf extracts were cytotoxic toward HeLa (cervical cancer) and MDA-MB-468 (breast cancer) cell lines (Jafarian et al. 2014). Z. spina-christi honey possesses cytotoxic activity toward HCT-116 (colon cancer), HTB-26 (breast cancer) and HepG2 (liver cancer) (El-Gendy 2010).

Aim of the study

In this study, we validated the ancient knowledge regarding anti-inflammatory effects of Z. spina-christi originated from ancient Egyptian prescriptions and identified its active compounds with anti-inflammatory activity toward leukemia cells. Thus, selected compounds found in Z. spina-christi were investigated in terms of their anti-inflammatory effect via inhibition of the NF-[kappa]B pathway and their interaction with NF-[kappa]B, 1-[kappa]K ([kappa]B kinase) -NEMO (NF-[kappa]B essential modulator) complex and I-[kappa]K by molecular docking. Epigallocatechin, gallocatechin, spinosin, 6"' feruloylspinosin and 6'" sinapoylspinosin exerted anti-inflammatory activity. Western blot experiments yielded supportive results. Using EMSA, we corroborated the extracts to affect the DNA-binding activity of NF-[kappa]B. Molecular docking studies on NF-[kappa]B pathway proteins demonstrated that epigallocatechin, gallocatechin, spinosin, 6"' feruloylspinosin and 6"' sinapoylspinosin shared comparable docking poses and binding energies with MG-132, a known NF-[kappa]B inhibitor. We examined the interrelationship of the IC50 values of two of the ingredients of Z. spina-christi (gallocatechin and epigallocatechin) in a panel of 60 cell lines of the National Cancer Institute, USA. The mean [log.sub.50] [IC.sub.50] values of gallocatechin and epigallocatechin were correlated with the baseline mRNA expression levels of 79 genes in the panel of cell lines of the NCI, USA.

Materials and methods

Chemicals and extracts

Plant material of Z. spina-christi was collected from a local market in Sudan, Khartoum on February 2012. The botanical identity was verified by one of the authors (H.K.). Voucher specimens are stored at the Department of Pharmaceutical Biology, University of Mainz, Germany (registration no. 1907). 2. spina-christi seed extracts were macerated separately in dichloromethane-DcM, ethyl acetate-EA and 80% methanol-Met. Leaf, root and stem extracts were macerated in DcMrMet (1:1). Powdered plant extracts were air-dried. Stocks (10mg/ml) were prepared in dimethyl sulfoxide (DMSO) and stored at -20 [degrees]C. The plant name (Ziziphus spinachristi (L) Desf.) has been verified at www.theplantlist.org. Gallocatechin (purity [greater than or equal to] 99%) (PubChem C1D:65084) and epigallocatechin (purity [greater than or equal to] 99%) (PubChem CID:72277) were purchased from Enzo Life Sciences GmbH (Lorrach, Germany). 6'" feruloylspinosin (PubChem CID: 21597353), 6"' sinapoylspinosin (PubChem CID: 44258337) and spinosin (PubChem CID:155692) were identified via HPLC-MS from Ziziphus seed extract prepared by using 80% methanol. Stock solutions (10 mM) were prepared in DMSO for epigallocatechin, gallocatechin and MG-132 whereas 10 mM stock solutions were prepared in Met for spinosin, 6"' sinapoylspinosin and 6"' feruloylspinosin. Stock solutions were stored at -20[degrees]C. TNF-[alpha] was purchased from Sino Biological Inc (Beijing, China). A 100 [micro]g/ml stock solution was prepared in sterile double distilled water and the aliquots were stored at -20 [degrees]C.

HPLC fractionation and profiling of crude extracts

The crude extract of Ziziphus spina-christi was analyzed by high pressure liquid chromatography (HPLC, Agilent 1100 Series) equipped with a LiChrospher RP 18 column (3 x 125 mm; 5 [micro]m, Merck, Darmstadt, Germany). The column was used at 40 [degrees]C and a flow rate of 1 ml/min. An elution gradient was used composed of [H.sub.2]O + 0.1% (v/v) trifluoroacetic acid and acetonitrile, starting from 100% [H.sub.2]O + 0.1% (v/v) trifluoroacetic acid to 100% acetonitrile over a period of 23 mins. The compounds were detected via a diode array detector. For separation of the crude extract, we used the same method with a subsequent located fraction collector (Agilent 1100 Series). The use of 96-well plates for collecting the flow resulted in 92 different fractions of 250 [micro]l each. These fractions could be used in biological assays.

The molecular weight of the selected peaks was determined using a HPLC-MS (Agilent 1260 Series LC and 6130 Series Quadrupole MS System, Agilent, Santa Clara, CA, USA). The mass spectra were recorded using atmospheric pressure chemical ionization (APCI) with positive and negative polarization. A Superspher RP 18 (125 x 2mm; 4 [micro] m, Merck) column was used at 40 [degrees]C. For every run 1 [micro]l of a sample at a concentration of 1 mg/ml was injected. The elution was performed with a gradient of [H.sub.2] 0+0.1% (v/v) formic acid and acetonitrile and a flow rate of 0.45 ml/min. The 3D-tool of the program Chemstation (Agilent) was used to create 3D graphics.

Cell culture

Human Jurkat T leukemia cells were obtained from the Institute of Pharmaceutical Sciences (Albert-Ludwigs-University Freiburg, Germany). Cells were maintained under standard conditions (37[degrees]C, 5% C[O.sub.2]) in RPMI 1640 medium (Gibco BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (100 U/ml penicillin, 100[micro]g/ml streptomycin). Cells were passaged twice weekly. All experiments were performed with cells in the logarithmic growth phase (~70% confluency).

Resazurin assay

Z. spina-christi extracts (10 [micro]g/ml) were evaluated in terms of their cytotoxicity toward Jurkat T cells by resazurin assay after 72 h treatment. Cell viabilities were evaluated as stated before (O'Brien et al. 2000; Saeed et al. 2015).

Electromobility shift assay (EMSA)

We tested different time points, 72 h treatment with the compounds yielded the best results, therefore we conducted our experiments accordingly. Jurkat T cells (100,000 cells) were plated in 5 ml wells. After 24 h the cells were treated with 4 ng/ml TNF-[alpha] alone for 1 h and then treated for 72 h with the compound (10[micro]M) or the extract (10 [micro]g/ml). As a control, cells left untreated or were only treated for 73 h with 4 ng/ml TNF-[alpha]. Subsequently, the cells were harvested by centrifugation and the total NF-[kappa]B protein extract was prepared. The extract was incubated with labelled oligonucleotide ([sup.33]P-labeled ATP), which contains the NF-[micro]B binding sequence and separated by electrophoresis. After drying the gel, a Phosphorlmager was used to detect the labelled NF-[micro]B oligonucleotide complex. Only the active NF-[kappa]B can bind to labelled oligonucleotide, not the inactive complex. Detailed conditions are described (Lyss et al. 1998).

Western blotting

Jurkat T cells were treated by the following approach: 1 h TNF-[alpha] induction followed by 72 h treatment with indicated concentrations of compounds or 10[micro]g/ml extracts. TNF-[alpha] is kept for a total of 73 h. Cytoplasmic and nuclear protein extracts were prepared with NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific, Rockford, USA) supplemented with EDTA-Free Halt Protease Inhibitor Cocktail (Thermo Scientific) according to the manufacturer's protocol. Protein concentrations were determined by triplicate Nanodrop measurement. Nuclear p65 levels were determined using rabbit anti-NF-[kappa]B p65 polyclonal antibody (1:1000; Thermo Scientific). Histone H3 protein levels determined with rabbit anti-histone H3 polyclonal antibody (1:2000; Cell Signaling, Danvers, USA) served as loading control. MG-132 (0.1 [micro]M) was used as the positive control. DMSO was used as control for epigallocatechin, gallocatechin and MG-132, methanol was used as control for spinosin, 6"' sinapoylspinosin and 6"' feruloylspinosin. Quantification of the protein band intensities were performed by ImageJ software (http://imagej.nih.gov/ij/). Normalized p65 protein levels and percentage protein levels were calculated for each experiment. Mean [+ or -] standard deviation (SD) values were provided, significance was evaluated with student's t-test (two tails and unequal variance).

Molecular docking

Molecular docking was performed with AutoDock 4 on target proteins to identify potential anti-inflammatory Ziziphus compounds. VMD and AutoDock bioinformatics tools were used to yield images. The compounds identified by HPLC analysis from Z. spina-christi were selected for molecular docking calculations. MG-132 was used as the control compound. The selected proteins, their PDB ID's, target regions on the proteins and their relevant residues for the dockings are represented in Table 5. Grid parameters for dockings are depicted in Table 6. Defined molecular docking with 2,500,000 energy evaluations and 250 runs covering the regions of interest as shown in Table 6 were performed three times and the average of the lowest binding energies, mean binding energies and predicted inhibition constants were taken into account.

Correlation analysis of microarray data

The mRNA microarray hybridization of the NCI cell lines has been reported and deposited at the NCI website (http://dtp.nci.nih.gov) (Amundson et al. 2008; Scherf et al. 2000). The compilation of genes involved in inflammatory processes is based on the PCR assays from SABiosciences (Qiagen, Hilden, Germany). Correlations coefficients (R-values) and significance values (P-values) were calculated from [log.sub.10] [IC.sub.50] values of gallocatechin and epigallocatechin and microarray-based mRNA expression values by using Pearson's correlation test as relative measure for the linear dependency of two variables. This test was implemented into the WinSTAT Program (Kalmia Co, MA, USA).

Statistical tests

In order to evaluate the statistical significance, student's t test was applied with two tails and unequal variance. Experiments yielding p values lower than 0.05 were accepted as statistically significant.

Results

HPLC profiling

3D HPLC-peaks were prepared for root, seed (Met) and stem extracts. The 3D graphics for root (Fig. 1A), seed (Met) (Fig. IB) and stem (Fig. 1C) extracts depicted 6"' feruloylspinosin, 6'" sinapoylspinosin, spinosin, epigallocatechin and gallocatechin presence within the extracts. The ratios of the pure compound within the extracts were estimated by areas of the UV spectra:

Seed (Met): 15,2% of extract is spinosin; 4,6% of extract is 6"' sinapoylspinosin and 9,7% of extract is 6'" feruloylspinosin.

Root: 1,0% of extract is gallocatechin and 6,8% of the extract is epigallocatechin.

Stem: 2,3% of the extract is gallocatechin and 6,2% of the extract is epigallocatechin.

The approximate concentrations of compounds in 10[micro]g/ml seed (Met) extract: 2.50 [micro]M spinosin, 0.56 [micro]M 6'" sinapoylspinosin, 1.24 p,M 6'" feruloylspinosin.

The approximate concentrations of compounds in 10 [micro]g/ml root extract: 0.33 [micro]M gallocatechin, 2.22 [micro]M epigallocatechin.

The approximate concentrations of compounds in 10 [micro]g/ml stem extract: 0.75 [micro]M gallocatechin, 2.03 [micro]M epigallocatechin.

Resazurin assay

Z. spina-christi extracts (10 [micro]g/ml) did not show cytotoxicity toward Jurkat T cells. The cell viability graph is depicted in Fig. 2.

EMSA

The involvement of NF-[kappa]B in the anti-inflammatory effects of the extracts and compounds was validated by EMSA in terms of their ability to inhibit its DNA binding. Root and seed (DcM) treatments caused a slight inhibition in the range of 15-20% as shown in Fig. 3. No effect was observed for 6'" feruloylspinosin, 6"' sinapoylspinosin, spinosin and gallocatechin (data not shown).

Western blotting

The effect of the extracts and compounds on NF-[kappa]B was validated by Western blot in terms of their ability to inhibit the nuclear translocation of NF-[kappa]B-p65. Extracts induced p65 inhibition in the following order: seed (DcM) (39.0% [+ or -] 5.8%), stem (36.4% [+ or -] 7.7%), root (32.7% [+ or -] 4.7%), seed (EA) (26.4% [+ or -] 8.3%), leaf (24.2% [+ or -] 6.9), and seed (Met) (15.4% [+ or -] 4.5%). Compounds induced p65 inhibition in the following order: 10 [micro]M 6'" sinapoylspinosin (34.8% [+ or -] 11.2%), 10 [micro]M gallocatechin (28.7% [+ or -] 3.3%), 10 [micro]M epigallocatechin (28.2% [+ or -] 3.6%), 10 [micro]M spinosin (25.2% [+ or -] 7.7%), 1 [micro]M 6"' feruloylspinosin (23.2% [+ or -] 2.3%). MG-132 (0.1 [micro]M) induced an inhibition of (36.9% [+ or -] 10.1%). The results from three independently repeated Western blots are summarized in Fig. 4.

Molecular docking

In order to further validate the anti-inflammatory activity of the selected compounds, in silico molecular docking calculations were conducted on NF-[kappa]B pathway proteins. The residues that the compounds form hydrogen bond with and residues at the pharmacophore regions mentioned at Table 5 are labeled bold. The molecular docking results are summarized in Table 7. The docking poses of the compounds are depicted in Fig. 5. All compounds showed the highest affinity on the NF-[kappa]-DNA complex with a binding energy of -8.98 [+ or -] 0.72 kcal/mol for 6"' sinapoylspinosin, -8.91 [+ or -] 0.01 kcal/mol for epigallocatechin, -8.44 [+ or -] 0.01 kcal/mol for gallocatechin, -8.02 [+ or -] 0.16 kcal/mol for spinosin, -7.92 [+ or -] 0.59 kcal/mol for 6"' feruloylspinosin. MG-132 revealed the highest affinity with a binding energy of 10.25 [+ or -] 0.09 kcal/mol on the NF-[kappa]B-DNA complex. The compounds formed hydrogen bonds with the bound DNA, but not with the amino acid residues. Epigallocatechin and gallocatechin formed hydrogen bonds with residues at the ATP binding site of I-[kappa], as does MG-132. Taken together, the compounds docked to similar sites on target proteins with comparable binding energies as MG-132.

Correlation analysis of microarray-based mRNA expression profiling

The mean [log.sub.10] [IC.sub.50] values of gallocatechin and epigallocatechin were correlated with the baseline mRNA expression levels of 79 genes in the panel of cell lines of the NCI, USA, represented by 507 different microarray hybridization experiments. These genes have been selected, because they are involved in inflammatory processes. The mRNA expression of 17 genes represented by 19 different DNA clones significantly correlated to the [log.sub.10] [IC.sub.50] values for gallocatechin or epigallocatechin with a significant level of P < 0.05 and a correlation coefficient of R < -0.2 and R > 0.2, respectively (Table 8).

Discussion

In this study, we evaluated the effect of NF-[kappa]B being involved in inflammatory processes of selected Ziziphus compounds (epigallocatechin, gallocatechin, spinosin, 6'" feruloylspinosin and 6"' sinapoylspinosin) and crude extracts both in vitro and in silico. We provided evidence that this plant exerts anti-inflammatory activity by inhibiting the NF-[kappa]B pathway. Molecular docking calculations indicated that selected compounds interacted with the NF-[kappa]B pathway proteins with comparable binding energies and similar docking poses as the known inhibitor, MG-132. NF-[kappa]B has a central role in inflammation as transcription factor by regulating the expression of various pro-inflammatory and proliferative proteins such as interleukins, TNF-[alpha], interferons and COX-2 upon response to carcinogens, growth factors and inflammatory stimuli. Overexpression of NF-[kappa]B represents an important factor for upregulated cell proliferation and cancer progression (Dhanalakshmi et al. 2002; Ghosh and Hayden 2008). Moreover, hyperactivity of the NF-[kappa]B pathway may lead to tumorigenesis (Jiang et al. 2012; Jin et al. 2013; Rial et al. 2012; Zhou et al. 2014). Therefore, inhibition of the NF-[kappa]B pathway in cancer cells may be a good strategy to halt the tumor growth and carcinogenesis.

Many compounds of Ziziphus were known to possess anticancer and anti-inflammatory effects, e.g. gallocatechin and epigallocatechin. Gallocatechins are potential inhibitors of 1L-8 expression, and the possible mechanism involves the I[kappa]-K inhibition (Aneja et al. 2004). Epigallocatechin-3-gallate (EGCG) is well known as a green tea polyphenol having a similar structure with epigallocatechin with an additional gallate. It decreased COX-1 activity possibly by stimulating the adenylate cyclase/cAMP/Akinase/VASP-Serl57 phosphorylation pathway (Lee et al. 2013). Secretion of TNF-[alpha], IL-6 and IL-8 was inhibited by EGCG in HMC-1 human mast cells through the attenuation of extracellular signalregulated kinases (ERK) and NF-[kappa]B (Shin et al. 2007). Furthermore, it influences various signaling pathways, including activator protein 1 (AP-1) or the synthesis of eicosanoids and prostaglandins E2 (PGE2) (Porath et al. 2005). EGCG possessed anti-carcinogenic effects in epidemiological and animal studies. Administration of green tea, green tea extract or EGCG reduced tumor formation and growth and also revealed anti-angiogenic and anti-mutagenic effects (Crespy and Williamson 2004; Fujiki et al. 1998; Wang et al. 1989). Three flavonoids (spinosin, 6"' sinapoylspinosin and 6"' feruloylspinosin) previously found in Ziziphus jujuba (Cheng et al. 2000; Kim et al. 2014; Woo et al. 1980) were identified via HPLC-MS for the first time by us in Z. spina-christi seed extract. Spinosin possessed anxiolytic-like effect (Liu et al. 2014), memory-ameliorating effect (Jung et al. 2014) and sleep inducing effect (Wang et al. 2010) by implication on GABA and serotonin systems.

The concentration used for the compounds (10 [micro]M) is not very different from the amounts present in the extracts. In addition, we observed similar effects on the p65 protein level for root, stem and epigallocatechin/gallocatechin. The effect of methanolic seed extract and spinosin compounds was also similar in terms of p65 protein levels implying similar anti-inflammatory effects of separated compounds and extracts. We evaluated these compounds using EMSA and Western blot experiments in terms of their effects on NF-[kappa]B-DNA binding and NF[kappa]B-p65 nuclear translocation, respectively. Epigallocatechin yielded slight decrease of nuclear NF-[kappa]/p65 protein levels. Seed, root and leaf extracts showed slight inhibition of NF-[kappa]B-DNA binding. The spinosin compounds showed inhibitory activity for nuclear p65 protein level.

Further validation of the results for the compounds was performed with in silico molecular docking analyses on NF-[kappa]B pathway proteins. Our results indicated that epigallocatechin, gallocatechin, spinosin, 6"' feruloylspinosin and 6'" sinapoylspinosin strongly bound to NF-[kappa]B and I-[kappa]K with comparable binding energies and similar docking poses as MG-132. Epigallocatechin and gallocatechin also strongly interacted with the I-[kappa]K-NEMO association domain. The fact that the compounds revealed even higher affinities to DNA-bound NF-[kappa]B than free NF-[kappa]B implies inhibition of DNA binding. Indeed, this was supported by EMSA. The observation that these compounds strongly bound to NF-[kappa]B related proteins (I-[kappa]K-NEMO association domain and I-[kappa]K), which are involved in nuclear translocation of NF-[kappa]B indicates that the active compounds of Z spina-christi may target different sites of the NF-[kappa]B pathway to inhibit NF-[kappa]B-mediated inflammation. The docking analyses are not presented as standalone results. Rather, they have been supported by EMSA and western blots. MG-132 is a well-known NF-[kappa]B inhibitor and has been frequently used as control drug (Snyder et al. 2002; Zanotto-Filho et al. 2010; Nakajima et al. 2011) independent of whether this compound acts in a direct or indirect manner. We aimed to evaluate the binding modes of the compounds on NF-kB pathway proteins and used MG-132 as the control compound, since it has been used widely as a known NF-[kappa]B inhibitor. Molecular docking is an established in silico method to perform such studies and evaluate interaction of ligands with target proteins. We indeed observed that the compounds possess similar docking poses and comparable binding energies with those of MG-132. This implies that the compounds may target the NF-[kappa]B pathway proteins. Furthermore, the microarray-based expression of genes involved in inflammatory processes correlated the [log.sub.10][IC.sub.50] values for two compounds of Z. spina-christi, i.e. gallocatechin and epigallocatechin. We focused on these two compounds, only, because other constituents of this plant were not included in the NCI database. The two compounds were correlated with the mRNA expression of a number of inflammation-related genes, indicating that these compounds from Z. spina-christi might indeed affect inflammatory processes.

Our results are compatible with general observations in phytotherapy that medicinal plants act rather in a multifactorial fashion than addressing single targets (Efferth and Koch 2011). This is reasonable with an evolutionary perspective, since multi-target approaches were more effective for plants during evolution than single target approaches in terms of protection from microbials and herbivores. The active ingredients revealed anti-inflammatory effects, albeit in a weak manner. Concentrations higher than 10 [micro]M (for the compounds)/10 [micro]g/ml (for the extracts) will probably yield more satisfactory outputs. Hence, our results serve as a good starting point to further evaluate the effects of those compounds

As suggested by the reading of ancient Egyptian prescriptions. Z. spina-christi revealed anti-inflammatory effects in our experiments correlating with the ancient Egyptian prescriptions. The frequency of topical (external) applications against swellings, pain, and heat as opposed to internal use in ancient Egyptian remedies suggests a potential for future applications to treat inflamed tissues.

Taken together, it was not the intention of this investigation to come up with new compounds. Rather, the concept of the manuscript is to prove, whether a plant described in archeological sources as being anti-inflammatory can be proven as such with modern pharmacological methods. This is an interdisciplinary project between archeological sciences and pharmacy. The confirmation of medical uses described in ancient Egyptian documents by pharmacological and biological methods represents a pioneer for the successful collaboration of the life sciences and the humanities. This is the unique and innovative character of the present project. The fact that the phytochemical determination revealed known inflammatory compounds makes the paper even stronger, because this further supports the correctness of the archeological description of the usefulness of Z. spina-christi. Our intention is to evaluate the effects of an ancient medicinal plant from Egypt, Ziziphus spina Christi, and investigate its anti-inflammatory properties. We found out that this plant might indeed possess anti-inflammatory effects, since it contains active ingredients such as epigallocatechin, gallocatechin and spinosins in that regard.

Our results add experimental evidence to the historical use of Z. spina-christi in ancient Egypt, and to the broader medicinal range of applications by Bedouins and Nubians in Egypt, as well as inhabitants of the Arabian Peninsula, Jordan, Iraq, and Morocco. Further preclinical and clinical studies are warranted to confirm the therapeutic potential of the identified Ziziphus compounds for clinical use.

Conflict of interest

The authors declare that they have no conflict of interest.

http://dx.doi.org/10.1016/j.phymed.2016.01.004

ARTICLE INFO

Article history:

Received 1 December 2015

Revised 8 January 2016

Accepted 16 January 2016

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Onat Kadioglu (a), Stefan Jacob (b), Stefan Bohnert (b), Janine Nass (a), Mohamed E.M. Saeed (a), Hassan Khalid (c), Irmgard Merfort (d), Eckhard Thines (b,f), Tanja Pommerening (e), **, Thomas Efferth (a),*

(a) Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany

(b) Institut fur Biotechnologie und Wirkstoff Forschung gGmbH, Erwin-Schrodinger-Strasse 56, 67663 Kaiserslautern, Germany

(c) Department of Pharmacognosy, University of Khartoum, Khartoum, Sudan

(d) Department of Pharmaceutical Biology and Biotechnology, Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Stefan-Meier-StraJSe 19, 79104 Freiburg, Germany

(e) Department of Egyptology, Institute of Ancient Studies, Johannes Gutenberg University, Hegelstrasse 59, 55122 Mainz, Germany

(f) Institute of Biotechnology and Drug Research, Johannes Gutenberg University, Duesbergweg 10-14, 55128 Mainz, Germany

Abbreviations: COX, cyclooxygenase; DcM, dichloromethane; DMSO, dimethyl sulfoxide; EMSA, electromobility shift assay; HPLC, high pressure liquid chromatography; I[kappa]B, inhibitor of NF-[kappa]B; IKK, I[kappa]B kinase; IL, interleukin; Met, methanol; NEMO, NF-[kappa]B essential modulator; NF-[kappa], nuclear factor kappa-light-chain-enhancer of activated B cells; TNF- a, tumor necrosis factor-[alpha].

* Corresponding author. Tel.: +49 6131 3924322; fax +49 6131-3923752.

** Co-Corresponding author. Tel.+49 0 6131 39 38348; fax: +49 0 6131 39 38338.

E-mail addresses: tpommere@uni-mainz.de (T. Pommerening), efferth@unimainz.de (T. Efferth).

Table 1
Ancient Egyptian papyri with prescriptions containing Ziziphus
spina-christi ('nebes').

Papyrus         Time of record   Content

pRamesseum V    around 1900 BC   Exclusively prescriptions for
                                 metu-vessels: 20 prescriptions

pEdwin Smith    around 1550 BC   Recto: "Book of wounds", style of
                                 language 2300 BC; 48 teaching texts

pEbers          around 1550 BC   Compendium: 44 teaching texts, 28
                                 short teaching versions, 776
                                 prescriptions, 11 prescriptions with
                                 spell, 10 spells with treatment, 3
                                 spells, 4 prognoses, 4 excerpts

pHearst         around 1550 BC   Compendium: 10 abbreviated teaching
                                 texts, 236 prescriptions, 6
                                 prescriptions with spell, 8 spells

pBerlin 3038    around 1250 BC   Compendium: 3 teaching texts, 5
                                 abbreviated teaching texts. 185
                                 prescriptions, 1 prescription with
                                 spell, 1 spells with treatment, 1
                                 spells, 7 prognoses, 1 excerpt

pBrooklyn       around           Book against snake bites 1 prognosis,
47.218.48/.85   600-300 BC       113 prescriptions, 9 prescriptions
                                 with spell, 4 abbreviated teaching
                                 texts, 4 spells, 25 excerpts

Papyrus         Prescriptions with Ziziphus
                (Abbreviations cf. Westendorf
                1999)

pRamesseum V    Ram V, Nr. XII

pEdwin Smith    Sm Fall 48

pEbers          Eb 159, 208, 210, 213, 226, 228,
                272, 479, 480, 536, 582, 616,
                631, 663,766c

pHearst         H 14, 84, 95, 134, 173b, 191,
                221, 226, 238

pBerlin 3038    Bln 131, 140, 141, 153, 159, 168

pBrooklyn       Brk [section] 8a
47.218.48/.85

Table 2
Three examples of ancient Egyptian Ziziphus-containing prescriptions.

Prescription   Medical information

H 134          Driving out illness from all body parts of a man or a
               woman bread of Ziziphus (fruits) in water
               (The body parts) must be bandaged with (this drug).

Eb 536         Healing all things from which a man suffers, namely any
               Setscha' bread of Ziziphus (fruits) must be boiled in
               water (and the 'setscha'-swelling) must be bandaged
               with (this drug) in a pleasant warmth.

Bln 140        Drug for driving out slimy substances (setet), if one
               suffers in any body part in winter Ischedu fruit 1 dja;
               bread of Ziziphus (fruits) 1 dja; oil 1/4 dja; honey
               1/4 dja (The body parts) must be bandaged with (this
               drug).

Table 3
Overview about ancient Egyptian Ziziphus-containing
prescriptions for external use.

                                                           Ziziphus
Code         Indication                                    part

Bln 131      Another (drug) [for driving out swellings     2. Leaves
             in the legs]

Bln 140      Drug for driving out slimy substances         2. Bread 1
             (setet), if one suffers in any body part      dja
             in winter

Bln 141      Another (drug) [for driving out slimy         1. Leaf 1
             substances (setet), if one suffers in any     dja
             body part in winter]

Brk          Drug for snake bite, if it is small           3. Leaf
[section]
87a

Eb 208       Another drug for driving out obstruction      1. Bread
             in the area of the stomach                    1 dose

Eb 213       Another drug for driving out obstruction      1. Bread
             in the area of the stomach                    1 dose

Eb 272       Another (drug) [to control the urine]         1. Wood
                                                           1 dose

Eb 536       Healing all things from which a man           1. Bread
             suffers, namely any 'setscha'-swelling

Eb 582       Another (drug) [for driving out a swelling    4. Fruits
             in any body part]                             1 dose

Eb 616       Beginning of drugs for a finger, if it is     2. Leaf
             ill, or for a toe. Afterwards you should      1/4 dja
             make a drug for him for cooling

Eb 663       Another (drug) for weakness of a              8. Sawdust
             metu-vessel                                   1 dose 12.
                                                           Leaf 1 dose

Eb 766c      If the opening is moist, then you should      2. Leaf
             make a powder for it for drying wounds

H14          Making smooth [a bone if it is broken]        4. Fruits
                                                           1 dose

H 95         Cooling a metu-vessel                         1. Leaf
                                                           1 dose

H 134        Drive out illness from all body parts of a    1. Bread
             man or a woman

H 173b       Drug for treating a finger or a toe           2. Leaf
             Afterwards you should make a cooling drug     1/2 dja
             for him

H 191        Another (drug) [for a nail of a toe]          7. Leaf
                                                           1/8 dja

H 221        Another (drug) [for attaching a bone, if      3. Leaf
             it is broken, on the first day]               1 dose

H 226        Drug for cooling a bone, after it has been    3. Leaf
             attached, in any body part of a man           1 dose

H 238        Another drug for cooling a metu-vessel in     1. Leaf
             any body part                                 1 dose

Ram V Nr.    Cooling a metu-vessel, strengthening          1. Leaves
XII          weakness                                      1 dose

Sm Fall      Therefore you make a remedy for him to        2. Not
41/1         cool and to tow the warmth from the           specified
             opening of the wound

Code         Other ingredients           Preparation       Application

Bln 131      1. Acacia leaves            --                Bandaging
             3. Ochre
             4. Honey

Bln 140      1. Ischedu-Fruit l dja      --                Bandaging
             3. Oil 1/4 Dja
             4. Honey 1/4 dja

Bln 141      2. Khet-des-tree leaf 1     --                Bandaging
             dja
             3. Mash 1/4 dja
             4. Rind fat 1/2 dja
             5. Conifer sawdust

Brk          1. Acacia leaves            Grinding          Powdering
[section]    2. Ima-tree leaves          finely
87a          4. Ibsa-plant

Eb 208       2. Gourd 1 dose             Making as         Bandaging
             3. Discharge of a           one thing
             tomcat 1 dose
             4. Sweet beer 1 dose
             5. Vine 1 dose

Eb 213       2. Excrements of a tomcat   Making as         Bandaging
             3. Red ochre                one thing
             4. Gourd
             5. Sweet beer
             6. Vine

Eb 272       2. Mesta-liquid, third      1 triturating     Anointing
             part                        in 2              the glans

Eb 536       2. Water                    1 boiling         Bandaging
                                         in 2              in pleasant
                                                           warmth

Eb 582       1. Mortar                   1 grinding        Bandaging
             2. Water of gum 1 dose      with 2 making
             3. Sycomore fruits 1 dose   as one thing
             5. Willow fruits
             6. Mimi-corn

Eb 616       1. Leaf of acacia 1/4 dja   Grinding          Bandaging
             3. Ochre 1/32 dja
             4. Green pigment
             (schesait) from malachite
             1/32 dja
             5. Interior of freshwater
             clam 1/8 dja

Eb 663       37 ingredients              Making as         Bandaging
                                         one thing

Eb 766c      1. Acacia leaf              Grinding          Giving the
             3. Willow fruit                               powder on it
             4. Cumin

H14          1. [] of the builder 1      Sprinkling the    Bandaging
             dose                        fingers with
             2. Gum 1 dose               it in honey
             3. Sycamore fruits 1 dose
             5. Ima-tree fruits [...]
             6. [...]

H 95         2. Willow leaf 1 dose       Grinding finely   Bandaging
             3. Acacia leaf 1 dose       making as one
             4. Dschais-plant 1 dose     thing
             5. North salt 1 dose
             6. Onion 1 dose

H 134        2. Water                    --                Bandaging

H 173b       1. Acacia leaf Vi dja       Grinding          Bandaging
             3. Ochre 1/32 dja
             4. Green pigment
             (schesait) from malachite
             1/32 dja
             5. Interior of freshwater
             clam 1/8 dja

H 191        8 ingredients.              Boiling           Bandaging

H 221        1. Mortar of a builder 1    Making as         Bandaging
             dose                        one thing
             2. Sycamore leaf 1 dose
             4. Ima-tree leaf 1 dose
             5. Acacia leaf 1 dose
             6. Honey 1 dose
             7. Acacia gum

H 226        1. Dscharet pulp 1 dose     --                Bandaging
             2. Ima-tree leaf 1 dose
             4. Sycamore leaf 1 dose
             5. Mimi-corn 1 dose
             6. Water 1 dose

H 238        2. Willow leaf              Grinding          Bandaging
             3. Acacia leaf              finely
             4. North salt
             5. Leek fruits

Ram V Nr.    2. Acacia leaves 1 dose     Beating           Bandaging
XII          3. Honey 1 dose             with honey

Sm Fall      1. Willow leaves            --                Giving
41/1         3. Quesenti-mineral

Explanations:
[] these parts are amended due to the headings introducing the
respective group of prescriptions.

The ingredients have been registered only for drugs with a maximum of
7 drugs.

For measurements (dja/oipe) cf. Pommerening 2010b

Table 4
Overview about ancient Egyptian Ziziphus-containing prescriptions for
internal use.

                                                           Ziziphus
Code      Indication                                       part

Bln 153   Know-how for (healing the) migration of many     7. bread
          wechedu-substances in his body parts Then        1/8 dja
          you make remedies for him to kill the
          wechedu-substances, and remedies for (healing
          the) migration of wechedu-substances in the
          belly

Bln 159   Another (drug) [for a man (with) an inflation    3. leaf
          in his belly. To rub out his nourishment]        1/8 dja

Bln 168   Drug for removing wechedu-substances             4. leaf
                                                           1/8 dja

Eb 159    Another (drug) for cooling, the art of the       3. leaf
          physician                                        1 dose

Eb 210    Another (drug) for driving out obstruction in    15. leaves
          the right side, when it extinguishes             1/32 dja

Eb 226    Another (drug) [for driving out aaa-semen of a   8. bread
          god or a dead man from the belly of a man]       1/16 dja

Eb 228    Another (drug) [for driving out aaa-semen        3. bread
          from the heart, driving out forgetfulness from   1/16 dja
          the heart/mind, fleeing from the heart/mind
          and injury from the heart/mind]

Eb 479    Another (drug) [for treating the liver]          3. pulp of
                                                           the fruits
                                                           1/8 dja

Eb 480    Another (drug) (for treating the liver]          4. bread
                                                           1/8 dja

Eb 631    Another (drug) for treating metu-vessels in      13. bread
          the left side                                    1/8 dja

H 84      Another (drug) [for driving out aaa-semen of a   9. bread
          god or a dead man from the belly of a man]       1/16 dja

Code      Other ingredients          Preparation       Application

Bln 153   1. Fat living meat 1 dja   Grinding finely   Eating from
          2. Inenek-plant 1/8 dja    and boiling       the man with
          3. Celery from foreign     making as         sweet beer
          land 1/16 dja              schait-cake
          4. Incense 1/64 dja
          5. Fresh bread 1/64 dja
          6. Sechpet-liquid 1/16
          oipe

Bln 159   1. Behen-oil 1/8 dja       Spending the      Pouring into
          2. Honey 1/8 dja           night in the      the hinder
          4. Acacia leaf 1 /8 dja    dew beating       part
          5. Khet-ds leaf 1/8 dja.   with water

Bln 168   1. Fresh behen-oil 1/8     --                Pouring into
          dja                                          the hinder
          2. Honey 1/8 dja                             part
          3. Acacia leaf 1/8 dja
          5. Khet-ds-tree 1/8 dja
          6. Sweet beer
          1/16+1/64 oipe

Eb 159    1. Water of                --                Pouring into
          dscharet-plant 1 dose                        the hinder
          2. Acacia leaf 1 dose                        part
          4. Mehui-liquid

Eb 210    17 ingredients             --                Drinking

Eb 226    10 ingredients             Straining         Drinking before
                                                       going to bed

Eb 228    1. Grapes 1/16 dja         [Spending the     [Drinking]
          2. Chufa 1/8 dja           night in the
          4. Ibu-plant 1/16 dja      dew]
          5. Celery 1/32 dja
          6. Inset-plant 1/16 dja
          7. Water 1/16 oipe

Eb 479    8 ingredients              [Spending the     Drinking
                                     night in the
                                     dew] straining

Eb 480    10 ingredients             spending the      Drinking
                                     night in the
                                     dew straining

Eb 631    15 ingredients             Spending the      Drinking
                                     night in the
                                     dew straining

H 84      11 ingredients             Spending the      Drinking
                                     night in the
                                     dew

Explanations:

[] these parts are amended due to the headings introducing the
respective group of prescriptions.

The ingredients have been registered only for drugs with a maximum of
7 drugs

For measurements (dja/oipe) cf. Pommerening 2010b

Table 5
NF-[kappa]B and I[kappa]K protein structures used for molecular
docking studies.

Target proteins                    PDB ID   Target region

NF-[kappa]B p52-RelB complex       3D07     DNA binding site
(Fusco et al. 2009)

NF-[kappa]B p50-p65 heterodimer    1VKX     Bound DNA and DNA
complexed to kappa B DNA                    binding site
(5'-TGGGGACTTTCCAGGAAAGTCCCC-3')
(Chen et al. 1998)

I[kappa]K-NEMO association         3BRT     I[kappa]K-NEMO interaction
domain (Rustle et al. 2008)                 site

I[kappa]K (Xu et al. 2011)         3RZF     ATP binding site

Target proteins                    Relevant residues in that region

NF-[kappa]B p52-RelB complex       on p52 : Arg52, Arg54, Tyr55, Cys57,
(Fusco et al. 2009)                Glu58, Ser61, His62, Thr142, Lysl43,
                                   Lys252, Cln254, Lys255, Gln284 on
                                   RelB : Argll7, Argll9, Tyrl20,
                                   Cysl22, Glul23, Argl25, Ser129,
                                   Arg209, Lys210, Gln307, Lys308,
                                   Arg333, Cln334

NF-[kappa]B p50-p65 heterodimer    on p50 : Arg54, Arg56, Tyr57, Cys59,
complexed to kappa B DNA           Glu60, His64, Cly65, Cly66, Lysl44,
(5'-TGGGGACTTTCCAGGAAAGTCCCC-3')   Lysl45, Lys241, Lys272, Gln274,
(Chen et al. 1998)                 Lys275, Arg305, Arg306 on p65 :
                                   Arg33, Arg35, Tyr36, Cys38, Glu39,
                                   Lysl22, Lysl23, Argl87, Lys218,
                                   Gln220, Lys221, Arg246, Gln247

I[kappa]K-NEMO association         on I[kappa]K : Phe734, Leu737,
domain (Rustle et al. 2008)        Asp738, Trp739, Ser740, Trp741 on
                                   NEMO : Leu51, Cys54, Asn58, Leu61,
                                   Ile65, Phe97, Vall04, Leul07

I[kappa]K (Xu et al. 2011)         Gly24, Phe26, Lys44, Glu97, Cys99,
                                   Lysl47, llel65, Aspl66

Table 6
Grid parameters used for molecular docking studies.

                    Spacing   Axis
                              X         y         z

NF-[kappa]B and NF-[kappa]B-DNA complex:

Number of points     0.375    114        94        90
Grid center                    27.376    60.420    75.564

I[kappa]K-NEMO:
Number of points     0.664    126       104       126
Grid center                     5.385    15.537     4.597

I[kappa]K:
Number of points     0.419     98        80       104
Grid center                    88.492   -29.695    56.299

Table 7
Molecular docking results for the selected Ziziphus compounds.

                          Lowest binding       Mean binding energy
                         energy (kcal/mol)         (kcal/mol)

NF-[kappa]B (RelB/p52):

MC132                   -6.05 [+ or -] 0.38    -5.34 [+ or -] 0.80

Hpigallocatechin        -7.20 [+ or -] 0.02    -6.71 [+ or -] 0.37

Gallocatechin           -6.71 [+ or -] 0.08    -6.16 [+ or -] 0.26

6"' Feruloylspinosin    -5.00 [+ or -] 0.82    -4.70 [+ or -] 0.70

6"' Sinapoylspinosin    -4.95 [+ or -] 0.16    -4.79 [+ or -] 0.43

Spinosin                -4.92 [+ or -] 0.21    -4.78 [+ or -] 0.33

NF-[kappa]B-DNA (p65/p50):

MG132                   -10.25 [+ or -] 0.09   -9.62 [+ or -] 0.02

Epigallocatechin        -8.91 [+ or -] 0.01    -8.32 [+ or -] 0.10

Gallocatechin           -8.44 [+ or -] 0.01    -7.97 [+ or -] 0.02

6"' Feruloylspinosin    -7.92 [+ or -] 0.59    -7.41 [+ or -] 0.44

6"' Sinapoylspinosin    -8.98 [+ or -] 0.72    -8.98 [+ or -] 0.72

Spinosin                -8.02 [+ or -] 0.16    -7.55 [+ or -] 0.44

I[kappa]K-NEMO:

MG132                   -3.98 [+ or -] 0.37    -3.83 [+ or -] 0.64

Epigallocatechin        -7.26 [+ or -] 0.07    -6.50 [+ or -] 0.14

Gallocatechin           -7.49 [+ or -] 0.03    -7.05 [+ or -] 0.09

6"' Feruloylspinosin    -3.00 [+ or -] 0.35    -3.00 [+ or -] 0.35

6"' Sinapoylspinosin    -3.62 [+ or -] 0.31    -3.62 [+ or -] 0.31

Spinosin                -3.63 [+ or -] 0.52    -3.39 [+ or -] 0.82

I[kappa]K:

MG132                   -6.26 [+ or -] 0.20    -5.42 [+ or -] 0.24

Epigallocatechin        -7.15 [+ or -] 0.02    -6.52 [+ or -] 0.02

Gallocatechin           -7.05 [+ or -] 0.02    -6.53 [+ or -] 0.03

6"' Feruloylspinosin    -6.20 [+ or -] 0.53    -6.20 [+ or -] 0.53

6"' Sinapoylspinosin    -6.44 [+ or -] 0.75    -6.00 [+ or -] 0.60

Spinosin                -5.82 [+ or -] 0.34    -3.91 [+ or -] 0.19

                        Residues forming       pKi ([micro]M)
                        H-bond

NF-[kappa]B (RelB/p52):

MC132                   on RelB: Tyrl20,       42.41 [+ or -] 29.46
                        Lys210

Hpigallocatechin        on RelB: Clyll5,       5.31 [+ or -] 0.15
                        Argll7, Argl19.
                        Serl29, Ilel30,
                        Lys273

Gallocatechin           on p52: Arg52.         12.08 [+ or -] 1.61
                        Asp219, Lys221,
                        Lys252

6"' Feruloylspinosin    on RelB: Argll7,       424.69 [+ or -] 567.52
                        Clul23, Clu238

6"' Sinapoylspinosin    on RelB: Asn242,       242.05 [+ or -] 69.83
                        Lys274, Lys305

Spinosin                on RelB: Hisl74.       255.20 [+ or -] 78.70
                        Arg209

NF-[kappa]B-DNA (p65/p50):

MG132                   on p65: Lysl23         0.03 [+ or -] 0.01

Epigallocatechin        --                     0.29 [+ or -] 0.01

Gallocatechin           --                     0.65 [+ or -] 0.01

6"' Feruloylspinosin    --                     2.04 [+ or -] 1.36

6"' Sinapoylspinosin    on p65: Lysl23,        0.44 [+ or -] 0.52
                        Argl87, Gln220

Spinosin                --                     1.36 [+ or -] 0.38

I[kappa]K-NEMO:

MG132                   on NEMO: Arg87         1370.17 [+ or -] 927.11
                        on I[kappa]K: Asp725

Epigallocatechin        on NEMO: Glu89         4.79 [+ or -] 0.62
                        on I[kappa]K: Asp725

Gallocatechin           on NEMO: Gln86,        3.26 [+ or -] 0.16
                        Glu89 on I[kappa]K:
                        Asp725

6"' Feruloylspinosin    on NEMO: Gln83
                                               7080.00 [+ or -] 4323.93

6"' Sinapoylspinosin    on NEMO: Gln83 on
                        I[kappa]K: Asp725      2410.00 [+ or -] 1076.98

Spinosin                on NEMO: Arg87,
                        Clu89, Lys90           2675.22 [+ or -] 1633.33

I[kappa]K:

MG132                   Cys99, Aspl03          26.75 [+ or -] 8.90

Epigallocatechin        Thr23, Glu97, Cys99,   5.79 [+ or -] 0.17
                        Aspl03, Ile165

Gallocatechin           Thr23, Glu97,          6.83 [+ or -] 0.20
                        Aspl03, Ilel65

6"' Feruloylspinosin    Glul9, Thr23,          35.04 [+ or -] 21.68
                        Lysl47, Thrl85

6"' Sinapoylspinosin    Arg31, GlulO0,         29.44 [+ or -] 27.62
                        Glul49, Aspl66

Spinosin                Arg20, Leu21, Arg31,   60.06 [+ or -] 35.69
                        Glu97, Aspl03

Table 8
Microarray-based expression of genes correlating with [log.sub.10]
[IC.sub.50] values of gallocatechin and epigallocatechin in the NCI
panel of cell lines.

Pearson correlation test         Gene

          Gallo-     Epigallo-
          catechin   catechin    Symbol

Chemokines and chemokine receptors:

R-value     0,095    * 0,301     C5
P-value     0,250    * 0,013
R-value     0,095    * 0,302     C5
P-value     0,250    * 0,013
R-value     0,297      0,215     CCL13
P-value     0,015      0,059
R-value     0,244      0,176     CCL22
P-value     0,039      0,102
R-value    -0,258     -0,340     CCL5
P-value     0,032    * 0,006
R-value     0,198    * 0,303     CCL7
P-value     0,077    * 0,013
R-value    -0,063     -0,263     CCL7
P-value     0,327    * 0,027
R-value     0,219    * 0,253     CCL8
P-value     0,057    * 0,032
R-value     0,391    * 0,316     CX3CL1
P-value     0,002    * 0,010
R-value     0,333    * 0,294     CX3CL1
P-value   * 0,007    * 0,015
R-value   * 0,330      0,167     CX3CL1
P-value     0,008      0,114
R-value   * 0,277    * 0,277     CX3CL1
P-value   * 0,022    * 0,021
R-value   * 0,358    * 0,339     CX3CL1
P-value   * 0,004    * 0,006
R-value   * 0,382    * 0,304     CXCL13
P-value   * 0,002    * 0,013
R-value    -0,103     -0,276     CCR3
P-value     0,231    * 0,022

Interleukins and interleukin receptors:

R-value   * 0,258    * 0,275     1L16
P-value   * 0,031    * 0,022
R-value   * 0,231    * 0,289     IU7c
P-value   * 0,048    * 0,017
R-value     0,147    * 0,296     ILlb
P-value     0,146    * 0,015
R-value   * 0,296      0,112     IL1M
P-value   * 0,016      0,211
R-value    -0,231     -0,201     IL1M
P-value   * 0,048      0,073
R-value    -0,266     -0,219     IL33
P-value   * 0,027      0,056
R-value    -0,296     -0,253     IL7
P-value   * 0,016    * 0,032
R-value     0,158      0,287     ILIORB
P-value     0,129    * 0,018
R-value   * 0,263     -0,043     IL9R
P-value   * 0,029      0,378

Other inflammation-related cytokines and receptors:

R-value    -0,465     -0,383     FASLG
P-value     0,001    * 0,002
R-value   * 0,282      0,226     MIF
P-value   * 0,020      0,050
R-value   * 0,244      0,160     NAMPT
P-value   * 0,039      0,123
R-value   * 0,229      0,192     NAMPT
P-value   * 0,050      0,082
R-value    -0,244     -0,176     TNFSF10
P-value   * 0,039      0,102
R-value    -0,242     -0,180     TNFSF10
P-value   * 0,041      0,096
R-value     0,254    * 0,286     TNFSF13
P-value   * 0,033    * 0,018
R-value     0,270      0,252     TNFSF13
P-value   * 0,025    * 0,033
R-value   * 0,280    * 0,283     TNFSF13
P-value   * 0,021    * 0,019
R-value   * 0,375    * 0,366     TNFSF13
P-value     0,003    * 0,003
R-value    -0,404   * -0,406     VEGFA
P-value   * 0,001    * 0,001

Gene name                        Genebank    Experimental   Microarray
                                 Accession   ID no.         Platform

Complement component 5           H52518      GC13136        Stanford

Complement component 5           W80640      GC17567        Stanford

Chemokine (C-C motif)            AJ001634    GC27857        Affymetrix
ligand 13                                                   U95Av2

Chemokine (C-C motif)            NM_002990   GC182172       Affy U133
ligand 22

Chemokine (C-C motif) ligand 5   M21121      GC89615        Affymetrix
                                                            U95

Chemokine (C-C motif) ligand 7   AA040170    GC18974        Stanford

Chemokine (C-C motif) ligand 7   NM_006273   GC184544       Affy U133

Chemokine (C-C motif) ligand 8   Y16645      GC101414       Affymetrix
                                                            U95

Chemokine (C-X3-C motif)         U84487      GC32714        Affymetrix
ligand 1                                                    U95Av2

Chemokine (C-X3-C motif)         U84487      GC97784        Affymetrix
ligand 1                                                    U95

Chemokine (C-X3-C motif)         U84487      GC191655       Affy U133
ligand 1

Chemokine (C-X3-C motif)         U84487      GC192669       Affymetrix
ligand 1                                                    U133A/B

Chemokine (C-X3-C motif)         NM.002996   GC234123       Affymetrix
ligand 1                                                    U133A/B

Chemokine (C-X-C motif)          AF044197    GC31541        Affymetrix
ligand 13                                                   U95Av2

Chemokine (C-C motif)            NM.001837   GC181283       Affy U133
receptor 3

Interleukin 16                   AL109669    GC83315        Affymetrix
                                                            U95

Interleukin 17c                  AF152099    GC213907       Affymetrix
                                                            U133A/B

Interleukin 1 [beta]             W47101      GC15780        Stanford

Interleukin 1 receptor           X52015      GC28008        Affymetrix
antagonist                                                  U95Av2

Interleukin 1 receptor           BE563442    GC173484       Affymetrix
antagonist                                                  U133

Interleukin 33                   AB024518    GC151165       Affymetrix
                                                            U133

Interleukin 7                    J04156      GC88604        Affymetrix
                                                            U95

Interleukin 10 receptor          AA044391    GC19091        Stanford
subunit [beta]                                              Affymetrix

Interleukin 9 receptor           NM_002186   GC181555       U133

Fas ligand                       D38122      GC85840        Affymetrix
                                                            U95

Macrophage migration             AA045695    GC10185        Stanford
inhibitory factor

Nicotinamide                     AA045438    GC19397        Stanford
phosphoribosyltransferase

Nicotinamide                     BF575514    GC176400       Affymetrix
phosphoribosyltransferase                                   U133

Tumor necrosis factor            AW474434    GC170228       Affymetrix
superfamily member 10                                       U133

Tumor necrosis factor            NM_003810   GC182763       Affymetrix
superfamily member 10                                       U133

Tumor necrosis factor            AF046888    GC38298        Affymetrix
superfamily member 13                                       U95Av2

Tumor necrosis factor            AF055872    GC56055        Affymetrix
superfamily member 13                                       U95

Tumor necrosis factor            NM_003809   GC182762       Affymetrix
superfamily member 13                                       U133

Tumor necrosis factor            NM_003809   GC232183       Affymetrix
superfamily member 13                                       U133A/B

Vascular endothelial growth      W19225      GC15550        Stanford
factor A

* R > 0.20; P < 0.05
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Author:Kadioglu, Onat; Jacob, Stefan; Bohnert, Stefan; Nass, Janine; Saeed, Mohamed E.M.; Khalid, Hassan; M
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:7EGYP
Date:Mar 15, 2016
Words:10523
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