Evaluating ancient Egyptian prescriptions today: anti-inflammatory activity of Ziziphus spina-christi.
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.
Ancient Egyptian medicinal papyri
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.
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.
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).
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).
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 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).
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.
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.
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.
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).
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.
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).
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.
Received 1 December 2015
Revised 8 January 2016
Accepted 16 January 2016
Abdel-Wahhab, M.A., Omara, E.A., Abdel-Galil, M.M., Hassan, N.S., Nada, S.A., Saeed, A.. el-Sayed, M.M., 2007. Zizyphus spina-christi extract protects against aflatoxin Bl-initiated hepatic carcinogenicity. Afr. J. Tradit. Complement. Altern. Med. / Afr. Netw. Ethnomed. 4, 248-256.
Amin, A., Mahmoud-Ghoneim, D., 2009. Zizyphus spina-christi protects against carbon tetrachloride-induced liver fibrosis in rats. Food Chem. Toxicol. 47, 2111-2119.
Amundson, S.A., Do, K.T., Vinikoor, L.C., Lee, R.A., Koch-Paiz, C.A., Ahn, J., Reimers, M., Chen, Y., Scudiero, D.A., Weinstein, J.N., Trent, J.M., Bittner, M.L, Meltzer, P.S., Fornace Jr, A.J., 2008. Integrating global gene expression and radiation survival parameters across the 60 cell lines of the National Cancer Institute Anticancer Drug Screen. Cancer Res. 68, 415-424.
Aneja, R., Hake, P.W., Burroughs, T.J., Denenberg, A.G., Wong, H.R., Zingarelli, B., 2004. Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats. Mol. Med. 10, 55-62.
Barta, W., 1963. Die aMgyptische Opferliste, von der Friihzeit bis zur griechischromischen Epoche, Berlin (= Munchner Agyptologische Studien, Bd. 3). S. 181, Abb. 4.
Boulos, L., 2000. Flora of Egypt, vol. 2, Cairo.
Chen, F.E., Huang, D.B., Chen, Y.Q., Ghosh, G., 1998. Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA. Nature 391, 410-413.
Cheng, G., Bai, Y.J., Zhao, Y.Y., Tao, J., Lin, Y., Tu, G.Z., Ma, L.B., Liao, N.. Xu, X.J., 2000. Flavonoids from Ziziphus jujuba Mill var. spinosa. Tetrahedron 56, 8915-8920.
Crespy, V., Williamson, G., 2004. A review of the health effects of green tea catechins in in vivo animal models. J. Nutr. 134, 3431S-3440S.
Dafni, A., Levy, S., Lev, E., 2005. The ethnobotany of Christ's Thorn Jujube (Ziziphus spina-christi) in Israel. J. Ethnobiol. Ethnomed. 1, 8.
Dhanalakshmi, S., Singh, R.P., Agarwal, C., Agarwal, R., 2002. Silibinin inhibits constitutive and TNFalpha-induced activation of NF-kappaB and sensitizes human prostate carcinoma DU145 cells to TNFalpha-induced apoptosis. Oncogene 21, 1759-1767.
Efferth, T., Koch, E., 2011. Complex interactions between phytochemicals. The multi-target therapeutic concept of phytotherapy. Curr. Drug Targets 12,122-132.
El-Gendy, M.M.A., 2010. In vitro, evaluation of medicinal activity of Egyptian honey from different floral sources as anticancer and antimycotic infective agents. J. Microb. Biochem. Technol. 02, 118-123.
Fujiki, H., Suganuma, M., Okabe, S., Sueoka, N., Komori, A., Sueoka, E., Kozu, T., Tada, Y., Suga, K., Imai, K., Nakachi, K., 1998. Cancer inhibition by green tea. Mutat. Res. 402, 307-310.
Fusco, A.J., Huang, D.B., Miller, D., Wang, V.Y., Vu, D., Ghosh, G., 2009. NF-kappaB p52:RelB heterodimer recognizes two classes of kappaB sites with two distinct modes. EMBO Rep. 10, 152-159.
Garcia, L., Hernandez, I., Sandoval, A., Salazar, A., Garcia, J., Vera, J., Grijalva, G., Muriel, P., Margolin, S., Armendariz-Borunda, J., 2002. Pirfenidone effectively reverses experimental liver fibrosis. J. Hepatol. 37, 797-805.
Gasparini, C., Feldmann, M., 2012. NF-kappa B as a target for modulating inflammatory responses. Curr. Pharma. Des. 18, 5735-5745.
Gertsch, J., 2009. How scientific is the science in ethnopharmacology? Historical perspectives and epistemological problems. J. Ethnopharmacol. 122, 177-183.
Ghosh, S., Hayden, M.S., 2008. New regulators of NF-kappaB in inflammation. Nat. Rev. Immunol. 8, 837-848.
Guizani, N., Waly, M.I., Singh, V., Rahman, M.S., 2013. Nabag (Zizyphus spina-christi) extract prevents aberrant crypt foci development in colons of azoxymethanetreated rats by abrogating oxidative stress and inducing apoptosis. Asian Pac. J. Cancer Prev. 14, 5031-5035.
Jafarian, A., Zolfaghari, B., Shirani, K., 2014. Cytotoxicity of different extracts of arial parts of Ziziphus spina-christi on Hela and MDA-MB-468 tumor cells. Adv. Biomed. Res. 3, 38.
Jiang, Z., Wu, W., Qian, M.L., 2012. Cellular damage and apoptosis along with changes in NF-kappa B expression were induced with contrast agent enhanced ultrasound in gastric cancer cells and hepatoma cells. Cancer Cell Int. 12, 8.
Jin, R., Sterling. J.A., Edwards, J.R., DeGraff, D.J., Lee, C., Park, S.I., Matusik, R.J., 2013. Activation of NF-kappa B signaling promotes growth of prostate cancer cells in bone. PloS One 8, e60983.
Jung, I.H., Lee, H.E., Park, S.J., Ahn, Y.J., Kwon, G., Woo, H., Lee, S.Y., Kim, J.S., Jo, Y.W., Jang, D.S., Kang, S.S., Ryu, J.H., 2014. Ameliorating effect of spinosin, a C-glycoside flavonoid, on scopolamine-induced memory impairment in mice. Pharmacol. Biochem. Behav. 120, 88-94.
Kim, W.I., Zhao, B.T., Zhang, H.Y., Lee, J.H., Son, J.K., Woo, M.H., 2014. Quantitative and pattern recognition analyses of magnoflorine, spinosin, 6'"-feruloyl spinosin and jujuboside A by HPLC in Zizyphi Semen. Arch. Logic. Res. 37, 1139-1147.
Lee, D.H., Kim, Y.J., Kim, H.H., Cho, H.J., Ryu, J.H., Rhee, M.H., Park, H.J., 2013. Inhibitory effects of epigallocatechin-3-gallate on microsomal cydooxygenase-1 activity in platelets, Biomol. Therap. 21, 54-59.
Liu, J., Zhai, W.M., Yang, Y.X., Shi, J.L., Liu, Q.T., Liu, G.L., Fang, N., Li, J., Guo, J.Y., 2014. GABA and 5-HT systems are implicated in the anxiolytic-like effect of spinosin in mice. Pharmacol. Biochem. Behav. 128C, 41-49.
Luqman, S., Pezzuto, J.M., 2010. NFkappaB: a promising target for natural products in cancer chemoprevention. Phytother. Res. 24, 949-963.
Lyss, G., Knorre, A., Schmidt, T.J., Pahl, H.L., Merfort, I., 1998. The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65. J. Biol. Chem. 273, 33508-33516.
Michel, C.G., Nesseem, D.I., Ismail, M.F., 2011. Anti-diabetic activity and stability study of the formulated leaf extract of Zizyphus spina-christi (L.) Willd with the influence of seasonal variation. J. Ethnopharmacol. 133, 53-62.
Moursi, H., 1992. Die Heilpflanzen im Land der Pharaonen. Agyptisch-Nubische Volksmedizin, Kairo.
Nakajima, S., Kato, H., Takahashi, S., Johno, H., Kitamura, M., 2011. Inhibition of NF-kappaB by MG132 through ER stress-mediated induction of LAP and LIP. FEBS Lett. 585, 2249-2254.
Nicholson, P.T., Shaw, I., 2000. Ancient Egyptian materials and technology. Cambridge University Press, Cambridge; New York, pp. 346-347.
O'Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur.J. Biochem. 267, 5421-5426.
Pommerening, T, 2015. Bdume, Strducher und Friichte in altagyptischen Listen--eine Betrachtung zur Kategorisierung und Ordnung. In: Susanne, D., Erik, M. (Eds.), Die Liste. Ordnungen von Dingen und Menschen in Agypten. Kulturverlag Kadmos, Berlin, pp. 125-166.
Pommerening, T., 2012. Altagyptische Rezepte - Eine diachrone Betrachtung. Geschichte der Pharmazie 64, 33-38.
Pommerening, T., 2010a. Von Impotenz und Migrane - eine kritische Auseinandersetzung mit Obersetzungen des Papyrus Ebers. In: Imhausen, A., Pommerening, T. (Eds.), Writings of Early Scholars in the Ancient Near East, Egypt, Rome and Greece. Translating Ancient Scientific Texts. Beitrage zur Altertumskunde; 286. De Gruyter, Berlin/New York, pp. 153-174.
Pommerening, T., 2010b. Healing measures: dja and oipe in Ancient Egyptian pharmacy and medicine, in: Cockitt, J., David, R. (Eds.), Pharmacy and Medicine in Ancient Egypt. Proceedings of the conferences held in Cairo (2007) and Manchester (2008). (British Archaeological Reports; S2141). Archaeopress, Oxford. pp. 132-137.
Pommerening, T., 2006. Uberlegungen zur Beurteilung der Wirksamkeit altagyptischer Arzneimittel aus heutiger Sicht. In: Zibelius Chen, K., Fischer-Elfert, H.W. (Eds.), Von reichlich agyptischem Verstande. Festschrift fiir Waltraud Guglielmi zum 65. Geburtstag. (Philippika. Marburger altertumskundliche Abhandlungen; 11). Harrassowitz, Wiesbaden, pp. 103-112.
Pommerening, T., 2005. Altagyptische Heilpflanzen - eine Perspektive fiir die moderne Phytotherapie? Zeitschrift fUr Phytotherapie 26, 61-65.
Porath, D., Riegger, C., Drewe, J., Schwager, J., 2005. Epigallocatechin-3-gallate impairs chemokine production in human colon epithelial cell lines. J. Pharmacol. Exp. Ther. 315, 1172-1180.
Rial, N.S., Choi, K., Nguyen, T., Snyder, B., Slepian, M.J., 2012. Nuclear factor kappa B (NF-kappaB): a novel cause for diabetes, coronary artery disease and cancer initiation and promotion? Med. Hypotheses 78, 29-32.
Rushe, M., Silvian, L., Bixler, S., Chen, L.L., Cheung, A., Bowes, S., Cuervo, H., Berkowitz, S., Zheng, T., Guckian, K., Pellegrini, M., Lugovskoy, A., 2008. Structure of a NEMO/IKK-associating domain reveals architecture of the interaction site. Structure 16, 798-808.
Saeed, M.E., Abdelgadir, H., Sugimoto, Y., Khalid, H.E., Efferth, T., 2015. Cytotoxicity of 35 medicinal plants from Sudan towards sensitive and multidrug-resistant cancer cells. J. Ethnopharmacol 174, 644-658.
Scherf, U., Ross, D.T., Waltham, M., Smith, L.H., Lee, J.K., Tanabe, L., Kohn, K.W., Reinhold, W.C., Myers, T.G., Andrews, D.T., Scudiero, D.A., Eisen, M.B., Sausville, EA, Pommier, Y., Botstein, D., Brown, P.O., Weinstein, J.N., 2000. A gene expression database for the molecular pharmacology of cancer. Nat. Genet. 24, 236-244.
Shin, H.Y., Kim, S.H., Jeong, H.J., Kim, S.Y., Shin, T.Y., Urn, J.Y., Hong, S.H., Kim, H.M., 2007. Epigallocatechin-3-gallate inhibits secretion of TNF-alpha, IL-6 and IL-8 through the attenuation of ERK and NF-kappaB in HMC-1 cells. Int. Arch. Allergy Immunol. 142, 335-344.
Snyder, J.G., Prewitt, R., Campsen, J., Britt, L.D., 2002. PDTC and Mg132, inhibitors of NF-kappaB, block endotoxin induced vasodilation of isolated rat skeletal muscle arterioles. Shock 17, 304-307.
Wang, L.E., Cui, X.Y., Cui, S.Y., Cao, J.X., Zhang, J., Zhang, Y.H., Zhang, Q.Y., Bai, Y.J., Zhao, Y.Y., 2010. Potentiating effect of spinosin, a C-glycoside flavonoid of Semen Ziziphi spinosae, on pentobarbital-induced sleep may be related to postsynaptic 5-HT(1A) receptors. Phytomedicine 17, 404-409.
Wang, Z.Y., Cheng, S.J., Zhou, Z.C., Athar, M., Khan, W.A., Bickers, D.R., Mukhtar, H., 1989. Antimutagenic activity of green tea polyphenols. Mutat. Res. 223, 273-285.
Woo, W.S., Kang, S.S., Wagner, H., Seligmann, O., Chari, V.M., 1980. Structure of flavone-C-glycosides 0.18. acylated flavone-C-glycosides from the seeds of Zizyphusjujuba. Phytochemistry 19, 2791-2793.
Xu, G.. Lo, Y.C., Li, Q., Napolitano, G., Wu, X., Jiang, X., Dreano, M., Karin, M., Wu, H., 2011. Crystal structure of inhibitor of kappaB kinase beta. Nature 472, 325-330.
Zanotto-Filho, A., Delgado-Canedo, A., Schroder, R., Becker, M., Klamt, F., Moreira, J.C., 2010. The pharmacological NFkappaB inhibitors BAY117082 and MG132 induce cell arrest and apoptosis in leukemia cells through ROS-mitochondria pathway activation. Cancer Lett. 288, 192-203.
Zhou, X.L, Fan, W., Yang, G., Yu, M.X., 2014. The clinical significance of PR, ER, NFkappa B, and TNF-alpha in breast cancer. Dis. Markers, 494581.
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: email@example.com (T. Pommerening), firstname.lastname@example.org (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|
|Date:||Mar 15, 2016|
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