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Effect of herbal melanin on IL-8: A possible role of Toll-like receptor 4 (TLR4)
BBRC Biochemical and Biophysical Research Communications 344 (2006) 1200–1206 www.elsevier.com/locate/ybbrc
Effect of herbal melanin on IL-8: A possible role of Toll-like receptor 4 (TLR4) Adila El-Obeid a
a,*
, Adil Hassib b, Fredrik Ponte´n a, Bengt Westermark
a
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University Hospital, SE-751 85 Uppsala, Sweden b Faculty of Science, King Saud University, Riyadh, Saudi Arabia Received 3 April 2006
Abstract The production of IL-8 can be induced by LPS via TLR4 signaling pathway. In this study, we tested the effect of a herbal melanin (HM) extract, from black cumin seeds (Nigella sativa L.), on IL-8 production. We used HM and LPS in parallel to induce IL-8 production by THP-I, PBMCs, and TLR4-transfected HEK293 cells. Both HM and LPS induced IL-8 mRNA expression and protein production in THP-1 and PBMCs. On applying similar treatment to HEK293 cells that express TLR4, MD2, and CD14, both HM and LPS significantly induced IL-8 protein production. We have also demonstrated that HM and LPS had identical effects in terms of IL-8 stimulation by HEK293 transfected with either TLR4 or MD2–CD14. Melanin extracted from N. sativa L. mimics the action of LPS in the induction of IL-8 by PBMC and the other used cell lines. Our results suggest that HM may share a signaling pathway with LPS that involves TLR4. 2006 Elsevier Inc. All rights reserved. Keywords: Herbal melanin; Toll-like receptor 4; Nigella sativa L; IL-8; Lipopolysaccharide; MD2; CD14
Melanin is a pigment of plants and animals. It occurs both externally and internally in tissues and organs (e.g., in seed coats, hair, inner ear, Substantia nigra, and fertilized ova). In humans, the absence of melanin is correlated with the onset of various diseases like albinism and Parkinson’s disease [1,2]. Melanins have also been associated with many protective roles in biological systems [3]. The exact chemical structure and molecular weight of melanin are not yet fully established. It has been determined that melanins are built up of indolequinones at various degrees of combination and attachments to each other and other molecules [4]. Most authors describe melanins as macromolecules composed of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHIC) and their various oxidized forms [5,6]. A set of unique physical and chemical properties is generally used to characterize melanins [7]. Melanin has been extracted from a few plants [8,9] *
Corresponding author. Fax: +46 18 552739. E-mail address: [email protected] (A. El-Obeid).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.04.035
and recently it has been extracted from the seed coats of black cumin (Nigella sativa L.) (NS) [10]. A generally accepted belief in Muslim countries is that black cumin is a herb with special healing powers. A companion article to this paper provides a detailed physico-chemical characterization of the herbal melanin (HM) extracted from this seed (submitted for publication). The innate immune system in drosophila and mammals senses the invasion of microorganisms by using the family of Toll and Toll-like receptors (TLRs), respectively. In mammals, eleven members of the TLR family have been identified [11]. They recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity [12]. TLRs are members of the interleukin-1 receptor (IL-1R) superfamily and share significant homology in their cytoplasmic regions [13]. Each TLR recognizes a restricted subset of molecules such as peptidoglycan for TLR2, unmethylated DNA for TL9, and lipopolysaccharide (LPS) for TLR4 [14–16]. At present, only LPS, a predominant glycolipid in the outer membrane of
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Gram-negative bacteria, and taxsol, a product of the Western yew (Taxus brevifolia), have been identified as exogenous ligands for TLR4 [17]. LPS is made of hydrophilic polysaccharide regions with certain structural variability and less variable regions of hydrophobic lipid moieties [18]. It has now been established that the lipid moiety (lipid A) is the part responsible for the reactions of the host and responsible for the endotoxic, adjuvancy, and other immune system responses [19]. Recently, different herbal melanins have been shown to posses many LPS-like properties in regard to the induction of cytokines [20,21]. In spite of the fact that the structure of LPS is quite different from melanin, both are exogenous chemical systems eliciting a similar response from the host system. The mechanism of action and receptors of LPS has been analyzed by several investigators whereas, the action, cellular receptors, and signaling molecules for melanins have not been considered before. Activation of TLR4 by LPS initiates a cascade of intracellular events including the transcription factor nuclear factor KB (NF-jB) and causes the release of a number of proinflammatory mediators, such as interleukin (IL)-1, IL-6, and IL-8 [13]. IL-8 is a member of the a-chemokine family and produced by a wide variety of cell types in response to LPS [22]. Recognition of LPS by TLR4 requires three other extracellular proteins, the acute-phase LPS-binding protein (LBP), CD14, and myeloid differentiation protein-2 (MD2), in addition to TLR4. LBP is a 60 kDa serum LPSbinding protein and is essential for the rapid induction of an inflammatory response to LPS [23]. CD14 is an important 55 kDa that lacks transmembrane and cytoplasmic domains and needed for LPS sensing. It is present both as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood [24]. MD2 is the third and essential accessory protein for TLR4. MD2 is a 20– 25 kDa glycoprotein, is physically associated with TLR4, and confers LPS responsiveness to TLR-expressing cells [25]. In a previous study [21], we have shown that herbal melanin induces TNF-a and IL-6 production. In this study, we have tested whether the HM induced cytokine production via TLR4. HM and LPS were used in parallel and IL-8 production was tested in the presence and absence of TLR4 expression. Our results reveal similarities between HM and LPS in TLR4-expressing cells, THP-1 and PBMCs, and in HEK293 cells transfected with isolated TLR4, MD2, and CD14 or the three proteins. HM induced IL-8 mRNA and protein expression by THP-1 and PBMCs and protein production by 293-hTLR4A/MD2–CD14 cell line. Our results demonstrate that the signaling events by which HM modulates cytokine production are, at least in part, similar to that elicited by LPS via TLR4 activation. Materials and methods Cell lines and cell culture conditions. Human monocytic THP-1 cells and human embryonic kidney 293 (HEK293) cells were obtained from American
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Type Culture Collection (ATCC, Rockville, MD, USA). The stably transfected cell lines, 293-hTLR4A, 293-hMD2-CD14, and 293-hTLR4/MD2– CD14 cell lines were obtained from InvivoGen (3950 Sorrento Valley Blvd. Suite A, San Diego, CA, USA). THP-1 cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL) and antibiotics (100 U penicillin and 50 lg/ml streptomycin). HEK293 cells were grown in standard DMEM, 4.5 g/l glucose, and 10% FBS. 293-hTLR4A and 293-hMD2-CD14 cells were grown in standard DMEM with 10% FBS supplemented with blasticidin (10 lg/ml) and hygrogold (50 lg/ml), respectively. 293-hTLR4/MD2–CD14 clones were grown in the same media supplemented with both blasticidin (10 lg/ml) and hygrogold (50 lg/ml). For all cells, the culture medium was replaced 24 h prior to experiment by serum-free medium to avoid effects of serum on gene expression. Viability of the cells was regularly tested using Trypan blue. Blood cell isolation. Buffy coats from standard whole blood units (420 ml) of peripheral blood were obtained from healthy blood donors at the Uppsala University Hospital Blood Center, Uppsala, Sweden. Informed consent was obtained from each subject. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by means of Ficoll–Paque (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation following manufacturer’s instructions. PBMCs were seeded at a density of 2 · 106 cells/ml in 24-well microtiter plates in RPMI-1640 medium. Induction and analysis of IL-8 mRNA levels. In order to test IL-8 mRNA expression by THP-1 and PBMC, cells were treated with HM solutions at concentrations of 5 and 10 lg/ml. LPS was used as positive stimulus for cytokines induction at 10 lg/ml (Escherichia coli LPS 026:B5 Sigma). Untreated THP-1 cells and PBMCs taken from the same object were used as controls. Total cellular RNA was extracted from THP-1 and PBMC using TRIZOL reagent (Invitrogen) according to manufacture’s instructions. Two micrograms of total RNA was reverse transcribed into single-stranded cDNA as described previously [26]. The primers used for IL-8 cDNA amplification were ATG-ACT-TCC-AAG-CTG-GCC-GTGGCT (sense) and TCT-CAG-CCC-TCT-TCA-AAA-ACT-TCT-C (antisense). Conditions for RT-PCR were 30 cycles of 94 C for 30 s, 68 C for 1 min, and 72 C for 2 min. b-Actin was used as an internal control for RT-PCR. The primers used for b-actin were ATC-TGG-CAC-CAC-ACCTTC-TAC-AAT-GAG-CTG-CG and CGT-CAT-ACT-CCT-GCT-TGCTGA-TCC-ACA-TCT-GC. Conditions for RT-PCR were 40 cycles of 94 C for 45 s, 61 C for 45 s, and 72 C for 2 min. The products were separated on 2% agarose gel using electrophoresis and visualized by ethidium bromide staining. Induction and analysis of IL-8 protein levels. THP-1, PBMCs (2 · 106 cells/ml), HEK293, 293-hTLR4, 293-hMD2-CD14, and 293hTLR/MD2–CD14 (1 · 106 cells/ml) were seeded in appropriate media in 24-well dishes. Cells were treated with various concentrations of HM (5,10, 25, and 50 lg/ml) or LPS (10 lg/ml) for 24 h at 37 C and 100% humidity in 5% CO2 before collecting the supernatants for cytokine assays. Untreated cells were used as controls. Culture media supplemented with 50 lg/ml HM were used as additional controls. The concentrations of IL-8 produced in the supernatant were assayed by ELISA (R&D Systems Inc. Minneapolis, MN 55413, USA) following manufacturer’s instructions. To verify that HM did not interfere with the absorbance readings, the concentrations of IL-8 in the supernatants were determined from standard curves where cytokine standards were incubated with 50 lg/ml HM. HM did not interfere with absorbance and showed no effect on the outcome of cytokine assay (data not shown). Statistical analysis. Results were analyzed using Mann–Whitney test. All values were expressed as means ± standard error. Values of p < 0.05 were considered significant.
Results IL-8 mRNA expression in THP-1 cells and PBMC To evaluate whether THP-1 and PBMCs expressed IL-8 in response to HM treatment, we first tested the expression
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Fig. 1. Effect of HM on mRNA expression. THP-1 and PBMCs (1 · 106 cells/ml) were treated with HM for two (THP-1) and 24 h (PBMCs) at the indicated doses. LPS-treated cells (10 lg/ml) were used as positive control. Untreated cells were used as normal control. The total RNA was extracted from the cells and then analyzed by RT-PCR, as described in Materials and methods. The PCR products were separated on 2% agarose gel and were visualized by staining with ethidium bromide. (A) THP-1 expression of IL-8 mRNA (a) and b-actin mRNA (b). (B) PBMCs expression of IL-8 mRNA (a) and b-actin mRNA (b).
of IL-8 mRNA by RT-PCR. As shown in Fig. 1A, IL-8 mRNA (289 bp) was detected in all the lanes. Untreated THP-1 cells displayed very low level of IL-8 mRNA that was induced by LPS. Similarly, addition of HM (5 and 10 lg/ml) clearly increased IL-8 expression in a dose dependent manner. Fig. 1B shows that untreated PBMCs expressed detectable level of IL-8 transcripts. Similar increase in IL-8 expression was observed when treating the cells with HM (5 and 10 lg/ml) or LPS (10 lg/ml). The housekeeping gene, b-actin, indicated comparable levels of the internal mRNA in the control and treated THP-1 and PBMCs (Figs. 1A and B). IL-8 secretion in THP-1 cells and PBMC Fig. 2 shows that control THP-1 cells produced low level of IL-8 protein with a mean of 48 pq/ml (0.00–96 pq/ml). LPS treatment (10 lg/ml) increased the production of IL8 after 24 h stimulation as previously reported [27]. Under the same conditions, treatment with HM (5, 10, and 25 lg/ ml) significantly induced IL-8 production as compared to untreated cells (p = 0.046). Next, we investigated the effect of HM on PBMCs in comparison with LPS. Fig. 3 shows that untreated PBMC constitutively produced IL-8 protein that was significantly induced by LPS treatment (p = 0.042). Addition of HM increased IL-8 production in a dose-dependent manner.
Fig. 2. Effect of HM on IL-8 production by THP-1. THP-1 cells (2 · 106 cells/ml) were treated with HM and LPS as indicated concentrations for 24 h. LPS-treated cells were used as positive control, untreated cells as normal control, and culture media supplemented with 50 lg/ml as negative control. Concentration of IL-8 in the supernatants was determined by ELISA as specified by manufacturer. Data represent means from three independent experiments that yielded similar results. Values are presented as means ± SE. p < 0.05 is significant.
Fig. 3. Effect of HM on IL-8 production by PBMC. PBMCs (2 · 106 cells/ ml) were treated with HM and LPS indicated concentrations for 24 h. LPS-treated cells were used as positive control, untreated cells as normal control, and culture media supplemented with 50 lg/ml as negative control. Concentration of IL-8 in the supernatants was determined by ELISA as specified by manufacturer. Data represent means from three independent experiments that yielded similar results. Values are presented as means ± SE. p < 0.05 is significant.
Both HM (50 lg/ml) and LPS (10 lg/ml), however, induced the same amounts of IL-8 protein. IL-8 secretion in HEK293, 293-hTLR4A, 293-hMD2-CD14, and 293-hTLR4/MD2–CD14 LPS-TLR4 signaling pathway requires a receptor complex that is composed of TLR4, MD2, and CD14 [28]. HEK293 lacks the expression of TLR4, MD2, and CD14 and produces very low levels of IL-8 [29,30]. To carry out an analysis of the effect of HM on TLR4 signaling, we used 293-hTLR4A, 293-hMD2-CD14, and 293-hTLR4/MD2–CD14
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cell lines that were stably transfected with human TLR4A, MD2-CD14, and TLR4/MD2–CD14 plasmids, respectively. Cells were incubated in serum-free medium with either LPS (10 lg/ml), or HM (5, 10, and 25 lg/ml) for 24 h. Untreated cells were used as controls. To determine whether or not HM activates TLR4 signaling we tested initially its effect on 293-hTLR MD2/ CD14 cells that express TLR4 together with MD2 and CD14 proteins, i.e., the complete receptor complex needed for LPS/TLR4 signaling. Fig. 4A shows that addition of HM at 5 and 10 lg/ml significantly induced IL-8 production (p = 0.005 and p = 0.003, respectively). Similarly, LPS highly induced IL-8. The amount of IL-8 induced by HM (5 and 10 lg/ml) was slightly higher than that induced by LPS (10 lg/ml) with means of 3.691, 3.658, and 3.575 pq/ml, respectively. The LPS results agree with previous results showing an increase in IL-8 production by LPS and HEK293 cells in the presence of TLR4, MD2, and CD14 [28,31]. Transfecting the cells with TLR4, MD2, and CD14 increased in the basal level of IL-8 production by 293-hTLR4/MD2–CD14 as compared to HEK293 even in the absence of stimulation (data not shown). Subsequently, we examined the effect of HM on TLR4 in the absence of MD2 and CD14 by using 293-hTLR4transfected cells (Fig. 4B). Treatment of the cells with HM (5 and 10 lg/ml) or LPS (10 lg/ml), did not significantly induce IL-8 production (p = 0.590, p = 0.590 and p = 0.332, respectively). Much lower amounts of IL-8 were detected in the supernatants of HM-treated (1.790 and 1.759 pq/ml) and LPS-treated cells (1.826 pq/ml) as compared to 293-hTLR4/MD2–CD14 when similarly treated (Figs. 4A and B). Previous studies were controversial in respect to the role of MD2 in LPS/TLR4 signaling and different effects of MD2 on TLR4 signaling had been reported [25,32–35]. However, our data have shown very identical effects, for HM and LPS, on 293-hTLR4 which suggests a similar signaling pathway. Furthermore, we tested the effect of HM on IL-8 production by 293-hMD2-CD14transfected cells. The presence of MD2 and CD14 did
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not induce IL-8 production in HM-treated cells in the absence of TLR4. Fig. 4C shows that neither HM (5 and 10 lg/ml) nor LPS (10 lg/ml) had an effect on IL-8 production (p = 0.439, p = 1.000, and p = 0.439, respectively). Our result on LPS induced IL-8 production agrees with previous results [36]. Discussion A large body of studies has claimed an immunological role for the widely used herb, Nigella sativa L. [37,38]. None of these studies has ascribed the biological effects to the herbal melanin, which is a constituent of the plant. In this study, we investigated the effects of a herbal melanin (HM), extracted from the seed coats of the plant on cytokine production by PBMCs, THP-1 cells and by TLR4-transfected HEK293 cells. Our findings are: (1) Nigella sativa L. HM induced IL-8 mRNA expression and protein production in PBMCs and THP-1 cells, (2) HM induced IL-8 protein production in HEK293 cells co-transfected with TLR4 and its accessory molecules MD2 and CD14, and (3) the expression of IL-8 is dependent on the presence of MD2 and CD14. These results suggest that HM is acting as a TLR4 ligand. It is known that TLRs transmit signals in response to a wide range of microbial products that activate different TLR members, for example, LPS and TLR4 [16]. Ligand activation of TLRs induces cytokines production via activation of the NF-jB signaling pathway [13]. We have characterized first the activation of TLR4 by HM on cells that express TLR4, human THP-1, and PBMC [39,40], using production of IL-8 as an index of the cellular response. Cells were treated in parallel with different concentrations of HM and LPS. THP-1 and PBMC are known to be highly sensitive to LPS, responding by upregulating the secretion of cytokines [41,42]. Quantitation of IL-8 released by THP-1 and PBMCs by ELISA confirmed the stimulatory effects of HM and LPS. High levels of IL-8 were detectable in the supernatants of both cell types. Recently it has been
Fig. 4. Effect of HM on IL-8 production by 293-hTLR4/MD2–CD14 (A), 293-hTLR4A (B), and 293-hMD2-CD14 (C) cell lines. Cells (1 · 106 cells/ml) were treated with HM and LPS as indicated concentrations for 24 h. LPS-treated cells were used as positive control, untreated cells as normal control, and culture media supplemented with 50 lg/ml as negative control. Concentration of IL-8 in the supernatants of 293-hTLR4/MD2–CD14 (A—left), 293hTLR4A (B—middle), and 293-hMD2-CD14 (C—right) was determined by ELISA as specified by manufacturer. Data represent means from three independent experiments that yielded similar results. Values are presented as means ± SE. p < 0.05 is significant.
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reported that LPS up-regulates IL-8 production in PBMCs and THP-1 through TLR4 activation [43,44]. Our results have shown that on using similar concentrations of HM and LPS, induction of IL-8 by HM was stronger than by LPS in THP-1 and was close to LPS in PBMCs. The known TLR4 ligands, such as LPS, require accessory molecules, MD2 and CD14, to turn on and augment TLR4 signaling [25,24]. To determine whether HM can activate TLR4 we used transfected HEK 293 cells that express plasmids for TLR4, MD2, and CD14. Subsequently we challenged the transfected cells with HM or LPS. LPS, in the presence of a TLR4/MD2–CD14 complex, produced the expected, previously reported, strong response of production of high levels of IL-8 by the cells [28,31]. Similar results were also obtained on treating the cells with HM giving rise to a significantly increased production of IL-8. Next, we explored the possible roles for MD2 and CD14 in HM-TLR4-dependent signaling. Both HM and LPS had no significant effect on IL-8 production by 293-hTLR4. Our results have provided additional data supporting previous assumption that the response to LPS requires the presence MD2 in LPS/TLR4 signaling pathway [25,34,35] though other previous studies had reported TLR4-activation by LPS in the absence of MD2 [36]. We also clearly present the similarity between HM and LPS in IL-8 stimulation by TLR4-expressing cells. Whether TLR4 recognized HM by direct binding or through MD2 signaling is yet to be clarified. Similar unanswered questions were raised concerning LPS/TLR4 which merits, in both cases, further detailed investigations [45,46]. Eventually, we tested the effects of both of HM and LPS on 293-hMD2-CD14 cells. Both HM and LPS had no effect on IL-8 production in the absence of TLR4. Similar results were obtained by Heine et al. who showed the activation of TLR4 by LPS in HEK293-TLR4-transfected cells in the absence of CD14 and Garcia-Rodriguez and co-workers reported that transfection of isolated CD14, MD2 or both of them in HEK293 did not confer NF-jb activation by LPS [47,36]. Our results present the similarity in action between LPS and HM in the presence of MD2 and CD14 and absence of TLR4. Melanins are built up of repeated indolic units plus other molecular groups [4] and LPS are made of carbohydrates and fats [18]. Melanins, therefore, have on the basic chemical level, a different structure from that of LPS. However, in this study, HM have shown LPS-like properties with respect to the induction of different cytokines. Previously, we have shown that HM induced TNF-a and IL-6 mRNA and protein production in THP-1, human monocytes, and PBMCs in a way similar to the LPS stimulation of these cells [21]. In this study, we report that both of HM and LPS induce IL-8 production in THP-1 and PBMCs and that they show exactly the same pattern of 1L-8 protein expression in HEK293, 293-hTLR4A, 293-hMD2CD14, and 293-hTLR4/MD2–CD14. Taking together these facts we suggest that the increase in IL-8 induced
by LPS or HM in THP-1, PBMCs, 293-hTLR4, and 293hTLR4-MD2-CD14 cells is via TLR4 activation pathways. Recently Pasco and co-workers [20] reported that melanins extracted from different botanicals other than N. sativa L. activate NF-jB and induce IL-1b production and they tentatively proposed TLR2 as a receptor of ‘‘botanical’’ melanins. Our results demonstrate that HM is a powerful immunostimulant of TLR4-expressing cells. Recently, the immunostimulatory activity of some TLR ligands has led to the proposition of their use as adjuvants for immunotherapy of cancer [48]. Seya and co-workers [49] reported that BCGCWS induced TNF-a secretion from DC via TLR2 and TLR4 and they suggested its use as a potent adjuvant in cancer therapy. HM have induced TNF-a in THP-1, monocytes, and PBMC [21] and IL-8 in HEK-TLR4 transfected cells. One may, therefore, consider HM as an alternative adjuvant for cancer immunotherapy. TLR4 agonists elicit the innate immune system to act against various viral, bacterial, and fungal infections. HM is a non-toxic herbal extract that is safe for oral and/or other methods of application. There are potential applications of HM, as a TLR4 agonist, in many clinical applications such as infectious diseases and cancer. Acknowledgments This work has been supported by the grant of the Knut and Alice Wallenberg Foundation for the Swedish HPR project. We thank Dr. Gamal Mohamed for assistance with statistical analysis. References [1] J. Stinchcombe, G. Bossi, G.M. Griffiths, Linking albinism and immunity: the secrets of secretory lysosomes, Science 305 (2004) 55–59. [2] H. Fedorow, G.M. Halliday, C.H. Rickert, M. Gerlach, P. Riederer, K.L. Double, Evidence for specific phases in the development of human neuromelanin, Neurobiol. Aging 27 (2006) 506–512. [3] M.A. Pathak, Functions of melanin and protection by melanin, in: L. Zeise, M.R. Chedekel, T.B. Fitzpatrick (Eds.), Melanin: Its Role in Human Photoprotection, American Society for Photobiology, Valdenmar Publishing Co, Overland Park, KS, 1995, pp. 125–140. [4] P.A. Riley, Melanin, Int. J. Biochem. Cell Biol. 29 (1997) 1235–1239. [5] G. Prota, Progress in the chemistry of melanins and related metabolites, Med. Res. Rev. 8 (1988) 525–556. [6] A. Pezzella, M. d’Ischia, A. Napolitano, A. Palumbo, G. Prota, An integrated approach to the structure of sepia melanin. Evidence for a high proportion of degraded 5,6-dihydroxyindole-2-carboxylic acid units in the pigment backbone, Tetrahedron 53 (1977) 8281–8286. [7] L. Zeise, Analytical methods for characterization and identification of eumelanins, in: L. Zeise, M.R. Chedekel, T.B. Fitzpatrick (Eds.), Melanin: Its Role in Human Photoprotection, Valdenmar Publishing, Overland Park, KS, 1995, pp. 65–79. [8] E. Buszman, M. Kopera, T. Wilczok, Electron spin resonance studies of chloroquine–melanin complexes, Biochem. Pharmacol. 33 (1984) 7–11. [9] V.M. Sava, S.M. Yang, M.Y. Hong, P.C. Yang, G.S. Huang, Isolation and characterization of melanic pigments derived from tea and tea polyphenols, Food Chem. 73 (2001) 177–184.
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Effect of herbal melanin on IL-8: A possible role of Toll-like receptor 4 (TLR4) Adila El-Obeid a
a,*
, Adil Hassib b, Fredrik Ponte´n a, Bengt Westermark
a
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University Hospital, SE-751 85 Uppsala, Sweden b Faculty of Science, King Saud University, Riyadh, Saudi Arabia Received 3 April 2006
Abstract The production of IL-8 can be induced by LPS via TLR4 signaling pathway. In this study, we tested the effect of a herbal melanin (HM) extract, from black cumin seeds (Nigella sativa L.), on IL-8 production. We used HM and LPS in parallel to induce IL-8 production by THP-I, PBMCs, and TLR4-transfected HEK293 cells. Both HM and LPS induced IL-8 mRNA expression and protein production in THP-1 and PBMCs. On applying similar treatment to HEK293 cells that express TLR4, MD2, and CD14, both HM and LPS significantly induced IL-8 protein production. We have also demonstrated that HM and LPS had identical effects in terms of IL-8 stimulation by HEK293 transfected with either TLR4 or MD2–CD14. Melanin extracted from N. sativa L. mimics the action of LPS in the induction of IL-8 by PBMC and the other used cell lines. Our results suggest that HM may share a signaling pathway with LPS that involves TLR4. 2006 Elsevier Inc. All rights reserved. Keywords: Herbal melanin; Toll-like receptor 4; Nigella sativa L; IL-8; Lipopolysaccharide; MD2; CD14
Melanin is a pigment of plants and animals. It occurs both externally and internally in tissues and organs (e.g., in seed coats, hair, inner ear, Substantia nigra, and fertilized ova). In humans, the absence of melanin is correlated with the onset of various diseases like albinism and Parkinson’s disease [1,2]. Melanins have also been associated with many protective roles in biological systems [3]. The exact chemical structure and molecular weight of melanin are not yet fully established. It has been determined that melanins are built up of indolequinones at various degrees of combination and attachments to each other and other molecules [4]. Most authors describe melanins as macromolecules composed of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHIC) and their various oxidized forms [5,6]. A set of unique physical and chemical properties is generally used to characterize melanins [7]. Melanin has been extracted from a few plants [8,9] *
Corresponding author. Fax: +46 18 552739. E-mail address: [email protected] (A. El-Obeid).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.04.035
and recently it has been extracted from the seed coats of black cumin (Nigella sativa L.) (NS) [10]. A generally accepted belief in Muslim countries is that black cumin is a herb with special healing powers. A companion article to this paper provides a detailed physico-chemical characterization of the herbal melanin (HM) extracted from this seed (submitted for publication). The innate immune system in drosophila and mammals senses the invasion of microorganisms by using the family of Toll and Toll-like receptors (TLRs), respectively. In mammals, eleven members of the TLR family have been identified [11]. They recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity [12]. TLRs are members of the interleukin-1 receptor (IL-1R) superfamily and share significant homology in their cytoplasmic regions [13]. Each TLR recognizes a restricted subset of molecules such as peptidoglycan for TLR2, unmethylated DNA for TL9, and lipopolysaccharide (LPS) for TLR4 [14–16]. At present, only LPS, a predominant glycolipid in the outer membrane of
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Gram-negative bacteria, and taxsol, a product of the Western yew (Taxus brevifolia), have been identified as exogenous ligands for TLR4 [17]. LPS is made of hydrophilic polysaccharide regions with certain structural variability and less variable regions of hydrophobic lipid moieties [18]. It has now been established that the lipid moiety (lipid A) is the part responsible for the reactions of the host and responsible for the endotoxic, adjuvancy, and other immune system responses [19]. Recently, different herbal melanins have been shown to posses many LPS-like properties in regard to the induction of cytokines [20,21]. In spite of the fact that the structure of LPS is quite different from melanin, both are exogenous chemical systems eliciting a similar response from the host system. The mechanism of action and receptors of LPS has been analyzed by several investigators whereas, the action, cellular receptors, and signaling molecules for melanins have not been considered before. Activation of TLR4 by LPS initiates a cascade of intracellular events including the transcription factor nuclear factor KB (NF-jB) and causes the release of a number of proinflammatory mediators, such as interleukin (IL)-1, IL-6, and IL-8 [13]. IL-8 is a member of the a-chemokine family and produced by a wide variety of cell types in response to LPS [22]. Recognition of LPS by TLR4 requires three other extracellular proteins, the acute-phase LPS-binding protein (LBP), CD14, and myeloid differentiation protein-2 (MD2), in addition to TLR4. LBP is a 60 kDa serum LPSbinding protein and is essential for the rapid induction of an inflammatory response to LPS [23]. CD14 is an important 55 kDa that lacks transmembrane and cytoplasmic domains and needed for LPS sensing. It is present both as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood [24]. MD2 is the third and essential accessory protein for TLR4. MD2 is a 20– 25 kDa glycoprotein, is physically associated with TLR4, and confers LPS responsiveness to TLR-expressing cells [25]. In a previous study [21], we have shown that herbal melanin induces TNF-a and IL-6 production. In this study, we have tested whether the HM induced cytokine production via TLR4. HM and LPS were used in parallel and IL-8 production was tested in the presence and absence of TLR4 expression. Our results reveal similarities between HM and LPS in TLR4-expressing cells, THP-1 and PBMCs, and in HEK293 cells transfected with isolated TLR4, MD2, and CD14 or the three proteins. HM induced IL-8 mRNA and protein expression by THP-1 and PBMCs and protein production by 293-hTLR4A/MD2–CD14 cell line. Our results demonstrate that the signaling events by which HM modulates cytokine production are, at least in part, similar to that elicited by LPS via TLR4 activation. Materials and methods Cell lines and cell culture conditions. Human monocytic THP-1 cells and human embryonic kidney 293 (HEK293) cells were obtained from American
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Type Culture Collection (ATCC, Rockville, MD, USA). The stably transfected cell lines, 293-hTLR4A, 293-hMD2-CD14, and 293-hTLR4/MD2– CD14 cell lines were obtained from InvivoGen (3950 Sorrento Valley Blvd. Suite A, San Diego, CA, USA). THP-1 cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL) and antibiotics (100 U penicillin and 50 lg/ml streptomycin). HEK293 cells were grown in standard DMEM, 4.5 g/l glucose, and 10% FBS. 293-hTLR4A and 293-hMD2-CD14 cells were grown in standard DMEM with 10% FBS supplemented with blasticidin (10 lg/ml) and hygrogold (50 lg/ml), respectively. 293-hTLR4/MD2–CD14 clones were grown in the same media supplemented with both blasticidin (10 lg/ml) and hygrogold (50 lg/ml). For all cells, the culture medium was replaced 24 h prior to experiment by serum-free medium to avoid effects of serum on gene expression. Viability of the cells was regularly tested using Trypan blue. Blood cell isolation. Buffy coats from standard whole blood units (420 ml) of peripheral blood were obtained from healthy blood donors at the Uppsala University Hospital Blood Center, Uppsala, Sweden. Informed consent was obtained from each subject. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by means of Ficoll–Paque (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation following manufacturer’s instructions. PBMCs were seeded at a density of 2 · 106 cells/ml in 24-well microtiter plates in RPMI-1640 medium. Induction and analysis of IL-8 mRNA levels. In order to test IL-8 mRNA expression by THP-1 and PBMC, cells were treated with HM solutions at concentrations of 5 and 10 lg/ml. LPS was used as positive stimulus for cytokines induction at 10 lg/ml (Escherichia coli LPS 026:B5 Sigma). Untreated THP-1 cells and PBMCs taken from the same object were used as controls. Total cellular RNA was extracted from THP-1 and PBMC using TRIZOL reagent (Invitrogen) according to manufacture’s instructions. Two micrograms of total RNA was reverse transcribed into single-stranded cDNA as described previously [26]. The primers used for IL-8 cDNA amplification were ATG-ACT-TCC-AAG-CTG-GCC-GTGGCT (sense) and TCT-CAG-CCC-TCT-TCA-AAA-ACT-TCT-C (antisense). Conditions for RT-PCR were 30 cycles of 94 C for 30 s, 68 C for 1 min, and 72 C for 2 min. b-Actin was used as an internal control for RT-PCR. The primers used for b-actin were ATC-TGG-CAC-CAC-ACCTTC-TAC-AAT-GAG-CTG-CG and CGT-CAT-ACT-CCT-GCT-TGCTGA-TCC-ACA-TCT-GC. Conditions for RT-PCR were 40 cycles of 94 C for 45 s, 61 C for 45 s, and 72 C for 2 min. The products were separated on 2% agarose gel using electrophoresis and visualized by ethidium bromide staining. Induction and analysis of IL-8 protein levels. THP-1, PBMCs (2 · 106 cells/ml), HEK293, 293-hTLR4, 293-hMD2-CD14, and 293hTLR/MD2–CD14 (1 · 106 cells/ml) were seeded in appropriate media in 24-well dishes. Cells were treated with various concentrations of HM (5,10, 25, and 50 lg/ml) or LPS (10 lg/ml) for 24 h at 37 C and 100% humidity in 5% CO2 before collecting the supernatants for cytokine assays. Untreated cells were used as controls. Culture media supplemented with 50 lg/ml HM were used as additional controls. The concentrations of IL-8 produced in the supernatant were assayed by ELISA (R&D Systems Inc. Minneapolis, MN 55413, USA) following manufacturer’s instructions. To verify that HM did not interfere with the absorbance readings, the concentrations of IL-8 in the supernatants were determined from standard curves where cytokine standards were incubated with 50 lg/ml HM. HM did not interfere with absorbance and showed no effect on the outcome of cytokine assay (data not shown). Statistical analysis. Results were analyzed using Mann–Whitney test. All values were expressed as means ± standard error. Values of p < 0.05 were considered significant.
Results IL-8 mRNA expression in THP-1 cells and PBMC To evaluate whether THP-1 and PBMCs expressed IL-8 in response to HM treatment, we first tested the expression
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Fig. 1. Effect of HM on mRNA expression. THP-1 and PBMCs (1 · 106 cells/ml) were treated with HM for two (THP-1) and 24 h (PBMCs) at the indicated doses. LPS-treated cells (10 lg/ml) were used as positive control. Untreated cells were used as normal control. The total RNA was extracted from the cells and then analyzed by RT-PCR, as described in Materials and methods. The PCR products were separated on 2% agarose gel and were visualized by staining with ethidium bromide. (A) THP-1 expression of IL-8 mRNA (a) and b-actin mRNA (b). (B) PBMCs expression of IL-8 mRNA (a) and b-actin mRNA (b).
of IL-8 mRNA by RT-PCR. As shown in Fig. 1A, IL-8 mRNA (289 bp) was detected in all the lanes. Untreated THP-1 cells displayed very low level of IL-8 mRNA that was induced by LPS. Similarly, addition of HM (5 and 10 lg/ml) clearly increased IL-8 expression in a dose dependent manner. Fig. 1B shows that untreated PBMCs expressed detectable level of IL-8 transcripts. Similar increase in IL-8 expression was observed when treating the cells with HM (5 and 10 lg/ml) or LPS (10 lg/ml). The housekeeping gene, b-actin, indicated comparable levels of the internal mRNA in the control and treated THP-1 and PBMCs (Figs. 1A and B). IL-8 secretion in THP-1 cells and PBMC Fig. 2 shows that control THP-1 cells produced low level of IL-8 protein with a mean of 48 pq/ml (0.00–96 pq/ml). LPS treatment (10 lg/ml) increased the production of IL8 after 24 h stimulation as previously reported [27]. Under the same conditions, treatment with HM (5, 10, and 25 lg/ ml) significantly induced IL-8 production as compared to untreated cells (p = 0.046). Next, we investigated the effect of HM on PBMCs in comparison with LPS. Fig. 3 shows that untreated PBMC constitutively produced IL-8 protein that was significantly induced by LPS treatment (p = 0.042). Addition of HM increased IL-8 production in a dose-dependent manner.
Fig. 2. Effect of HM on IL-8 production by THP-1. THP-1 cells (2 · 106 cells/ml) were treated with HM and LPS as indicated concentrations for 24 h. LPS-treated cells were used as positive control, untreated cells as normal control, and culture media supplemented with 50 lg/ml as negative control. Concentration of IL-8 in the supernatants was determined by ELISA as specified by manufacturer. Data represent means from three independent experiments that yielded similar results. Values are presented as means ± SE. p < 0.05 is significant.
Fig. 3. Effect of HM on IL-8 production by PBMC. PBMCs (2 · 106 cells/ ml) were treated with HM and LPS indicated concentrations for 24 h. LPS-treated cells were used as positive control, untreated cells as normal control, and culture media supplemented with 50 lg/ml as negative control. Concentration of IL-8 in the supernatants was determined by ELISA as specified by manufacturer. Data represent means from three independent experiments that yielded similar results. Values are presented as means ± SE. p < 0.05 is significant.
Both HM (50 lg/ml) and LPS (10 lg/ml), however, induced the same amounts of IL-8 protein. IL-8 secretion in HEK293, 293-hTLR4A, 293-hMD2-CD14, and 293-hTLR4/MD2–CD14 LPS-TLR4 signaling pathway requires a receptor complex that is composed of TLR4, MD2, and CD14 [28]. HEK293 lacks the expression of TLR4, MD2, and CD14 and produces very low levels of IL-8 [29,30]. To carry out an analysis of the effect of HM on TLR4 signaling, we used 293-hTLR4A, 293-hMD2-CD14, and 293-hTLR4/MD2–CD14
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cell lines that were stably transfected with human TLR4A, MD2-CD14, and TLR4/MD2–CD14 plasmids, respectively. Cells were incubated in serum-free medium with either LPS (10 lg/ml), or HM (5, 10, and 25 lg/ml) for 24 h. Untreated cells were used as controls. To determine whether or not HM activates TLR4 signaling we tested initially its effect on 293-hTLR MD2/ CD14 cells that express TLR4 together with MD2 and CD14 proteins, i.e., the complete receptor complex needed for LPS/TLR4 signaling. Fig. 4A shows that addition of HM at 5 and 10 lg/ml significantly induced IL-8 production (p = 0.005 and p = 0.003, respectively). Similarly, LPS highly induced IL-8. The amount of IL-8 induced by HM (5 and 10 lg/ml) was slightly higher than that induced by LPS (10 lg/ml) with means of 3.691, 3.658, and 3.575 pq/ml, respectively. The LPS results agree with previous results showing an increase in IL-8 production by LPS and HEK293 cells in the presence of TLR4, MD2, and CD14 [28,31]. Transfecting the cells with TLR4, MD2, and CD14 increased in the basal level of IL-8 production by 293-hTLR4/MD2–CD14 as compared to HEK293 even in the absence of stimulation (data not shown). Subsequently, we examined the effect of HM on TLR4 in the absence of MD2 and CD14 by using 293-hTLR4transfected cells (Fig. 4B). Treatment of the cells with HM (5 and 10 lg/ml) or LPS (10 lg/ml), did not significantly induce IL-8 production (p = 0.590, p = 0.590 and p = 0.332, respectively). Much lower amounts of IL-8 were detected in the supernatants of HM-treated (1.790 and 1.759 pq/ml) and LPS-treated cells (1.826 pq/ml) as compared to 293-hTLR4/MD2–CD14 when similarly treated (Figs. 4A and B). Previous studies were controversial in respect to the role of MD2 in LPS/TLR4 signaling and different effects of MD2 on TLR4 signaling had been reported [25,32–35]. However, our data have shown very identical effects, for HM and LPS, on 293-hTLR4 which suggests a similar signaling pathway. Furthermore, we tested the effect of HM on IL-8 production by 293-hMD2-CD14transfected cells. The presence of MD2 and CD14 did
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not induce IL-8 production in HM-treated cells in the absence of TLR4. Fig. 4C shows that neither HM (5 and 10 lg/ml) nor LPS (10 lg/ml) had an effect on IL-8 production (p = 0.439, p = 1.000, and p = 0.439, respectively). Our result on LPS induced IL-8 production agrees with previous results [36]. Discussion A large body of studies has claimed an immunological role for the widely used herb, Nigella sativa L. [37,38]. None of these studies has ascribed the biological effects to the herbal melanin, which is a constituent of the plant. In this study, we investigated the effects of a herbal melanin (HM), extracted from the seed coats of the plant on cytokine production by PBMCs, THP-1 cells and by TLR4-transfected HEK293 cells. Our findings are: (1) Nigella sativa L. HM induced IL-8 mRNA expression and protein production in PBMCs and THP-1 cells, (2) HM induced IL-8 protein production in HEK293 cells co-transfected with TLR4 and its accessory molecules MD2 and CD14, and (3) the expression of IL-8 is dependent on the presence of MD2 and CD14. These results suggest that HM is acting as a TLR4 ligand. It is known that TLRs transmit signals in response to a wide range of microbial products that activate different TLR members, for example, LPS and TLR4 [16]. Ligand activation of TLRs induces cytokines production via activation of the NF-jB signaling pathway [13]. We have characterized first the activation of TLR4 by HM on cells that express TLR4, human THP-1, and PBMC [39,40], using production of IL-8 as an index of the cellular response. Cells were treated in parallel with different concentrations of HM and LPS. THP-1 and PBMC are known to be highly sensitive to LPS, responding by upregulating the secretion of cytokines [41,42]. Quantitation of IL-8 released by THP-1 and PBMCs by ELISA confirmed the stimulatory effects of HM and LPS. High levels of IL-8 were detectable in the supernatants of both cell types. Recently it has been
Fig. 4. Effect of HM on IL-8 production by 293-hTLR4/MD2–CD14 (A), 293-hTLR4A (B), and 293-hMD2-CD14 (C) cell lines. Cells (1 · 106 cells/ml) were treated with HM and LPS as indicated concentrations for 24 h. LPS-treated cells were used as positive control, untreated cells as normal control, and culture media supplemented with 50 lg/ml as negative control. Concentration of IL-8 in the supernatants of 293-hTLR4/MD2–CD14 (A—left), 293hTLR4A (B—middle), and 293-hMD2-CD14 (C—right) was determined by ELISA as specified by manufacturer. Data represent means from three independent experiments that yielded similar results. Values are presented as means ± SE. p < 0.05 is significant.
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reported that LPS up-regulates IL-8 production in PBMCs and THP-1 through TLR4 activation [43,44]. Our results have shown that on using similar concentrations of HM and LPS, induction of IL-8 by HM was stronger than by LPS in THP-1 and was close to LPS in PBMCs. The known TLR4 ligands, such as LPS, require accessory molecules, MD2 and CD14, to turn on and augment TLR4 signaling [25,24]. To determine whether HM can activate TLR4 we used transfected HEK 293 cells that express plasmids for TLR4, MD2, and CD14. Subsequently we challenged the transfected cells with HM or LPS. LPS, in the presence of a TLR4/MD2–CD14 complex, produced the expected, previously reported, strong response of production of high levels of IL-8 by the cells [28,31]. Similar results were also obtained on treating the cells with HM giving rise to a significantly increased production of IL-8. Next, we explored the possible roles for MD2 and CD14 in HM-TLR4-dependent signaling. Both HM and LPS had no significant effect on IL-8 production by 293-hTLR4. Our results have provided additional data supporting previous assumption that the response to LPS requires the presence MD2 in LPS/TLR4 signaling pathway [25,34,35] though other previous studies had reported TLR4-activation by LPS in the absence of MD2 [36]. We also clearly present the similarity between HM and LPS in IL-8 stimulation by TLR4-expressing cells. Whether TLR4 recognized HM by direct binding or through MD2 signaling is yet to be clarified. Similar unanswered questions were raised concerning LPS/TLR4 which merits, in both cases, further detailed investigations [45,46]. Eventually, we tested the effects of both of HM and LPS on 293-hMD2-CD14 cells. Both HM and LPS had no effect on IL-8 production in the absence of TLR4. Similar results were obtained by Heine et al. who showed the activation of TLR4 by LPS in HEK293-TLR4-transfected cells in the absence of CD14 and Garcia-Rodriguez and co-workers reported that transfection of isolated CD14, MD2 or both of them in HEK293 did not confer NF-jb activation by LPS [47,36]. Our results present the similarity in action between LPS and HM in the presence of MD2 and CD14 and absence of TLR4. Melanins are built up of repeated indolic units plus other molecular groups [4] and LPS are made of carbohydrates and fats [18]. Melanins, therefore, have on the basic chemical level, a different structure from that of LPS. However, in this study, HM have shown LPS-like properties with respect to the induction of different cytokines. Previously, we have shown that HM induced TNF-a and IL-6 mRNA and protein production in THP-1, human monocytes, and PBMCs in a way similar to the LPS stimulation of these cells [21]. In this study, we report that both of HM and LPS induce IL-8 production in THP-1 and PBMCs and that they show exactly the same pattern of 1L-8 protein expression in HEK293, 293-hTLR4A, 293-hMD2CD14, and 293-hTLR4/MD2–CD14. Taking together these facts we suggest that the increase in IL-8 induced
by LPS or HM in THP-1, PBMCs, 293-hTLR4, and 293hTLR4-MD2-CD14 cells is via TLR4 activation pathways. Recently Pasco and co-workers [20] reported that melanins extracted from different botanicals other than N. sativa L. activate NF-jB and induce IL-1b production and they tentatively proposed TLR2 as a receptor of ‘‘botanical’’ melanins. Our results demonstrate that HM is a powerful immunostimulant of TLR4-expressing cells. Recently, the immunostimulatory activity of some TLR ligands has led to the proposition of their use as adjuvants for immunotherapy of cancer [48]. Seya and co-workers [49] reported that BCGCWS induced TNF-a secretion from DC via TLR2 and TLR4 and they suggested its use as a potent adjuvant in cancer therapy. HM have induced TNF-a in THP-1, monocytes, and PBMC [21] and IL-8 in HEK-TLR4 transfected cells. One may, therefore, consider HM as an alternative adjuvant for cancer immunotherapy. TLR4 agonists elicit the innate immune system to act against various viral, bacterial, and fungal infections. HM is a non-toxic herbal extract that is safe for oral and/or other methods of application. There are potential applications of HM, as a TLR4 agonist, in many clinical applications such as infectious diseases and cancer. Acknowledgments This work has been supported by the grant of the Knut and Alice Wallenberg Foundation for the Swedish HPR project. We thank Dr. Gamal Mohamed for assistance with statistical analysis. References [1] J. Stinchcombe, G. Bossi, G.M. Griffiths, Linking albinism and immunity: the secrets of secretory lysosomes, Science 305 (2004) 55–59. [2] H. Fedorow, G.M. Halliday, C.H. Rickert, M. Gerlach, P. Riederer, K.L. Double, Evidence for specific phases in the development of human neuromelanin, Neurobiol. Aging 27 (2006) 506–512. [3] M.A. Pathak, Functions of melanin and protection by melanin, in: L. Zeise, M.R. Chedekel, T.B. Fitzpatrick (Eds.), Melanin: Its Role in Human Photoprotection, American Society for Photobiology, Valdenmar Publishing Co, Overland Park, KS, 1995, pp. 125–140. [4] P.A. Riley, Melanin, Int. J. Biochem. Cell Biol. 29 (1997) 1235–1239. [5] G. Prota, Progress in the chemistry of melanins and related metabolites, Med. Res. Rev. 8 (1988) 525–556. [6] A. Pezzella, M. d’Ischia, A. Napolitano, A. Palumbo, G. Prota, An integrated approach to the structure of sepia melanin. Evidence for a high proportion of degraded 5,6-dihydroxyindole-2-carboxylic acid units in the pigment backbone, Tetrahedron 53 (1977) 8281–8286. [7] L. Zeise, Analytical methods for characterization and identification of eumelanins, in: L. Zeise, M.R. Chedekel, T.B. Fitzpatrick (Eds.), Melanin: Its Role in Human Photoprotection, Valdenmar Publishing, Overland Park, KS, 1995, pp. 65–79. [8] E. Buszman, M. Kopera, T. Wilczok, Electron spin resonance studies of chloroquine–melanin complexes, Biochem. Pharmacol. 33 (1984) 7–11. [9] V.M. Sava, S.M. Yang, M.Y. Hong, P.C. Yang, G.S. Huang, Isolation and characterization of melanic pigments derived from tea and tea polyphenols, Food Chem. 73 (2001) 177–184.
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