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ScientificstudiesonCamelurine
http://caller-to-islam.tk
Camel urine components display anti-cancer properties in vitro
Nujoud Al-Yousef a
, Ameera Gaafar b
, Basem Al-Otaibi c
, ...
2. Materials and methods
2.1. CU preparation and use
Urine was collected aseptically from three young (3–4 years
old) fema...
cells (PBMCs) were obtained by centrifugation over ficol-hypaque
gradients (Pharmacia, Uppsala, Sweden). 106
cells were cul...
2.12. Statistical analysis
Statistical analysis was performed by student’s t-test and p
values of 0.05 and less were consi...
shows that CU treatment (16 mg/mL) for 72 h triggered mainly
apoptosis (90%) with only slight proportion of necrosis. Inte...
On the other hand, the produced level of IL-6 was 5 fold reduced
by CU-treatment (Fig. 6B). Furthermore, CU strongly reduc...
mechanism in Hepa 1c1c7 cell line. Journal of Ethnopharmacology 133,
184–190.
Altieri, D.C., 2008. Survivin, cancer networ...
Original Articles
The Antiplatelet Activity of Camel Urine
Abdulqader Alhaidar, BPharm, MSc, PhD,1
Abdel Galil M. Abdel Ga...
was demonstrated that the ultrastructure and function of
camel platelets bears a high degree of dissimilarity as com-
pare...
Statistical analysis
Data were analyzed using the SSPS program (Version 15).
Differences in means between groups were comp...
samples treated with the lower dilutions (mean of < 300)
(Table 4).
Discussion
Urine therapy, or urotherapy, has been in p...
the putative antiplatelet properties of camel blood. Analysis
of platelet function using the PFA-100 platelet function an-...
20. Khorshid F, Moshref S, Heffny N. An ideal selective anti-
cancer agent in vitro, I: Tissue culture study of human lung...
379
RESEARCH OPINIONS IN ANIMAL & VETERINARY SCIENCES
PRINT ISSN 2221-1896, ONLINE ISSN 2223-0343
www.roavs.com
Preliminar...
Khogali et al roavs, 2011, 1(6), 379-381.
380
Khogali et al roavs, 2011, 1(6), 379-381.
381
CU at 0.1 ml/bath completely abolished the
spontaneous contractions of rabbi...
Journal of Natural Sciences Research www.iiste.org
ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
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Cyto...
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ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
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ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
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Journal of Natural Sciences Research www.iiste.org
ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
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Tab...
Journal of Natural Sciences Research www.iiste.org
ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
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Tab...
This academic article was published by The International Institute for Science,
Technology and Education (IISTE). The IIST...
POSTER PRESENTATION Open Access
The effect of camel urine on islet morphology
and CCL4-induced liver cirrhosis in rat
S Al...
African Journal of Agricultural Research Vol. 5(11), pp. 1331-1337, 4 June, 2010
Available online at http://www.academicjo...
Scientific studies on Camel urine
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Scientific studies on Camel urine

  1. 1. ScientificstudiesonCamelurine http://caller-to-islam.tk
  2. 2. Camel urine components display anti-cancer properties in vitro Nujoud Al-Yousef a , Ameera Gaafar b , Basem Al-Otaibi c , Ibrahim Al-Jammaz c , Khaled Al-Hussein b , Abdelilah Aboussekhra a,n a Department of Molecular Oncology, King Faisal Specialist Hospital and Research Center, MBC # 03, PO BOX 3354, Riyadh 11211, Saudi Arabia b Histocompatibility & Immunogenetics Research Unit, Stem Cell Therapy Program, King Faisal Specialist Hospital and Research Center, MBC # 03, PO BOX 3354, Riyadh 11211, Saudi Arabia c Department of Cyclotron and Radiopharmaceuticals, King Faisal Specialist Hospital and Research Center, MBC # 03, PO BOX 3354, Riyadh 11211, Saudi Arabia a r t i c l e i n f o Article history: Received 17 March 2012 Received in revised form 23 July 2012 Accepted 27 July 2012 Available online 16 August 2012 Keywords: Camel urine Cancer Apoptosis Immune response a b s t r a c t Ethnopharmacological relevance: While camel urine (CU) is widely used in the Arabian Peninsula to treat various diseases, including cancer, its exact mechanism of action is still not defined. The objective of the present study is to investigate whether camel urine has anti-cancer effect on human cells in vitro. Materials and methods: The annexinV/PI assay was used to assess apoptosis, and immunoblotting analysis determined the effect of CU on different apoptotic and oncogenic proteins. Furthermore, flow cytometry and Elispot were utilized to investigate cytotoxicity and the effect on the cell cycle as well as the production of cytokines, respectively. Results: Camel urine showed cytotoxicity against various, but not all, human cancer cell lines, with only marginal effect on non-tumorigenic epithelial and normal fibroblast cells epithelial and fibroblast cells. Interestingly, 216 mg/ml of lyophilized CU inhibited cell proliferation and triggered more than 80% of apoptosis in different cancer cells, including breast carcinomas and medulloblastomas. Apoptosis was induced in these cells through the intrinsic pathway via Bcl-2 decrease. Furthermore, CU down- regulated the cancer-promoting proteins survivin, b-catenin and cyclin D1 and increased the level of the cyclin-dependent kinase inhibitor p21. In addition, we have shown that CU has no cytotoxic effect against peripheral blood mononuclear cells and has strong immuno-inducer activity through inducing IFN-g and inhibiting the Th2 cytokines IL-4, IL-6 and IL-10. Conclusions: CU has specific and efficient anti-cancer and potent immune-modulator properties in vitro. & 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Cancer remains a worldwide public health concern. Although cancer incidence has increased over the past four decades, mortality has remained stable. This is probably reflecting the improvement in treatment options. Chemotherapy is a core modality for the treat- ment of a wide range of cancer types at different stages. However, most of the currently used chemotherapeutic regimens are highly toxic with long term side effects, morbidity and lethality (Rood et al., 2004; Rossi et al., 2008). Of 121 prescription drugs in use for cancer treatment, 90 are derived from plant species and 74% of these drugs were discovered by investigating a folklore claim (Craig, 1997; Craig and Beck, 1999). Among the natural products in the Arabic peninsula that are used for the treatment of various diseases is camel urine. Patients drink camel urine ($100 mL/day) either alone or mixed with milk. This prompted us to ask whether the urine of this extraordinary animal has anticancer properties? Camel urine urine has an unusual and unique biochemical composition. Indeed, Dr. Bernard Read published in 1925 a paper describing the chemical constituents of camel (Camelus bactrinus) urine (Read, 1925). He has reported that unlike all the other animals, including humans, camels excrete no ammonia and only very slight trace of urea, and these molecules are responsible for bad smell and toxicity of urine. However, a significant amount of creatine and creatinine was detected. Further studies have shown that camel urine contains about 10 folds more mineral salts than human urine. Furthermore, while human urine is acidic, camel urine is basic with a pHZ7.8 (Read, 1925). In a recent report, Alhaidar et al. have shown that camel urine has potent antiplatelet activity against ADP-induced (clopidogrel-like) and AA-induced (aspirin-like) platelet aggregation (Alhaidar et al., 2011). Several have claimed anti-cancer effects of camel urine. However, no clear scientific evidence has been pub- lished so far to confirm or refute these claims. Recently, it has been shown that camel urine inhibits the induction of Cyp1a1, a cancer activating gene, in Hepa 1c1c7 cell line (Alhaider et al., 2011). In the present report we have shown that camel urine has indeed several anti-cancer properties in vitro. Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.07.042 n Correspondence to: Department of Molecular Oncology, King Faisal Specialist Hospital and Research Center, MBC # 03-66, PO BOX 3354, Riyadh 11211, Saudi Arabia. Tel.: þ966 1 464 7272x32840; fax: þ966 1 442 7858. E-mail address: aboussekhra@kfshrc.edu.sa (A. Aboussekhra). Journal of Ethnopharmacology 143 (2012) 819–825
  3. 3. 2. Materials and methods 2.1. CU preparation and use Urine was collected aseptically from three young (3–4 years old) female camels (Camelus dromadaries), desert living healthy local red ones, in sterile bottles and urine from each camel was pooled separately. CU was lyophilized, weighted and immediately before utilization it was resuspended in PBS. Chemical character- ization of these samples has been performed using 1 H-NMR analysis using DMSO as solvent system. The obtained spectra indicate that these CU samples are of the same nature, and chemical shifts are as follow: CU1: 1 H-NMR (DMSO, 298 K) d: 7.85 (d), 7.52 (d), 7.46 (t), 7.27 (d), 7.04 (s), 3.66 (s), 3.35 (s), 2.91 (s), 2.50 (s). CU2: 1 H-NMR (DMSO, 298 K) d: 7.86 (d), 7.47 (d), 7.46 (t), 7.28 (d), 7.04 (s), 3.66 (s), 3.35 (s), 2.91 (s), 2.50 (s). CU3: 1 H-NMR (DMSO, 298 K) d: 7.85 (d), 7.48 (d), 7.46 (t), 7.28 (d), 7.03 (s), 3.66 (s), 3.34 (s), 2.91 (s), 2.50 (s). These results were confirmed by HPLC, which shows a major peak at around 11 min retention time for the 3 samples (Fig. 1). 2.2. Cell lines and cell culture MCF 10A, MDA-MB-231, U2OS, DAOY, LoVo and HCT-116 were obtained from ATCC and were cultured following the instructions of the company. MED-1, MED-8, MED-13 are primary medullo- blastoma cells that were cultured as previously described (Shinwari et al., 2011). HFSN1 cells are primary skin fibroblast cells that were routinely cultured in the DMEM:F12 (50:50) medium supplemented with 10% FBS. 2.3. Cellular lysate preparation Cells were washed and scraped in lysis buffer [150 mM NaCl, 1% NP40, 50 mM Tris–HCl (pH 7.5)] supplemented with 40 mg/ml aprotinin, 20 mg/ml leupeptin and 5 mg/ml pepstatin. Lysates were homogenized using a Polytron homogenizer and then centrifuged at 14000 rpm in an Eppendorf microcentrifuge tube for 20 min. The supernatant was removed, aliquoted and stored at À80 1C. 2.4. Immunoblotting SDS-PAGE was performed using 12% separating minigels as previously described (Al-Hujaily et al., 2011). The antibodies directed against b-Actin (C-11), GAPDH (FL-335), Bax (B-9), Bcl- 2 (C-2), survivin (C-19), p21 (F-5), and p53 (DO-1), b-Catenin (9F2) and Cyclin D1 (HD11) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. The antibodies against cleaved caspase3 (Asp175), cleaved caspase9 (Asp315) and cleaved PARP (Asp214) were purchased from Cell Signaling. 2.5. Quantification of protein expression level The expression levels of the immunoblotted proteins were measured using the densitometer (BIO-RAD GS-800 Calibrated Densitometer). X-ray films were scanned and protein signal intensity of each band was determined. Next, dividing the obtained value of each band by the values of the corresponding internal control allowed a correction of the loading differences. The fold of induction in the protein levels was determined by dividing the corrected values that corresponded to the treated samples by that of the non-treated one (time 0). 2.6. Annexin V and flow cytometry For each cell culture, cells were either not treated (control) or treated with CU. Detached and adherent cells were then har- vested after 72 h, unless otherwise stated, and treated as pre- viously described (Al-Hujaily et al., 2011). For each cell culture 3 independent experiments were performed using 104 cells in each experiment. 2.7. PBMC preparation and culture 10 ml of heparinised blood samples were obtained from healthy volunteer employers and then peripheral blood mononuclear Fig. 1. HPLC analysis of CU samples, 3 camel urine samples (CU1, CU2, CU3) were analyzed by HPLC and the corresponding chromatograms are shown. N. Al-Yousef et al. / Journal of Ethnopharmacology 143 (2012) 819–825820
  4. 4. cells (PBMCs) were obtained by centrifugation over ficol-hypaque gradients (Pharmacia, Uppsala, Sweden). 106 cells were cultured in RPMI-1640 medium supplemented with 10% FCS (Gibco, Island NY, PBS) and complements and incubated at 37 1C in 5% CO2 incubator. 2.8. PBMC cytotoxicity PBMCs were treated with different concentrations of CU for 3 days and cell death was measured with annexinV/PI-flow cytometry. 2.9. Elispot assay Elispot assay (Diaclone Research, France) was used as recom- mended by the manufacturer. 5.103 cells suspended in 100 ml complete media containing CU (16 mg/mL) and IL-2 and were incubated for 10 days in 96-well microtiter plates pre-coated with the appropriate antibody. Subsequently, cells were either treated with PHA (10 mg/ml) or LPS (1 mg/ml), for stimulating the produc- tion of IFN-g, IL-4 and IL-10 and IL-6, respectively. The plates were incubated at 37 1C in humidified atmosphere containing 5% CO2 for appropriate period of time according to the different cytokine kinetics. After washing, the plates were read using the Elispot AID reader version 3.0. 2.10. Cell proliferation analysis 2–4.103 cells were seeded in 96 well plate and 100 ml of complete medium was loaded in each well. The plate was incubated for at least 30 min in a humidified, 37 1C, 5% CO2 incubator, and then was inserted into the Real-Time Cell Electro- nic Sensing System (RT-CES system) (ACEA Biosciences Inc., San Diego, CA) for 16 h. Cells were then either PBS-treated or treated with CU (16 mg/mL) and cell proliferation was monitored for 24 h. 2.11. HPLC analysis HPLC analysis was carried out on Econosil C-18 reversed phase column (analytical, 250 mm  4.6 mm). The solvent system used was non-linear gradient (eluent A, water with 0.1% TFA; eluent B, ACN; gradient, 0–10% B, 10–90% B, 90–90% B and 90–0% B over 5 min each at flow rate of 1.0 mL/min). A Jasco chromatographic system equipped with a variable wavelength ultraviolet monitor and in tandem with a Canberra flow through radioactivity detector was used. Ultraviolet absorption was monitored at 254 nm. Chromatograms were acquired and analyzed using BORWIN software. Fig. 2. CU is cytotoxic against cancer cells but not against normal fibroblasts and non-tumorigenic epithelial cells. Cells were treated with CU as indicated and the cytotoxic effect was assessed by flow cytometry following PI staining. (A) Cells were treated with the indicated CU doses for 72 h. (B) Cells were treated with CU (16 mg/mL) for the indicated periods of time. (C and D) The indicated cells were treated with CU (16 mg/mL) for 72 h. (C) Flow cytometry charts. (D) Histogram, Error bars represent means7S.D. *: p valueo0.05. N. Al-Yousef et al. / Journal of Ethnopharmacology 143 (2012) 819–825 821
  5. 5. 2.12. Statistical analysis Statistical analysis was performed by student’s t-test and p values of 0.05 and less were considered as statistically significant. 3. Results 3.1. Camel urine is cytotoxic against various cancer cells We started this study by investigating the cytotoxic effect of CU against the MDA-MB-231 breast cancer cells as well as the non- tumorigenic breast epithelial cells (MCF 10A), using the Propidium Iodide (PI)/flow cytometry technique. Cells were treated with increas- ing concentrations of CU for 72 h. Fig. 2A shows dose-dependent increase in the proportion of death cells among MDA-MB-231 cells, more than 80% died in response to 16 mg/ml ($800 ml of urine). On the other hand, the same concentrations of CU had no effect on MCF 10A cells. Next, we investigated the effect of CU over time using 16 mg/ml. Fig. 2B shows that the effect of CU on breast cancer cells increased with time reaching its maximum at 72 h of treatment, while no effect was observed on MCF 10A cells. This shows that CU is cytotoxic, but with specific effect on breast cancer cells. Next, we investigated the specific cytotoxic effect of CU on other cancer cell lines. Fig. 1C shows that based on the response to CU, these cells can be grouped into 2 different sub-groups. Group 1 contains CU-resistant cells including MCF 10A and the normal fibroblast (HFSN-1) cells, as well as the osteosarcoma (U2OS), the breast cancer (MCF-7), the medulloblastoma MED-8 and the colon cancer (LoVo and HCT-116) cells. The second group is composed of CU-sensitive cells, with more than 50% cell death, and includes the breast cancer (MDA-MB-231) and the medullo- blastoma (DAOY, MED-4 and MED-13) cells (Figs. 2C, 1D). Inter- estingly, these effects were obtained with similar doses of urine collected from 2 other female camels (data not shown), indicating that these CU samples collected from different animals living in different regions and receiving different foods have similar chemical characteristics (materials and methods) and cytotoxic effect against cancer cells. Therefore, CU powders from these camels were pooled and used in the next experiments. 3.2. Camel urine triggers apoptosis in cancer cells In order to identify the cell death pathway that CU triggers in cancer cells, we first made use of the AnnexinV-PI/flow cytometry technique that can detect both apoptotic and necrotic cells. Fig. 3 Fig. 3. CU induces apoptosis through the mitochondrial pathway. Cells were treated either with PBS or with CU (16 mg/mL) for 72 h, and then cell death was assessed by annexin V/PI in association with flow cytometry. (A) Charts, the numbers into the boxes indicate the proportion of normal (lower, left), early apoptotic (lower, right), late apoptotic (higher, right) and necrotic (higher, left) cells. (B) Histograms presenting the proportions of induced apoptosis in the indicated normal and cancer cells. Error bars represent standard deviations of three different experiments, *: p valueo0.05. (C) MDA-MB-231 cells were treated with CU (16 mg/mL), and then were harvested after the indicated periods of time. 50 mg of extracted proteins were used for western blot analysis utilizing the indicated antibodies. The numbers below the bands represent the corresponding expression levels as compared to time 0 and after normalization against GAPDH. D. As in C, b-actin was used as internal control and the graph is showing the Bax/Bcl-2 ratios after normalization against b-actin. Error bars represent standard deviations of three different experiments. N. Al-Yousef et al. / Journal of Ethnopharmacology 143 (2012) 819–825822
  6. 6. shows that CU treatment (16 mg/mL) for 72 h triggered mainly apoptosis (90%) with only slight proportion of necrosis. Interest- ingly, the efficiency of CU in inducing apoptosis was variable among the various cancer cells. While the medulloblastoma DAOY and MED-4 cells showed high sensitivity, MED-8 showed clear resistance (Fig. 3B). Interestingly, Fig. 3 confirmed what has been shown in Fig. 2 for the sensitive cells. However, for the resistant ones, the annexin V assay showed higher proportion of cells dying through the apoptotic pathway. To confirm the induction of apoptosis, MDA-MB-231 cells were treated with CU (16 mg/mL) and harvested after different periods of time (0–72 h). Whole cell extracts were prepared and 50 mg of proteins were used to analyze the effect of CU on the cleavage of the important effector caspase 3 by immunoblotting. Fig. 3C shows that treatment by CU caused a time-dependent increase in the level of the active cleaved caspase 3, reaching at 72 h a level 18.6 fold higher than the basal level. Similarly, the level of cleaved PARP increased 3.4 fold as compared to the basal level after 48 h of treatment (Fig. 3C). Together, these results clearly show that CU triggers apoptosis in MDA-MB-231 cells. 3.3. Camel urine triggers apoptosis via the mitochondrial pathway Next, we evaluated the effect of CU on the levels of the pro- and anti-apoptotic proteins (Bax and Bcl-2). Fig. 3D shows that CU triggered a time-dependent decrease in the level of the anti- apoptotic protein Bcl-2 and a time-dependent increase in the level of the pro-apoptosis protein Bax. This led to a time- dependent increase in the Bax/Bcl-2 ratio, reaching its maximum (3 fold higher) after 72 h of treatment (Fig. 3D). This shows that CU induces apoptosis mainly through the mitochondrial pathway via Bcl-2 decrease. To confirm this we assessed the effect of CU on the level of caspase 9 and cleaved caspase 9. Fig. 3C shows that while the level of caspase 9 decreased 5 fold, a strong increase in the level of cleaved caspase 9 was observed, which confirms the induction of the apoptotic mitochondrial pathway by CU. Further- more, CU also decreased the expression of the anti-apoptotic survivin protein, reaching a level more than 33 fold lower after 72 h of treatment (Fig. 3C). 3.4. Camel urine efficiently inhibits the proliferation of breast cancer cells Next, we used the Real-Time Cell Electronic Sensing System to study the effect of CU on MCF 10A and MDA-MB-231 cell proliferation. Therefore, cells were cultured for 16 h and then were treated either with PBS or with CU (16 mg/mL) and were reincubated for 24 h during which their proliferation rate was assessed. Fig. 4 shows that while PBS-treated MDA-MB-231 and CU-treated MCF 10A cells continued to proliferate normally, the proliferation rate of CU-treated MDA-MB-231 cells decreased sharply and cells stopped proliferating immediately after adding CU. This shows that CU has great anti-proliferative effect on breast cancer cells. 3.5. Effect of camel urine on cancer-related genes MDA-MB-231 cells were treated with CU (16 mg/mL) for different periods of time (0–24 h), and then protein levels were monitored by immunoblotting. Interestingly, CU significantly down-regulated b-catenin, which reached a level 5 fold lower after 16 h of treatment (Fig. 5). To confirm the inhibitory effect of CU on b-catenin, we studied the effect of CU on its major target cyclin D1 (Rowlands et al., 2004). Indeed, the level of cyclin D1 decreased also more than two fold after 24 h of treatment (Fig. 5). In addition, CU decreased by 2 fold the level of survivin (Fig. 4). Together, these results show that CU inhibits the b-catenin- related cancer pathway. Furthermore, CU up-regulated the expression of the cyclin-dependent kinase inhibitor p21, with a maximum level (3.5 fold higher) reached after 16 h of treatment (Fig. 5). 3.6. Camel urine is not cytotoxic against blood cells and is a potent modulator of the immune system We first studied the cytotoxic effect of CU on peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals. Cells were treated with increasing CU concentrations, incubated for 6 h and then cell viability was assessed using AnnexinV/PI flow cytometry. Fig. 6A shows that CU was not cytotoxic against PBMCs. At high concentration (20 mg/mL) the viability decreased to about 45%. However, the level of CD3 did not decrease by increasing the CU dose. This indicates that the proportion of T cells did not change, showing that CU does not affect these important population of immune cells. Moreover, the CU acti- vated these cells as indicated by the increase of the CD3þ CD69þ and CD3þ HLA-DRþ . This activation was more pronounced at the high dose of 20 mg/mL (Fig. 6A). Next, we evaluated the effect of CU on the immunogenecity of PBMCs from normal controls. Interestingly, treatment of PBMCs with CU (20 mg/mL) stimulated the production of IFN-g, which reached a level 25 fold higher than that of resting PBMCs (Fig. 6B). Fig. 4. CU inhibits cell proliferation. Sub-confluent cells were treated either with PBS or with CU (16 mg/mL) for the indicated periods of time, and cell proliferation rate was determined using the Real-Time Cell Electronic Sensing System. Fig. 5. CU modulates the expression of several oncoproteins. MDA-MB-231 cells were treated with CU (16 mg/mL) for the indicated periods of time. Subsequently, cells were harvested and 50 mg of extracted proteins were used for western blot analysis using the indicated antibodies. The numbers under the bands represent the corresponding expression levels as compared to time 0 and after normal- ization against GAPDH. N. Al-Yousef et al. / Journal of Ethnopharmacology 143 (2012) 819–825 823
  7. 7. On the other hand, the produced level of IL-6 was 5 fold reduced by CU-treatment (Fig. 6B). Furthermore, CU strongly reduced the production of IL-4 and IL-10, which became almost undetectable. This indicates that CU is a potent immuno-modulator product. 4. Discussion An efficient anti-cancer agent is expected to trigger cell death and/or inhibit cell proliferation of cancer cells avoiding normal ones, and activates the immune system. In the present report we present evidence that camel urine collected from 3 different female camels presents all these features. Indeed, we have first shown that CU is cytotoxic against different human cancer cell lines, while it has only marginal effect on normal fibroblasts and non-tumorigenic epithelial cells. This specific anti-cancer effect was not observed when cells were exposed to rat urine, which killed both cancer as well as normal cells with similar effect (data not shown). Next, we used different techniques to elucidate the cell death pathway induced by CU, and we have shown that CU triggers mainly apoptosis through the mitochondrial pathway, via Bcl-2 decrease. Importantly, cancer cells exhibited differential response to the killing effect of CU. In fact, U2OS, MED-8, MCF-7 and MED-13 were resistant to CU. Furthermore, even tumors from the same organ showed different sensitivity to CU. For example, while the medulloblastoma DAOY cell line and primary cells MED-4 showed high sensitivity to CU, MED-8 and MED-13 were resistant to the same dose. Similarly, the breast cancer cell line MCF7 exhibited high resistance, whilst MDA-MB-231 was highly sensitive (Fig. 3). This suggests that CU-dependent induction of apoptosis is genetically regulated. Indeed, we have shown that CU modulates the expression of several cancer-related genes, such as b-catenin, cyclin D1 and the anti-apoptotic survivin protein. b-catenin is a transcription factor that has been found highly expressed in various types of cancer, including breast carcinomas (Prasad et al., 2007; Paul and Dey, 2008). Cyclin D1 is an oncogene that is over-expressed in about 50% of all breast cancer cases (Bartkova et al., 1995), and its down-regulation is an important target in breast cancer therapy (Yang et al., 2006). Furthermore, CU had a strong inhibitory effect on the two major apoptosis inhibitor proteins Bcl-2 and survivin, which are both related to breast cancer pathology and therapeutic outcome (Tanaka et al., 2000; Callagy et al., 2006; Altieri, 2008). Furthermore, it has been recently shown that CU significantly inhibits the induction of Cyp1a1, a well known cancer activating gene, in Hepa 1 C7 cell line (Alhaider et al., 2011). Therefore, CU seems to inhibit cancer through targeting several molecular signaling pathways. In addition, CU exhibited potent anti-proliferative effect on breast cancer cells but not on non-tumor epithelial cells (Fig. 4). This effect could be mediated through the induction of the cyclin- dependent kinase inhibitor p21. Indeed, we have shown that CU up-regulates p21 in the p53-defective MDA-MB-231 cells (Lacroix et al., 2006), indicating that this effect is p53-independent. Furthermore, CU enhanced the production of the main Th1 cytokine IFN-g and also has a great inhibitory effect on the production of the Th2 cytokines IL-4, IL-6 and IL-10, which has immunosuppressive and tumor growth stimulating functions. Cumulative evidence indicate that IL-4 is a key cytokine not only for Th2 type immune reactions but also for tumor cell growth itself in various human cancers, including breast carcinomas (Nagai and Toi, 2000). Similarly, high systemic levels of IL-10 correlated well with poor survival of patients suffering from different types of cancer (Mocellin et al., 2005). The IL-6 cytokine is a potent growth factor for breast cancer cells. Moreover, high levels of IL-6 were detected in breast cancer serums and the increase correlated with the stage of the tumors (Knupfer and Preiss, 2007). This indicates that IL-6 down-regulation holds promises as a potential therapeutic strategy to combat breast cancer. In conclusion, the present data provide clear indication that camel urine has anticancer effects on various human cancer cell lines. Therefore, we are currently searching for the active molecule(s) present in this natural animal product. Acknowledgments We are grateful to the Research Centre Administration for their continuous support. We also thank P.S. Manogaran for his help with the flow cytometry, and M. Velasco for his help with the figures. This work was performed under RAC # 2100018. References Al-Hujaily, E.M., Mohamed, A.G., Al-Sharif, I., Youssef, K.M., Manogaran, P.S., Al-Otaibi, B., Al-Haza’a, A., Al-Jammaz, I., Al-Hussein, K., Aboussekhra, A., 2011. PAC, a novel curcumin analogue, has anti-breast cancer properties with higher efficiency on ER-negative cells. Breast Cancer Research and Treatment 128, 97–107. Alhaidar, A., Abdel Gader, A.G., Mousa, S.A., 2011. The antiplatelet activity of camel urine. Journal of Alternative and Complementary Medicine 17, 803–808. Alhaider, A.A., El Gendy, M.A., Korashy, H.M., El-Kadi, A.O., 2011. Camel urine inhibits the cytochrome P450 1a1 gene expression through an AhR-dependent Fig. 6. CU is a potent immuno-modulator. (A) PBMCs were treated with the indicated concentrations of CU for 6 h and the cytotoxic effect was assessed with the annexin V/PI-flow cytometry assay. (B) PBMCs were treated either with PBS (control) or with CU (20 mg/mL), and then the production of the indicated cytokines was assessed by Elispot using the appropriate antibodies. Error bars represent means7SDs. N. Al-Yousef et al. / Journal of Ethnopharmacology 143 (2012) 819–825824
  8. 8. mechanism in Hepa 1c1c7 cell line. Journal of Ethnopharmacology 133, 184–190. Altieri, D.C., 2008. Survivin, cancer networks and pathway-directed drug discovery. Nature Reviews Cancer 8, 61–70. Bartkova, J., Lukas, J., Strauss, M., Bartek, J., 1995. Cyclin D1 oncoprotein aberrantly accumulates in malignancies of diverse histogenesis. Oncogene 10, 775–778. Callagy, G.M., Pharoah, P.D., Pinder, S.E., Hsu, F.D., Nielsen, T.O., Ragaz, J., Ellis, I.O., Huntsman, D., Caldas, C., 2006. Bcl-2 is a prognostic marker in breast cancer independently of the Nottingham Prognostic Index. Clinical Cancer Research 12, 2468–2475. Craig, W., Beck, L., 1999. Phytochemicals: health protective Effects. Canadian Journal of Dietetic Practice and Research 60, 78–84. Craig, W.J., 1997. Phytochemicals: guardians of our health. Journal of the American Dietetic Association 97, S199–204. Knupfer, H., Preiss, R., 2007. Significance of interleukin-6 (IL-6) in breast cancer (review). Breast Cancer Research and Treatment 102, 129–135. Lacroix, M., Toillon, R.A., Leclercq, G., 2006. p53 and breast cancer, an update. Endocrine-Related Cancer 13, 293–325. Mocellin, S., Marincola, F.M., Young, H.A., 2005. Interleukin-10 and the immune response against cancer: a counterpoint. Journal of Leukocyte Biology 78, 1043–1051. Nagai, S., Toi, M., 2000. Interleukin-4 and breast cancer. Breast Cancer 7, 181–186. Paul, S., Dey, A., 2008. Wnt signaling and cancer development: therapeutic implication. Neoplasma 55, 165–176. Prasad, C.P., Gupta, S.D., Rath, G., Ralhan, R., 2007. Wnt signaling pathway in invasive ductal carcinoma of the breast: relationship between beta-catenin, dishevelled and cyclin D1 expression. Oncology 73, 112–117. Read, B.E., 1925. Chemical constituents of camel’s urine. Journal of Biological Chemistry 64, 615–617. Rood, B.R., Macdonald, T.J., Packer, R.J., 2004. Current treatment of medulloblas- toma: recent advances and future challenges. Seminars in Oncology 31, 666–675. Rossi, A., Caracciolo, V., Russo, G., Reiss, K., Giordano, A., 2008. Medulloblastoma: from molecular pathology to therapy. Clinical Cancer Research 14, 971–976. Rowlands, T.M., Pechenkina, I.V., Hatsell, S., Cowin, P., 2004. Beta-catenin and cyclin D1: connecting development to breast cancer. Cell Cycle 3, 145–148. Shinwari, Z., Al-Hindi, H., Al-Shail, E., Khafaga, Y., Al-Kofide, A., Al-Kum, N., Aboussekhra, A., 2011. Response of medulloblastoma cells to vincristine and lomustine: role of TRKC, CTNNB1 and STK15. Anticancer Research 31, 1721–1734. Tanaka, K., Iwamoto, S., Gon, G., Nohara, T., Iwamoto, M., Tanigawa, N., 2000. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clinical Cancer Research 6, 127–134. Yang, C., Trent, S., Ionescu-Tiba, V., Lan, L., Shioda, T., Sgroi, D., Schmidt, E.V., 2006. Identification of cyclin D1- and estrogen-regulated genes contributing to breast carcinogenesis and progression. Cancer Research 66, 11649–11658. N. Al-Yousef et al. / Journal of Ethnopharmacology 143 (2012) 819–825 825
  9. 9. Original Articles The Antiplatelet Activity of Camel Urine Abdulqader Alhaidar, BPharm, MSc, PhD,1 Abdel Galil M. Abdel Gader, MD, PhD,1 and Shaker A. Mousa, PhD, MBA1,2 Abstract Background: For centuries, camel urine has been used for medicinal purposes and anecdotally proclaimed as a cure for a wide range of diseases. However, the apparent therapeutic actions of camel urine have yet to be subjected to rigorous scientific scrutiny. Recent preliminary studies from the authors’ laboratory have indicated that camel urine possesses potent antiplatelet activity, not found in human or bovine urines, suggesting a possible role for camel urine in inhibiting platelet function. The goal of the current study was to characterize the antiplatelet activity of camel urine against normal human platelets based on agonist-induced aggregation and platelet function analyzer (PFA-100) closure time. Materials and methods: Urine was collected from healthy virgin, pregnant, and lactating camels aged 2–10 years. Platelet-rich plasma (PRP) was prepared from blood collected from healthy individuals’ blood into ci- trated anticoagulant. Agonist-induced aggregometry using donor PRP and PFA-100 closure times in whole blood were carried out in the presence and absence of added camel urine. The responses of platelets to multiple doses of camel urine were also assessed. The experimental procedure was repeated in human and bovine urines. Results: Camel urine completely inhibited arachidonic acid (AA) and adnosine diphosphate (ADP)–induced aggregation of human platelets in a dose-dependent manner. PFA-100 closure time using human whole blood was prolonged following the addition of camel urine in a dose-dependent manner. Virgin camel urine was less effective in inhibiting ADP-induced aggregation as compared to urine from lactating and pregnant camels; however, all three showed comparable inhibitory activity. Neither human nor bovine urine exhibited antiplatelet activity. Conclusions: Camel urine has potent antiplatelet activity against ADP-induced (clopidogrel-like) and AA- induced (aspirin-like) platelet aggregation; neither human nor bovine urine exhibited such properties. These novel results provide the first scientific evidence of the mechanism of the presumed therapeutic properties of camel urine. Introduction The one-humped camel (Camelus dromedaries) survives and reproduces under conditions of extreme drought and heat that are unsustainable to most other species of domestic mammal. Desert dwellers have used the camel for transpor- tation and as a source of food, but just as importantly, its milk and urine have been used as medicines for centuries.1,2 Camel milk and urine, for example, have been used to treat various ailments such as cancer,3,4 chronic hepatitis,5 hepatitis C,6,7 and peptic ulcers.8 More recently, it has been reported that camel milk can be used to successfully treat severe food al- lergies in children who are unresponsive to more conven- tional treatments.9 Most of the claimed therapeutic benefits of camel milk and urine are attributed variously to anti-infective, anti- inflammatory, and anticancer properties; by comparison, very little information is available on the efficacy of camel urine and/or milk in treating cardiovascular diseases. Short- chain peptides prepared from bovine milk have been shown to have potent antihypertensive angiotensin-converting en- zyme inhibitory action, because they can significantly reduce blood pressure after intravenous or oral administration, but they show little or no effect in normotensive subjects.10,11 By contrast, none of the claims of therapeutic benefit of camel urine or milk have been subjected to rigorous scientific scrutiny, and as a result, skepticism about camel urine, in particular as a form of alternative therapy, is strong. Along with this, there is a severe shortage of information on the constituents of camel milk and urine. The authors’ interest in this area stems from recent work in our laboratory characterizing camel platelets, in which it 1 The Coagulation Research Laboratory, Department of Physiology, College of Medicine and King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia. 2 The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY. THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINE Volume 17, Number 9, 2011, pp. 803–808 ª Mary Ann Liebert, Inc. DOI: 10.1089/acm.2010.0473 803
  10. 10. was demonstrated that the ultrastructure and function of camel platelets bears a high degree of dissimilarity as com- pared to human platelets.12,13 In addition, camels exhibited markedly inhibited platelet function in terms of agonist- induced aggregation responses and platelet function ana- lyzer (PFA-100) closure times.12 Notably, the addition of camel platelet–poor plasma to packed human erythrocytes resulted in a prolongation of PFA-100 closure time in human blood samples (Abdul Gader, unpublished observations). These re- sults suggest that camel plasma has antiplatelet properties. The authors set out to investigate whether camel urine has similar antiplatelet activity, perhaps lending credence to the claims of the therapeutic benefit of camel urine. The aim of the current study was to characterize the an- tiplatelet actions of camel urine on normal human platelets based on agonist-induced aggregation responses and PFA- 100 closure times. Camel urine exhibited potent platelet in- hibitory activity, blocking both the prostaglandin pathway (aspirin-like activity) as well as the adenosine diphosphate (ADP) receptor–mediated pathway (clopidogrel-like activity). Neither type of activity was detectable in human or bovine urine. These novel findings offer the first scientific evidence in support of the putative therapeutic properties of camel urine. Materials and Methods Animals and urine collection Urine was collected from healthy virgin, pregnant, and lactating domesticated camels (Camelus dromedaries). All camels were females, aged 2–10 years. The camels were raised on a private farm, were disease-free, and had free access to water and camel feed. The collection of urine was usually carried out during feeding and was performed by experienced camel attendants. Urine was allowed to flow directly into stainless steel containers and then transferred to glass vials. Urine samples were transported to the laboratory as soon as practical ( < 4 hours) and were stored at - 80°C until use. Human and bovine urine was collected and stored in a similar manner. Collection of human blood for platelet aggregation and PFA-100 studies Healthy volunteers were recruited from among blood donors, staff, medical students, and residents of our institu- tion. Specific inquiry was made about the ingestion of aspi- rin, nonsteroidal anti-inflammatory drugs (NSAIDs), and any form of cold therapy, at least 2 weeks before blood collection. Whole blood was drawn by clean venipuncture directly into vacutainer plastic tubes (Terumu Co., Japan) containing 3.8% (0.129 M) or 3.2% (0.105 M) buffered sodium citrate to yield a blood:anti-coagulant ratio of 9:1. Preparation of platelet-rich plasma and platelet-poor plasma Platelet-rich plasma (PRP) was obtained by centrifugation of citrated whole blood at 800–1000 rpm for 5 minutes. PRP was removed and the remaining sample was subjected to a second round of centrifugation at 3000 rpm for 10 minutes to obtain platelet-poor plasma (PPP). The platelet count of PRP was in the range of 200–300 · 09 /L. PRP was adjusted to a concentration of 250,000 – 50,000 with PPP. Platelet aggregometry The processing of blood samples and agonist-induced platelet aggregation technique were carried out as previously described using a Platelet Aggregation ProfileÒ (PAP-4) system (BioData, Horsham, PA).14,15 Arachidonic acid (AA) (BioData) was reconstituted from a lyophilized preparation of sodium arachidonate using distilled water to yield a working concentration of 5 mg/mL. ADP (BioData) was re- constituted from a lyophilized preparation with distilled water to yield a working concentration of 2 · 10- 4 M. Special macrocuvettes (8.75 · 50 mm) were used for all experiments. Briefly, using plastic tips, 0.45 mL of PRP were pipetted into the cuvette. Raw camel urine (0.05 mL) was added and the mixture was stirred with a plastic-coated magnetic stirrer for 2 minutes, after which 0.05 mL of the aggregating agent was added and the recording was started. Aggregation parame- ters of maximum aggregation (%) versus control (PPP) and the slope of the aggregation curve were recorded. PFA-100 closure time The PFA-100 assay (PFA-100Ò ; Dade Behring, USA) was carried out as previously described.12 The PFA100 is a device that measures platelet-related primary hemostasis in citrated whole blood specimens. It uses two disposable cartridges fitted with a membrane with central aperture (147 lm) coated with aggregation agonists (collagen and epinephrine and collegen and ADP), through which platelets are passed at high shear rates (5000–6000 s- 1 ). The PFA-100Ò deter- mines in whole blood the time (in seconds) elapsed from the start of the test until a platelet plug occludes the aperture. This time interval is referred to as closure time (CT), and is an indicator of platelet function (adhesion and aggregation). The system was programmed to stop recording when the CT reached ‡ 300 seconds. Preparation of the test cartridges. The pouch containing the test cartridges was allowed to warm up to room tem- perature prior to opening (approximately 15 minutes). After removal of the cartridges, the pouch was immediately closed using the reclosable seal. The top foil seal was removed from the test cartridge and discarded, and then the test cartridge(s) was placed in the cassette of the PFA-100 and snapped se- curely into place. Sample loading. The following steps were performed in sequence without interruption. 1. The blood sample was mixed by inverting the collection tube gently by hand 3–4 times. While the cassette con- taining the test cartridge was held on a flat surface, 800 lL of blood was pipette into the sample reservoir by dispensing slowly along one of the inside corners. This reduces the risk of air entrapment in the sample reser- voir. 2. The cassette with test cartridge containing sample was placed into the incubation well(s) of the instrument such that the cassette was flush with the carousel sur- face, and then recording was started. The system was programmed to stop recording when the aperture closed, or 300 seconds, whichever came first. 804 ALHAIDAR ET AL.
  11. 11. Statistical analysis Data were analyzed using the SSPS program (Version 15). Differences in means between groups were compared using the Mann–Whitney test. Analysis of variance was conducted using the Kruskal–Wallis test. Proportions from two or more independent groups were compared using either the v2 test or Fisher’s Exact test, as appropriate. A p-value £ 0.05 was considered statistically significant. Results A comparison of aggregation responses of human PRP before (control) and after the addition of camel urine to the aggregation mixture revealed that urine from virgin, lactat- ing, and pregnant camels significantly inhibited aggregation responses to both ADP and AA ( p < 0.001) (Fig. 1). Overall, urine from lactating camels exhibited the most potent platelet inhibitory activity. However, close examination of the individual responses showed that in some cases, camel urine induced a complete block of the aggregation responses to ADP and AA, while in other cases, it had no effect. To identify the prevalence of antiplatelet inhibitory activity in camel urine, a cut-off value was selected for maximum ag- gregation response of £ 40%. Using this approach, it was possible to identify more clearly which camel urine had the most potent antiplatelet activity (Table 1). Urine from lac- tating camels exhibited the highest inhibitory activity against ADP-induced aggregation, followed by pregnant camel ur- ine, while virgin camel urine was the least potent. In terms of inhibition of AA-induced aggregation, only lactating camel urine exhibited potent antiplatelet effects. The antiplatelet activities of camel urine grouped accord- ing to maximal aggregation response in the presence of ADP and AA are shown in Table 2. Inhibition of both ADP- and AA-induced aggregation differed significantly between lac- tating (50%), pregnant (29.7%), and virgin (22.4%) urine samples ( p = 0.0151; v2 test). These results indicated that lactating camel urine is the most potent inhibitor of human platelet aggregation. Dose–response AA and ADP-induced aggregation by camel urine Serial dilutions (neat, 1:2, 1:4, 1:8) of camel urine samples that exhibited complete inhibition of either AA- or ADP- induced aggregation of normal human platelets were pre- pared and the aggregation protocol was repeated. Dilutions were added to human PRP before the addition of ADP or AA. For all samples, there was a clear dose–response effect of the camel urine such that as the concentration of urine de- creased, there was a gradual reduction in inhibition of ag- gregation (Table 3). The effect of human and bovine urine on ADP- and AA-induced aggregation When the platelet aggregometry assay was repeated using undiluted human (n = 20) and bovine (n = 24) urine, it was not possible to detect any inhibition of either AA- or ADP- induced aggregation (data not shown). The effect of camel urine on PFA-100 closure time Camel urine samples that caused a complete inhibition of both ADP- and AA-induced aggregation were diluted 1:10 and 1:20, and then added to human whole blood (Table 4). In the presence of the higher concentration of camel urine (1:10 dilution), closure times exceed the limit of the recording (300 second). When the test was repeated with a lower concentration of urine (1:20 dilution), a significant shortening of closure times was observed ( p < 0.001) as compared to FIG. 1. The effect of camel urine (virgin, lactating, and pregnant) on the aggregation of human platelets in response to arachidonic acid (Arch) and adenosine diphosphate (ADP). Data represent means – standard deviation. Max- imum aggregation is expressed as a percentage of control (untreated) platelets. Observations by Gader. Table 1. Antiplatelet Action of Camel Urine Collected from Virgin, Pregnant, and Lactating Animals on the Aggregation Responses to Adenosine Diphosphate (ADP) and Arachidonic Acid (AA) of Healthy Human Platelet-Rich Plasma Maximum aggregation response to ADP Maximum aggregation response to AA Study group £ 40% > 40% p-Value £ 40% > 40% p-Value Control (no urine) 0 (0.0) 42 (34.2) < 0.001* 0 (0.0) 42 (39.2) < 0.001* Virgin camels 14 (25.9) 44 (35.8) 0.2665 26 (37.1) 32 (29.9) 0.4014 Pregnant camels 19 (35.2) 18 (14.6) 0.0038* 18 (25.8) 19 (17.8) 0.2784 Lactating camels 21 (38.9) 19 (15.4) 0.0012* 26 (37.1) 14 (13.1) < 0.001* Total 54 (100.0) 123 (100.0) 70 (100.0) 107 (100.0) Results are expressed as percent maximum aggregation response to ADP and AA of healthy human platelet-rich plasma. Observations by Gader. *Statistically significant as compared to untreated samples. ANTIPLATELET ACTIVITY OF CAMEL URINE 805
  12. 12. samples treated with the lower dilutions (mean of < 300) (Table 4). Discussion Urine therapy, or urotherapy, has been in practice since early historic times. A search of multiple electronic literature databases yields a plethora of information on the use of ur- ine, particularly human urine, with claims of successful treatment of a wide range of human ailments. However, al- most all the available information can be categorized as al- ternative medical practice by healers in many countries, particularly those where the practice of alternative medicine is prevalent such as India and China, with scant reporting from the United States, United Kingdom, and other Euro- pean countries. The perceived success of such therapeutic efforts by those who believe in the efficacy of urine therapy, whether through practice or personal experience, has prompted several books on urine therapy that have found wide readership.16–18 Many of the books and reports on ur- otherapy advocate the use of human urine therapy, partic- ularly using the individual’s own urine. Despite numerous claims of efficacy, the practice of ur- otherapy has yet to be subjected to scientific research, and even in situations where this form of therapy was prescribed or advised by qualified physicians, there are no studies that offer scientific support of such a practice. Therefore, at present, the practice of urine therapy should be viewed as unorthodox medical practice based primarily on trial and error, and not a field that has been subjected to rigorous scientific scrutiny. There have been a few isolated references to the use of bovine urine in Tibet and India, and the use of llama urine (a member of the Camelidae family) in Tibet, Mongolia, and China.18 The use of camel urine for therapeutic purposes is practiced widely among tribes that raise camels, both in Asia and Africa. In the Middle East, there is credible evidence that Prophet Mohamed advised the use of camel urine for the treatment of a wide range of disease conditions.1,2 There are numerous claims of the success of camel urine therapy in the management of a range of diseases from liver cirrhosis to skin and hair ailments.17 Cancer is prominent among the diseases that are reportedly treatable by urine (human and camel). Recent studies have shown both in vitro (tissue cul- ture) and in vivo in humans and animals that a component isolated from camel urine inhibits the growth of cancer cells, and reduces the size of both primary tumors and secondary metastases.19,20 To date, there are no reports in the literature of the use of camel urine to treat cardiovascular disease. The authors were encouraged to investigate this possibility by recent results from their laboratory on the structure and function of camel platelets.12,13 An important finding of this earlier work was Table 2. Antiplatelet Action of Camel Urine from Virgin, Pregnant, and Lactating Animals Grouped According to Percent Maximum Aggregation Response to Adenosine Diphosphate (ADP) and Arachidonic Acid (AA) on Healthy Human Platelet-Rich Plasma Study groups Maximum aggregation response to ADP Maximum Aggregation response to AA Virgin camels Pregnant camels Lactating camels £ 40 £ 40 13 (22.4%) 11 (29.7%) 20 (50.0%) £ 40 > 40 1 (1.7%) 8 (21%) 1 (2.5%) > 40 £ 40 13 (22.4%) 7 (18.9%) 6 (15.0%) > 40 > 40 31 (53.5%) 11 (29.7%) 13 (32.5%) Total 58 (100.0%) 37 (100.0%) 40 (100.0%) Results are expressed as percent maximum aggregation response to ADP or AA of healthy human platelet-rich plasma and are grouped to show inhibition of aggregation in response to a single agent, or both aggregation agents. Observations by Gader. Table 3. The Effect of Different Concentrations of Camel Urine (Neat and Serial Dilutions) on Adenosine Diphosphate (ADP)– and Arachidonic Acid–Induced Platelet Aggregation (Expressed as Maximum Aggregation %) of Healthy Human Platelet-Rich Plasma ADP (10 lg) Arachidonic acid Neat 1:2 Neat 1:2 1:4 1:8 N 18 18 24 24 24 4 Mean 21.2 58.0* 10.4 28.6* 47.9* 58.1* SD 9.5 9.0 11.3 22.1 20.7 29.1 Observations by Gader. *p < 0.001 as compared to neat (Wilcoxon rank sum test). SD, standard deviation. Table 4. Summary of PFA-100 Closure Times of Human Whole Blood After the Addition of Camel Urine (1/10 and 1/20 Dilutions of Camel Urine Samples that Caused Complete Inhibition of Adenosine Diphosphate (ADP)– and Arachidonic Acid–Induced Aggregation) PFA-ADP- 1/10 PFA-ADP- 1/20 PFA-EPI- 1/10 PFA-EPI- 1/20 Number 3 3 3 3 Mean 276.7 131.3 300 227.3 SD 29.1 27 0 63 Min 244 102 300 188 Max 300 155 300 300 Observations by Gader. PFA-100, platelet function analyzer; PFA-ADP, collagen/ADP cartridge; PFA-EPI, collagen/epinephrine cartridge; SD, standard deviation. 806 ALHAIDAR ET AL.
  13. 13. the putative antiplatelet properties of camel blood. Analysis of platelet function using the PFA-100 platelet function an- alyzer demonstrated that camel blood induces a prolonga- tion of closure time of human blood.12 These results suggested that camel plasma may have a platelet inhibitory activity, and that this activity may be recoverable in urine. In the present study, camel urine displayed significant platelet inhibitory activity against human blood collected from healthy volunteers, blocking the aggregation responses of human platelets to ADP and AA, and inducing a pro- longation of PFA-100 closure time. A major advantage of aggregation studies is that they provide information about the mechanism of action of agents that modulate platelet aggregation. Thus, inhibition of ADP-induced aggregation by camel urine can be assumed to occur mostly at the level of ADP receptors (P2Y12 and P1Y1).21,22 This assumption is supported by the result of the present authors’ dose– response studies. The ADP inhibitory action of camel urine, therefore, resembles that of the widely used antiplatelet theinopyridine drugs, particularly clopidogrel, which selec- tively blocks the P2Y12 receptor. However, the possibility cannot be excluded that camel urine also blocks the second P2Y1 receptor as well. The inhibition of AA-induced aggregation by camel urine resembles that of aspirin, which blocks the prostaglandin pathway of platelet activation by irreversibly acetylating the enzyme cycloxygenase.23,24 Whether the action of camel ur- ine mimics that of aspirin or whether it acts at other sites along the prostaglandin pathway (e.g., thromboxane A2 re- ceptors) is open to speculation. Conclusions The current results are the first demonstration of the an- tiplatelet actions of camel urine and provide an important foundation of scientific evidence for the exploration of camel urine as a therapeutic antiplatelet agent. There is also the interesting possibility that the aspirin-like and clopidogrel- like actions of camel urine may be responsible for some of its other widely claimed therapeutic benefits. Clearly, continued study is needed to uncover the chemical nature of the anti- platelet effects of camel urine. For example, the current re- sults do not elucidate why the urine of some camels had significant antiplatelet effects while that of others did not or elicited only a partial response. The authors’ recent investi- gations of the proteome of camel urine (unpublished data) resulted in the identification of three compounds with known antiplatelet effects: syndecan-4, an antithrombin- binding cell surface heparan sulphate proteoglycan25 ; a-1- antichymotrypsin26 ; and lactoferrin.27 Whether these pro- teins constitute the platelet inhibitory action of camel urine remains to be elucidated. Lastly, the demonstration that camel urine is endowed with potent antiplatelet activity lends support to the claimed anticancer effects of camel urine. Numerous studies have shown that aspirin has growth-inhibitory action against cancer cells.28–31 This effect of aspirin is hypothesized to be through the inhibition of tumor angiogenesis, promotion of apoptosis, or other possible mechanisms. The potent anti- platelet activity of camel urine demonstrated in the current study suggests a putative mechanism for the claimed anti- cancer properties of camel urine. Acknowledgments We thank Lugman Gasmel Sid and Mohamed A. Hamid for technical assistance. Disclosure Statement No competing financial interests exist. References 1. Al-Azraq I. The Facilitation of Benefits in Medicine and Wisdom [in Arabic]. Online document at: http:// hadithexegesis.blogspot.com/2009/05/camels-urine-itscure .html Accessed May 25, 2009. 2. Ali J. In: Details of Arab History Before Islam [in Arabic]. Buirur, Lebanon: Dar Alsaqi, 1957. 3. Gauthier-Pilters H, Dagg I. The Camel. London: University of Chicago Press, 1981. 4. Kabarity A, Mazroee S, Gendi A. Camel urine as a possible anticarcinogenic agent. Arab Gulf J Sci Research Agric Biol Sci 1988;6:55–63. 5. Sharmanov T, Zhangabylov AK, Zhaksylykova RD. Me- chanism of the therapeutic action of whole mare’s and camel’s milk in chronic hepatitis [in Russian]. Vopr Pitan 1982;1:17–23. 6. Ikeda M, Nozaki A, Sugiyama K, et al. Characterization of antiviral activity of lactoferrin against hepatitis C virus in- fection in human cultured cells. Virus Res 2000;66:51–63. 7. Redwan el-RM, Tabll A. Camel lactoferrin markedly inhibits hepatitis C virus genotype 4 infection of human peripheral blood leukocytes. J Immunoassay Immunochem 2007;28: 267–277. 8. Sharmanov T, Kadyrova R, Salkhanov BA. Effectiveness of peptic ulcer diet therapy using rations containing whole ma- re’s and camel’s milk [in Russian]. Vopr Pitan 1981;3:10–14. 9. Shabo Y, Barzel R, Margoulis M, Yagil R. Camel milk for food allergies in children. Isr Med Assoc J 2005;7:796–798. 10. FitzGerald RJ, Meisel H. Milk protein-derived peptide in- hibitors of angiotensin-I-converting enzyme. Br J Nutr 2000; 84(suppl 1):S33–S37. 11. Saito T. Antihypertensive peptides derived from bovine ca- sein and whey proteins. Adv Exp Med Biol 2008;606:295–317. 12. Gader A, Ghumlas A, Hussain M, Al-Haidary A. Platelet aggregation and platelet function analyser 100 (PFA-100) closure time in camels: A comparative study with humans. Comp Clin Pathol 2006;15:31–37. 13. Gader AG, Ghumlas AK, Hussain MF, et al. The ultra- structure of camel blood platelets: A comparative study with human, bovine, and equine cells. Platelets 2008;19:51–58. 14. Gader A, Bahakim H, Awadalla S, Malaika S. Ethnic varia- tions in the haemostatic system: Comparison between Ar- abs, Westerners (Europeans and Americans), Asians and Africans. Blood Coagul Fibrinolysis 1995;6:537–542. 15. Gader A, Bahakim H, Malaika S. A study of the normal pattern of platelet aggregation in healthy Saudis: A popu- lation-based study. Platelets 1990;1:139–143. 16. Armstrong J. Water of Life. Varanasi, India: Pilgrims Pub- lishing, 2004. 17. Christy M. Your Perfect Medicine. Mesa, AZ: Wishland Publishing, 2000. 18. van der Kreoon N. The Golden Fountain. Mesa, AZ: Wish- land Publishing, 2005. 19. Khorshid F. Potential anticancer natural product against human lung cancer cells. Trends Med Res 2009;4:9–15. ANTIPLATELET ACTIVITY OF CAMEL URINE 807
  14. 14. 20. Khorshid F, Moshref S, Heffny N. An ideal selective anti- cancer agent in vitro, I: Tissue culture study of human lung cancer cells A549. JKAU Med Sci 2005;12:3–18. 21. Maree AO, Fitzgerald DJ. Variable platelet response to as- pirin and clopidogrel in atherothrombotic disease. Circula- tion 2007;115:2196–2207. 22. Weerakkody GJ, Brandt JT, Payne CD, et al. Clopidogrel poor responders: An objective definition based on Bayesian classification. Platelets 2007;18:428–435. 23. Bhatt DL, Topol EJ. Scientific and therapeutic advances in antiplatelet therapy. Nat Rev Drug Discov 2003;2:15–28. 24. Shantsila E, Watson T, Lip GY. Aspirin resistance: What, why and when? Thromb Res 2007;119:551–554. 25. Kaneider NC, Feistritzer C, Gritti D, et al. Expression and function of syndecan-4 in human platelets. Thromb Haemost 2005;93:1120–1127. 26. Renesto P, Chignard M. Tumor necrosis factor-alpha en- hances platelet activation via cathepsin G released from neutrophils. J Immunol 1991;146:2305–2309. 27. Leveugle B, Mazurier J, Legrand D, et al. Lactotransferrin binding to its platelet receptor inhibits platelet aggregation. Eur J Biochem 1993;213:1205–1211. 28. Borthwick GM, Johnson AS, Partington M, et al. Therapeutic levels of aspirin and salicylate directly inhibit a model of angiogenesis through a Cox-independent mechanism. FAS- EB J 2006;20:2009–2016. 29. Flossmann E, Rothwell PM. Effect of aspirin on long-term risk of colorectal cancer: Consistent evidence from rando- mised and observational studies. Lancet 2007;369:1603–1613. 30. Ou YQ, Zhu W, Li Y, et al. Aspirin inhibits proliferation of gemcitabine-resistant human pancreatic cancer cells and augments gemcitabine-induced cytotoxicity. Acta Pharma- col Sin 2010;31:73–80. 31. Schreinemachers DM, Everson RB. Aspirin use and lung, colon, and breast cancer incidence in a prospective study. Epidemiology 1994;5:138–146. Address correspondence to: Abdel Galil M. Abdel Gader, MD, PhD The Coagulation Research Laboratory Department of Physiology College of Medicine and King Khalid University Hospital King Saud University Riyadh 11461 Saudi Arabia E-mail: amagader@ksu.edu.sa 808 ALHAIDAR ET AL.
  15. 15. 379 RESEARCH OPINIONS IN ANIMAL & VETERINARY SCIENCES PRINT ISSN 2221-1896, ONLINE ISSN 2223-0343 www.roavs.com Preliminary pharmacological investigations on camel urine (Camelus dromedarius) Salwa M.E. Khogali1 , Samia .H. Abdrahman1 Baragob, A. E. A. 2 and Elhassan A. M3 1 Department of Biochemistry - Central Veterinary Research Lab - Khartoum, Sudan; 2 Department of pharmaceutics, Karai University, Omdurman, Sudan; 3 Department of Pharmacology - Alrabat University - Khartoum, Sudan Abstract Pharmacological effects of camel urine (CU), its protein precipitate (PP), diluted urine (DU) and chloroformic extract (CE) were investigated. The PP inhibited the spontaneous movements of the isolated rat duodenum at a dose rate of 0.1ml/bath. Diluted female camel urine (0.4 ml/bath) or its protein precipitate (0.8 ml/bath) on rat fundus and rabbit jejunum revealed serotonin like effect which was antagonized by serotonin blocker cypohyptadine (0.2 ml /bath). In addition crude female camel urine produced transient relaxation on rabbit jejunum followed by increased contraction on first washing. chloroformic extract produced no effect on rat duodenum, fundus and rabbit jejunum, whereas rabbit and chick rectum showed slight changes in the frequency and amplitude contractions. Key words: Pharmacological, Investigation, Camel, Urine Introduction Arabian camel urine was standard prescription in Arab medicine and remains stable for Bedouin natural remedies to this day, both as diuretic snuff and delousing hair detergent (Mona, 1989; Kabariti, 1988). The percentage of use of camel urine among five nomadic tribes in eastern Sudan were as follows: 72% use camel urine for internal problems in general, while 52%, 32%, 20% and 32% used it for malaria, ascitis, dental problems and hair shampoo respectively. Regarding the sex of the animal, 88% use female urine whereas only 12% use male urine. Seventy two percent drink it as pure urine, whereas twenty eight percent mix it with milk (Ohaj, 1993, 1998). Therapeutic uses of animal’s urine have a long history as that of human. Most of the earlier and current studies deal with pharmacological and therapeutic effects of human urine (Bersnyski, 1986; Kabariti, 1988; Kroon, 1996; Martha, 2000; Natalie, 2002). No detailed studies were done on the pharmacology and/or the possible mechanism(s) of action of animals urine, especially the dromedary. Regarding the positive results obtained from the experimental studies (antibacterial, antifungal, anticarcinogenic, antiparasitic and hepatoprotective), as reported by Ohaj, 1998; Wisal, 2002; Mona, 2003 and Salwa, 2005 respectively, necessitate its pharmaco- logical investigations. In this study the pharmacological effect of female camel urine (different extracts) were performed utilizing laboratory animals isolated strips. Materials and Methods Camel urine was collected from naturally grazing animals (normal urination/or by tashweel technique). Physiological saline solutions (Tyroid’s & Kerb’s) were prepared according to the method of Kitchen (1984), CE, PP of she-camel urine: native protein precipitate was performed by salt saturation using ammonium sulphate (40%) w/v and DU was obtained by adding distilled water to the urine in ratio 3:1. Bioassay of isolated tissues was prepared according to the method described by Kitchen (1984). Using duodenum and fundus strips from a Wister albino rats, jejunum and rectum strips from local rabbits and rectum strips from 15 day old chicks. Results A dose of 0.1 ml/bath of camel urine PP abolished the spontaneous contractions of rat duodenum as shown in Fig. (1). Female CU and PP at a dose rate of 0.4 and 0.8 ml/bath, respectively however, stimulated the rat fundus and rabbit jejunum as shown in Fig. 2 and 3. The stimulant effects were blocked by cyproheptadine and atropine at a dose of 0.2 and 0.25ml/bath, respectively.
  16. 16. Khogali et al roavs, 2011, 1(6), 379-381. 380
  17. 17. Khogali et al roavs, 2011, 1(6), 379-381. 381 CU at 0.1 ml/bath completely abolished the spontaneous contractions of rabbit jejunum. However, the inhibitory effect was followed by transient contraction on first washing. The CE showed slight effect on rabbit and chick rectum strips Fig.4. Discussion This study showed that the inherited knowledge of traditional usage of camel urine for treating various ailments in Sudan could be a guide for the discovery of important biological activities which might be of useful therapeutic effects. Moreover, the scientific evaluation and identification of the mechanism (s) of action of camel urine is important for justification of its employment in modern medicine, in view of its wide uses in different parts of Sudan and other Arab countries. The results of the present study demonstrate important biological activities of the CU, PP, CE and DU. DU and CU exerted dual effects on the rabbit jejunum isolated strips. DU stimulated the organ while CU abolished the spontaneous rhythmicity of the same organ. Similar findings were reported by Rodenburg (1937) using human urine. The stimulant effect appeared to be mediated via muscarinic receptors stimulation as the effect was blocked by atropine sulphate (0.25 ml/bath). This is in agreement with Vicher (1983) and Ali et al. (1991) findings using extracts of medicinal plants. The addition of PP directly stimulated rabbit jejunum at 0.8 µl/bath the effect was blocked by atropine sulphate (0.2 ml/bath) which suggests acetylcholine-like action. Rat fundus was markedly stimulated with PP and DU as did serotonin. The abolishment of the stimulant effects of both urine forms and 5-Hydroxytryptamine (5-HT) by the addition of the non-selective serotonin blocker, cyproheptadine, demonstrated the 5-HT like activity of PP and DU. This high sensitivity might be due to the fact that rat fundus was found to be enriched with the 5-HT2B receptors (Vane, 1957). This has been recently verified as subtype of the 5-HT2 receptor family by Cox et al. (1996). The addition of PP to rat duodenum directly inhibited the myogenic contractions, which may suggest a direct musclotropic relaxation of smooth muscles. Similar findings were reported by Guddum (1955) and Horton (1959) using human urine. CE produced slight changes on rabbit and chick rectum rhythm city, however, no effects were observed on other strips. It can concluded that camel urine (indifferent forms) can penetrate subepithelially and induce generation of mast cells with release of chemical mediators, followed by forceful peristaltic contractions caused by 5-HT and other newly formed mediators. References Ali, M.B., Mohamed, A.H., Salih, W.M. and Homeida, A.H. 1991. Effect of an aqueous extract of Hibiscus sabdariffa calyces on the gastrointestinal tract. Fitoterapia Voi. 1. XII. No. 6 Pp: 475-479. Berzynski, S.R. 1986. Anti neoplaston in cancer therapy. History of the research drugs. Experimental & Clinical Research, Supply 11: 1-9. Guddum, J.H. 1955 .Polypeptides which stimulates plain muscle. London, Livingstone. P:130. Horton, E.W. 1959. Human Urinary Kinin Excretion. Brit. J. Pharmacol., 14:125-132. Kabariti, A. Mazruai, S. and Elgendi, A. 1988. Camel’s urine: A possible anticarcinogenic agent. Arab Gulf Journal of Science and Research Agrc. Kitchen, L. 1984. Text Book of Experimental Pharmacology, Isolated small intestine, 102-103. Kroon, C.V. 1996. The Golden fountain, Autourine therapy. Gate Way Books, ISBNO 73:2:244-256. Martha, C. 2000. Clinically tested medicinal proved book. Your Own Perfect Medicine. Mona, A.K. 2003. Antibacterial effect of camel urine (Camelus dromedaries) M.V.Sc. Faculty of Vet. Medicine University of Khartoum, Sudan. Mona, S. 1989. Camel urine as a hair detergent. B.Sc. Dissertation, Ahfad University, Khartoum, Sudan. Natalie, B. 2002. Urine Therapy (Drinking urine). Journal of Berkeley medicine. www.ocf.berkele. edu. Ohaj, H.M. 1998. Clinical trial for treatment of ascitis with camel urine M.Sc. University of the Gezira, Sudan. Ohaj, H.M. 1993. Clinical urine as a medicament in Sudan. B.Sc. Dissertation, University Gezira, Sudan. Rang, H.P., Dale, M.M. and Ritter, J.M. 1995. Pharmacology. 5th (ed.) Churchill Livingstone, London. Rodenburg, G.L. and Nagy, S.M. 1937. Growth stimulating and inhibiting substances in human urine. American Journal of Cancer, 29:66. Salwa, M.E.K. 2005. Hepatoprotective and antiparasitic effect of female camel urine. PhD Thesis. University of Khartoum, Sudan. Vane, J.R. 1957. A sensitive method for the assay of 5- HT. British Journal of Pharmacology, 12:344-349. Wisal, G.A. 2002. Antibacterial and antifungal effect of camel urine (Camelus dromedaries) M.V.Sc. University of Khartoum, Sudan.
  18. 18. Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.2, No.5, 2012 9 Cytotoxicity of the Urine of Different Camel Breeds on the Proliferation of Lung Cancer Cells, A549 Zahraa Alghamdi1* Faten Khorshid2 1. Biology Department, Dammam University, PO box 1982, Dammam 31441, Kingdom of Saudi Arabia. 2. Biology Department, King Abdulaziz University, PO box 80216, Jeddah 21589, Kingdom of Saudi Arabia. * E-mail of the corresponding author: zhghamdi@yahoo.com Abstract Objective: Cancer is a disease characterized by uncontrolled cellular proliferation and differentiation. Nearly all conventional cancer treatments have undesirable negative impacts, and safer chemotherapeutics would be advantageous. Consequently, the goal of current study was to evaluate and compare the effects of urine derived from two different camel breeds on proliferation of cultured human cancer cells. Human lung adenocarcinoma cells (A549) were cultured in the presence or absence of varied dilutions of urine obtained from two different camel breeds (Magateer and Majaheem). Within breeds, we compared the effects of sex and age of donor camels on urine cytotoxicity to A549 cells. After 48 hrs, surviving A549 cells were enumerated using the sulfarhodamine assay. A549 cell survival was lower using urine from Magateer versus Majaheem camels (84.8% versus 94.2% of starting cell number, respectively; n=20 for both groups, p<0.001). When evaluating the effect of camel age, urine from older Magateer camels was significantly more effective in inhibiting A549 proliferation than was urine from younger camels of this breed. An age-related effect was not observed for Majaheem camels. When comparing sex-effects on camel urine inhibition of A549 proliferation (n=10 in each group), we observed a trend towards more A549 inhibition using female versus male urine, in both camel breeds; however, this difference did not reach statistical significance. The present study confirms previous studies that showed that camel urine can inhibit the growth of cancer cells. It also provides the first evidence that there are slight differences in the cancer cell growth-inhibitory effect of camel urine depending on the camel breed, age, and, possibly, sex. Keywords: Camel breeds, Urine, Cancer cells, Cytotoxicity. 1. Introduction Cancer is a disease characterized by uncontrolled cellular proliferation and differentiation. Nowadays, cancer is a very common disease with a high annual incidence rate (Parkin, et al ; 1999]. Ferlay et al. (2000) reported that worldwide more than 5 million people are diagnosed with cancer and more than 3.5 million people die from cancer each year. Managing human malignancies still constitutes a major challenge for contemporary medicine (Coufal et al., 2007 and Widodo et al., 2007). Although with progress in understanding cancer biology, many new antineoplastic therapies have been developed that rely primarily on surgery, chemotherapy, radiotherapy, hormone therapy, and immunotherapeutic approaches (Khorshid et al., 2010). However, all available therapies are still far from ideal, in which treatment would selectively kill the malignant cells while sparing healthy tissues and vital organ function (Grever and Charbner, 1997 and Moshref, 2007). chemotherapy resulted in an overall increase in the survival rate and longevity of patients with life-threatening tumors, On the other hand also mean increased exposure to toxic substances and harmful effects on different tissues ( Maino, et al.,2000). Natural products play an important role in our healthcare system (Pezzuto, 1997 and Schwartsmann, 2000). They offer a valuable source of potent compounds with a wide variety of biological activities and novel chemical structures, many of which might be important for novel drug development (Vuorela, et al., 2004). Animal studies have shown that green tea is a potent inhibitor of lung tumor development (Zhang et al., 2000). PM 701 is another natural product readily available, cheap, and non-toxic (Khorshid, 2008). PM 701 was proven to be an anticancer substrate (Khorshid et al., 2005, 2008, Moshref et al., 2006 and El-Shahawy et al., 2010), and was found to be effective in limiting the metastatic spread of leukemia cells in an animal model (Moshref et al., 2006). PM 701 is considered safe as a potential anti-cancer agent, and exerts negligible effects on vital organs (Khorshid, 2009). Camel urine, also a natural product, has been used traditionally in the treatment of many diseases in Arabic countries. Drinking camel urine was shown to be effective in treating numerous cancer cases (Alhaider et al.,
  19. 19. Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.2, No.5, 2012 10 2011). Moreover, according to Saudi Gazette.com, Dr. F.A. Khorshid has a potential cure for cancer based on camel urine. After 8 years of research she has announced that nano-particles in camel urine can be used to fight cancer. Moreover, The Saudi Center for Medical Research added that there is a tendency to start in the production of a medical capsule containing camel’s urine for use in the treatment of cancer. In the same respect, Alhaider et al. (2011) examined the ability of three different camel urine samples (virgin, lactating, and pregnant sources) to modulate a well-known cancer-activating enzyme, cytochrome P 450 1a1 (Cyp 1a1) in the murine hepatoma Hepa 1c1c7 cell line. They found that all types of camel urine, but not bovine urine, differentially inhibited the induction of Cyp 1a1 expression by TCDD, a potent Cyp 1a1 inducer and a known carcinogen. Virgin camel urine showed the highest degree of Cyp 1a1 inhibition, followed by lactating and pregnant camel urine. Khorshid (2001) stated that in vitro approaches are the best way to initially evaluate the effect of novel biological compounds, utilizing growing mammalian cells in tissue culture. Consequently, the main goals of current study were to: 1) evaluate the inhibitory effect of urine obtained from two different camel breeds on the growth of lung cancer cells (A549),in vitro; and 2) study whether urine’s effect is changed according to differences in the camel’s breed, age, or sex. 2. Materials and Methods 2.1. Study area: The main part of this study was carried out at yebreen region located in the southern west of the eastern region at the periphery of The Rub' alkali (Empty Quarter) included in Kingdom of Saudi Arabia. 2.2. Animals: This study was conducted on 40 camels from two different breeds (Magateer and Majaheem). Ten males and 10 females were selected from each breed. The males ranged between 1-8 years old, whereas the females ranged from 3 to 9 years old. 2.3. Urine sampling and storage: Twenty milliters of urine were collected from each camel, kept in insulated boxes using freezing packs, and transferred to the laboratory (Tissue Culture Unit, King Fahd Medical Research Center (KFMRC), King Abdul Aziz University in Jeddah, Saudi Arabia). 2.4. Methods: Human non-small-cell adenocarcinoma cells (A549) were obtained from the American Type Culture Collection (ATCC) and were stored in the cell bank of tissue culture laboratory, where cytotoxicity assays were also conducted, as pioneered by a research team working in the medical center (Khorshid et al.,2005; Khorshid and Alameri, 2011). Different concentrations of PM 701 were used (1.0, 2.5, 5.0, 7.5, and 10 g/ml) and were added to A549 cell monolayers. The control group of A549 cells was not treated with PM 701 and is indicated as 0 concentration. Cytotoxicity assays were performed using the method of Skehan et al. (1990). Cancer cells were suspended in DMEM medium and plated in 96-well plates (104 cells/well) for 24h in a 5% CO2 incubator adjusted at 37°C before treatment with PM701, to allow cell attachment to the bottom of the plate. Different concentrations of the test substance (0, 1, 2.5, 5, and 10 g/ml) were then added to the cells monolayer. Triplicate wells were prepared for each individual concentration. Cell monolayers were incubated with PM701 for 48 h at 37°C and in atmosphere of 5% C02. After 48 h, cells were fixed using 50 µl/well trichloroacetic acid, refrigerated at 8°C for 1 hour, washed with distilled water, and then stained with Sulforhodamine B (SRB) (50 µl/well) for 30 min. Excess stain was washed with off with acetic acid and remaining attached stain was recovered with Tris EDTA buffer (100 µl/well). Color intensity was measured immediately in an ELISA reader at wavelength 570 nm. The relation between surviving cells and drug concentration was plotted to get the survival curve of each cell line after the specified period.
  20. 20. Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.2, No.5, 2012 11 2.5. Statistical analysis Statistical analysis of the data was performed with SPSS for Windows (Version 17.0.0). Data were calculated as follows: The different urine samples were collected from the two camel breeds from both sexes. Five concentrations of urine were tested from each individual camel (1, 2.5, 5, 7.5, 10), with 0 concentration used as controls. Each experimental concentration was added to six tissue culture wells containing cancer cells. Forty total urine samples were collected from each camel with their detected concentrations mentioned above, so 40 camels × 5 concentrations equals 200 urine samples. Urine specimens at the listed concentrations were directly applied to the six wells of cultured cancer cells, so the total wells assayed equaled 1200. 3.Results and Discussion: 3.1.Differences between two camel breeds: Data shown in Table 1 revealed that, camel urine reduced lung cancer cells to 84.75% and 92.81%, in Magateer and Majaheem breeds, respectively, versus untreated controls (100%). Highly significant differences were noticed between treated and control cultures when comparing urine activity within each breed and between the different breeds (P=0.000 and 0.001, respectively). Magateer urine significantly reduced cancer cell numbers more than did Majaheem urine. These results are in accordance with those of Alhaider et al. (2011) who reported that drinking camel urine has been used traditionally to treat numerous cases of cancer. The authors attributed this anticancer effect to the ability of camel urine to modulate the well-known cancer-activating enzyme, Cyp 1a1. They found that all types of camel urine differentially inhibited the induction of Cyp 1a1 gene expression by TCDD, the most potent Cyp 1a1 inducer and a known carcinogenic chemical. In the same respect, Eldor (1997) hypothesized that because some cancer cell antigens are transferred through urine, through oral autourotherapy, these antigens could be introduced to the immune system that might then create antibodies. 3.2.Camel age effects on cancer cell proliferation: 3.2.1. In the same strain: Table 2 clarifies the effects of urine obtained from young and adult Magateer and Majaheem camels on the growth of lung cancer cells (A549) in vitro. Urine obtained from adult Magateer camels induced a highly significant reduction in A549 cell survival ( P≤0.004) than that obtained from the same younger breed (81.538% versus 87.947%, respectively), while urine obtained from adult Majaheem breed induced a non- significant (P≤ 0.179) reduction in cancer cells when compared to younger camels of the same breed (93.486% versus 96.974%, respectively). No available literature could be found regarding the influence of age on the anti-cancer effect of camel urine. However, Alhaider et al. (2011) studied the ability of three different camel urines (virgin, lactating and pregnant) to modulate the cancer-activating enzyme CyP 1a1. They found that virgin camel urine showed the highest degree of inhibition at the activity level, followed by lactating and pregnant camel urine. 3.2.2.Age effects between the different camel breeds: Table 3 shows a comparison between the anti-cancer effect of urine obtained from the two young camel breeds as well as the anti-cancer effect of that obtained from the two adult camel breeds. The results revealed that urine from young Magateer camels induced a significant (P≤0.01) reduction in the growth of cancer cells versus that obtained from young Majaheem camels (87.947% versus 96.974%, respectively). In addition, urine obtained from adult Magateer camels induced a significant higher reduction (P=000) of cancer cells versus that obtained with adult Majaheem camels (81.536% versus 93.486%, respectively). The reason for the variability in the anti-cancer efficacy of camel urine obtained from Magateer and Majaheen breeds is not yet known. Further study is needed to determine the specific differences in the urine constituents of each breed, to know which compound(s) is responsible for this variable effect.
  21. 21. Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.2, No.5, 2012 12 3.2.3.. Sex affects camel urine-mediated cancer cell proliferation: 3.2.3.1 In the same breed: Table 4 represents the effect of sex on the ability of camel urine to inhibit the growth of lung cancer cells in vitro. It appears that the sex of camels within the same breed did not significantly affect camel urine-inhibition of A549 cancer cell proliferation. However, urine of males induced a slight, though insignificant inhibition in cancer cell proliferation versus that of females of the same breed. 3.2.3.2. In the different breeds: Table 5 shows a comparison between the anti-cancer effect of urine obtained from males and females of the two different camel breeds. Urine from male Magateer camels caused a significantly greater reduction in cancer cells when compared to that induced by urine of male Majaheem camels (86.568 versus 94.014, respectively; P=.000). Urine of female Magateer camels also induced a significantly greater reduction in cancer cells compared to that induced by urine of female Majaheem camels (82.935 versus 91.368; P=.000). Urine from male and female Magateer camels were more efficient in reducing lung cancer cell numbers compared with that observed using Majaheem camel urine. 5. Conclusion The present study confirms the findings of previous studies that camel urine can inhibit the growth of cancer cells. It also provides the first evidence that there are differences in the cancer-inhibiting effect of camel urine depending on the camel breed, age, and sex. 6. Acknowledgements The authors gratefully thank King Faisal University, represented by Prof. Dr. AbdelGader Homeida and Mr.Khalid Borsais who helped in obtaining samples. The authors also appreciate the kind help of Prof. Dr. Hodallah Hatem, Head of the Physiology Department, Faculty of Veterinary Medicine, Cairo University, Egypt. References 1. Alhaider, A.A., El Gendy, M.A., Korashy, H.M. & El-Kadi, A.O.(2011). Camel Urine Inhibits the Cytochrome P 405 1a1 Gene Expression through an AhR- Dependent Mechanism in Hepa 1c1c7 cell line. Journal of Ethnopharmacology, 133 (1),184-190. 2. Coufal, M., Maxwell, M.M., Russel, D.E., Amore, A.M., Altmann, S.M., Hollingsworth, Z.R., Young, A.B., Housman, D.E. & Kazantsev, A.G. (2007). Discovery of Novel Small-Molecule Targeting Selective Clearance of Mutant Huntingtin Fragments. Journal of Biomolecular Screening, 12,351-360. 3. Eldor, J. (1997). Urotherapy for Patients with Cancer. Medical Hypothesis. 48(4), 309-315. 4. El-Shahawy, A., Elsawi, N.M., Baker, W.S., Khorshid, F. & Geweely, N.S. (2010). Spectral Analysis, Molecular Orbital Calculations and Antimicrobial Activity of PMF-G Fraction Extracted from PM-701. International Journal Pharma Bioscience, 1(2): 1-19. 5. Ferlay, J., Bray, F., Pisani, P. & Parkin, D.M. GLOBOCAN (2000): Cancer Incidence, Mortality and Prevalence Worldwide, Version 1.0. IARC Cancer Base No. 5. Lyon: IARC Press, 2001. 6. Grever. M. & Chabner, B. (1997): Cancer Drug Discovery and Development in Cancer Propels and Practice of oncology. Cytotoxicity of Some Medical Plant Extract Used in Tanzanian Traditional Medicine. Journal of Ethnopharmacology,70, 143-149. 7. Khorshid, F.A. (2001). The Effect of the Viscosity of the Medium in the Reaction of Cells to Topography. PhD Thesis, Glasgow University, UK. 8. Khorshid, F., Alshazly, H., Al Jefery, A. & Osman, A.M.M. (2010). Dose Escalation Phase I Study in Healthy Volunteers to Evaluate the Safety of a Natural Product PM 701. Journal Pharmacology Toxicology, 5(3), 91-97. 9. Khorshid, F.A., Rahimaldeen, S.A. & Alameri, J.S. ( 2011).Apoptosis Study on the Effect of PMF on
  22. 22. Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.2, No.5, 2012 13 Different Cancer Cells, International Journal of Biological Chemistry, 5(2),150-155. 10. Khorshid, F.A.( 2005). Comparative study of Keloids Formation in Human and Laboratory Animals. Medical science monitor, 11(7), BR212-219 11. Khorshid, F.A.( 2008). Preclinical Evaluation of PM 701 in Experimental Animals. International Journal of Pharmaceutics, 4(6), 443-451. 12. Khorshid, F.A.(2009). Potential Anticancer Natural Product Against Human Lung Cancer Cells. Trends in Medical Research, 4(1), 8-15. 13. Khorshid, F.A., Moshref, S.S. & Heffny, N.(2005). An Ideal Selective Anticancer Agent In Vitro, I- Tissue Culture Study of Human Lung Cancer Cells A549. JKAU- Medical Sciences, 12, 3- 18. 14. Khorshid, F.A., Rahimaldeen, S.A. & AL-Amri, J.S. (2011). Apoptosis Study on the Effect of PMF on Different Cancer Cells. International Journal of Biological Chemistry, 5, 150-155. 15. Maino, D.M., Tran, S. & Mehta, F.(2000). Side Effects of Chemotherapeutic Oculo-Toxic Agents: A Review. Clinical Eye and Vision Care, 12(3-4),113-117. 16. Moshref, S.S. (2007). PM 701 A Highly Selective Anti-Cancerous Against L1210 Leukemic Cells :II- In Vivo Clinical and Histopathological Study. JKAU. Medical Sciences, 14(1), 85-99. 17. Moshref, S.S., Khorshid, F.A.& Jamal, Y.( 2006). The Effect of PM 701 on Mice Leukemic Cells: I - Tissue Culture Study of L1210 (In Vitro) II - In Vivo Study on Mice, JKAU- Medical Sciences , 13 (1), 3-19. 18. Parkin, D., Pisani, P. & Ferlay, J. (1999). Global Cancer Statistics. A Cancer Journal for Clinicians, 49: 33-64. 19. Pezzuto, J.M. (1997). Plant-Derived Anticancer Agents. Biochemical Pharmacology, 53, 121-133. 20. Schwartsmann, G. (2000). Marine Organisms and Other Novel Natural Sources of New Cancer Drugs. Annals of Oncology, 11, 235-243. 21. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T,. Bokesch, H., Kenney, S. & Boyd, M.R. (1990). New Colorimetric Cytotoxicity assay for Anticancer-Drug Screening. Journal of the National Cancer Institute, 82,1107–1112. 22. Vuorela, P., Leinonen, M., Saikku, P., Tammela, P., Rauha, J-P, Wennberg, T.& Vuorela, H.( 2004).Natural Products in the Process of Finding New Drug Candidates. Current Medicinal Chemistry, 11,1375–1389. PMid,15180572. 23. Widodo, N., Kaur, K., Shrestha, B.G., Takagi, Y., Ishii, T., Wadhwa R. & Kaul, S.C. (2007). Selective killing of Cancer Cells by Leaf Extract of Ashwagandha: Identification of a Tumor-Inhibitory Factor and the First Molecular Insights to its Effect. Clinical Cancer Research, 13, 2298-2306. 24. Zhang, Z., Liu, Q., Lantry, L.E., Wang, Y., Kelloff, G.J., Anderson, M.W., Wiseman, R.W., Lubet, R.A. & You, M. (2000). A Germ-Line P53 Mutation Accelerates Pulmonary Tumorigenesis: P53-Independent Efficacy of Chemopreventive Agents Green Tea or Dexamethasone/Myo-inositol and Chemotherapeutic Agents Taxol or Adriamycin. Cancer Research, 60(4), 901-907.
  23. 23. Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.2, No.5, 2012 14 Table1: Effect of camel urine obtained from Magateer and Majaheem breeds on the growth of lung cancer cells in vitro. Group N o. Mean % SD Control Test Sig. T.te st Sig. Magateer 60 0 84.752 23.641 100.00 15.798 .000 * 6.15 6 .001 ** Majaheem 60 0 92.805 19.805 100.00 9.126 .000 * . No: number of samples. . Mean: percentage of the mean value of the number of living cancer cells. . SD: Standard deviation . Control: Tissue culture containing untreated cancer cells (100 cell ). . * Comparison between the same strain treated cancer cells and non-treated cancer cells ( control). . ** Comparison between two strains. Table 2: Effect of urine obtained from young and adult Magateer and Majaheer breeds on the growth of lung cancer cells (A549) in vitro. Group No. Mean % SD T.test Sig. Magateer (young) 150 87.947 16.592 2.911 .004* Magateer (adult) 150 81.536 24.454 Majaheem (young) 150 96.974 29.460 1.346 .179* Majaheem (adult) 150 93.486 11.810 . No: number of samples. . Mean: percentage of the mean value of the number of living cancer cells. . SD: Standard deviation. . * : Comparison between young and adult at same strain.
  24. 24. Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.2, No.5, 2012 15 Table 3: Comparison between the anti-cancer effect of urine obtained from the two young camel breeds as well as the anti-cancer effect of that obtained from the two adult camel breeds. Group No. Mean % SD T.test Sig. Magateer (young) 150 87.947 16.592 3.499 .001* Majaheem (young) 150 96.974 29.460 Magateer (adult) 150 81.536 24.454 5.476 .000* Majaheem (adult) 150 93.486 11.810 . No: number of samples. . Mean: percentage of the mean value of the number of living cancer cells. . SD: Standard deviation . * Comparison between the two strains. Table 4: Effect of sex on the ability of camel urine to inhibit growth of lung cancer cells in vitro. Sex No. Mean % SD T.test Sig. Male Magateer 300 86.568 15.288 1.886 .060* Female Magateer 300 82.935 29.653 Male Majaheem 300 94.014 23.369 1.595 .111* Female Majaheem 300 91.368 15.867 - No: number of samples. - Mean: percentage of the mean value of the number of living cancer cells. - SD: Standard deviation - *Comparison between the males and females within each breed.
  25. 25. Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.2, No.5, 2012 16 Table 5: In vitro comparison between the anti-cancer effects of urine obtained from females and males in the two different camel breeds (Magateer and Majaheer). Sex No. Mean % SD T.test Sig. Male Magateer 300 86.568 15.288 4.543 .000* Male Majaheem 300 94.014 23.369 Female Magateer 300 82.935 29.653 4.343 .000* Female Majaheem 300 91.368 15.867 . No: number of samples. . Mean: percentage of the mean value of the number of living cancer cells. . SD: Standard deviation . * Comparison between the two strains.
  26. 26. This academic article was published by The International Institute for Science, Technology and Education (IISTE). The IISTE is a pioneer in the Open Access Publishing service based in the U.S. and Europe. The aim of the institute is Accelerating Global Knowledge Sharing. More information about the publisher can be found in the IISTE’s homepage: http://www.iiste.org The IISTE is currently hosting more than 30 peer-reviewed academic journals and collaborating with academic institutions around the world. Prospective authors of IISTE journals can find the submission instruction on the following page: http://www.iiste.org/Journals/ The IISTE editorial team promises to the review and publish all the qualified submissions in a fast manner. All the journals articles are available online to the readers all over the world without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself. Printed version of the journals is also available upon request of readers and authors. IISTE Knowledge Sharing Partners EBSCO, Index Copernicus, Ulrich's Periodicals Directory, JournalTOCS, PKP Open Archives Harvester, Bielefeld Academic Search Engine, Elektronische Zeitschriftenbibliothek EZB, Open J-Gate, OCLC WorldCat, Universe Digtial Library , NewJour, Google Scholar
  27. 27. POSTER PRESENTATION Open Access The effect of camel urine on islet morphology and CCL4-induced liver cirrhosis in rat S Al Neyadi* , R Al Jaberi, R Hameed, J Shafarin, E Adeghate From International Conference for Healthcare and Medical Students 2011 Dublin, Ireland. 4-5 November 2011 Introduction Camel urine has been used for decades as a medication for several ailments in the Middle East. Folklore medi- cine of the Middle East has shown that, camel urine has a beneficial effect in conditions such as liver cirrhosis. Methods Camel urine was given as a drink daily to normal and trea- ted rats for 4 weeks. Glucose tolerance test was performed at the end of the experiment. Immunohistochemistry was used to determine the percentage distribution of insulin and glucagon immunoreactive cells. H & E stain was used to access liver cirrhosis in control and urine-treated rats. Results The administration of camel urine significantly increased the number of insulin-positive cells in pancreatic islets. CCL4-treated rats did not have impaired glucose toler- ance. CCL4 caused vacuolarization of hepatic cells. Rats treated with camel urine have improved hepatic morphol- ogy compared to untreated controls. Conclusions The study shows that camel urine may contain bioactive agents capable of preventing CCL4-induced hepatic and pancreatic islet lesions. Published: 9 July 2012 doi:10.1186/1753-6561-6-S4-P42 Cite this article as: Al Neyadi et al.: The effect of camel urine on islet morphology and CCL4-induced liver cirrhosis in rat. BMC Proceedings 2012 6(Suppl 4):P42. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submitDepartment of Anatomy, Faculty of Medicine & Health Sciences, United Arab Emirates Al Neyadi et al. BMC Proceedings 2012, 6(Suppl 4):P42 http://www.biomedcentral.com/1753-6561/6/S4/P42 © 2012 Al Neyadi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
  28. 28. African Journal of Agricultural Research Vol. 5(11), pp. 1331-1337, 4 June, 2010 Available online at http://www.academicjournals.org/AJAR DOI: 10.5897/AJAR09.686 ISSN 1991-637X © 2010 Academic Journals Full Length Research Paper The inhibitory effect of camel's urine on mycotoxins and fungal growth Amira Hassan Abdullah Al-Abdalall Department of Botany and Microbiology, Faculty of Science for Girls, King Faisal University, El-Dammam, Kingdom of Saudi Arabia. E -mail: Dr2000Amira@hotmail.com. Accepted 8 January, 2010 The effect of urine and camel milk in the inhibition of biological effects of mycotoxins produced by nine isolates of Aspergillus flavus and one isolate of Aspergillus niger isolated from pulse seeds was studied. Where these toxins lost their ability to inhibit Bacillus subtilus growth, milk could not. Also, our study records the effect of camel urine on mycelial growth of some roots rot fungi isolated from seeds of pulses like Rhizoctonia solani, Fusarium moliniform, Aschocayta sp., Pythium aphanidermatum, Sclerotinia sclerotiorum studies, also included are some storage fungi (Aspergillus sp) isolated from coffee beans. Results proved that camel urine at low concentrations has no significant inhibitory effect on fungal growth, while inhibition can be obviously recorded after using high concentrations. Key words: Camel urine, mycotoxins, mycelial growth, inhibitory effect on fungal growth. INTRODUCTION It is mentioned in Islam online that camel's milk and urine have medical effects, so Islam encourages and permits the drinking of camel milk, and camel urine is permitted in case of necessary medical treatment (Al-Bukhhari). The Saheeh Hadeeth says that some people came to Madeenah and fell sick. The Prophet (peace and blessings of Allaah be upon him) told them to drink the milk and urine of camels, and when they drank it they recovered and grew fat. This was narrated by Al- Bukhaari. There are many well known health benefits, with regard to drinking the milk and urine of camels, to the earlier generations of medical science and they have been proven by modern scientific researches. For example swollen abdomen, which may indicate oedema and liver disease (jaundice), or cancer, and thin bodies which indicate extreme weakness, and which often accompanies hepatitis or cancer. This may be due to the effectiveness of camel's urine, as against all other cattle to the active substances contained in desert plants which benefited more of them; this was summed up by the Prophet (peace be upon him). Many researches have been conducted on a variety of desert plants and a strong effect against bacteria, yeast and fungi has been found. Kaul et al. (1976) and Zaki et al. (1984) have conducted researches on the wormwood plant, and results have shown strong effectiveness against bacteria, yeast and fungi. The chemical composition and nutritional quality of camel milk was studied. Results showed 11.7% total solids, 3.0% protein, 3.6% fat, 0.8% ash, 4.4% lactose, 0.13% acidity and a pH of 6.5. It contains low level of cholesterol and sugar and is rich in the levels of Na, K, Zn, Fe, Cu, Mn, niacin and vitamin C (Knoess, 1979). Besides, camel milk contains low level of protein and high concentration of insulin, and could be safely taken by people who have high sensitivity to lactose and have immune deficiency (Gast, 1969). Camel milk is pure white and sugary. Camels who feed on certain diets may produce salty milk when feed on desert weeds. There are physiological and genetical factors affecting milk production. Percentage of water in camel milk varies according to the doses of water which camel drinks; it may reach 89% in the milk if camels drink water every day, or 91% if camels drink one hour weekly. It seems

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