INTRODUCTION:

The use of natural products to treat pathologies and several health disorders is nowadays a very popular means. Besides medicinal plants, human and animal urine are used in traditional medicine under the name of urine-therapy with the using of some the human urines¹ or animal urines to heals some health disorders such as cow and camel^2,3. Camel“ Camelus dromedaries” urine (CU) has been widely used in the biomedical field in several Arabian countries such as Algeria especially in the Saharan regions as healing biological liquids for wounds and burns, against eczema and allergies, anti-dermatophytes and antitumor, without harmful side effects^4,5,

where of the effected researches and biochemical analysis and studies carried out on CU that focused on nitrogen metabolites has found that those metabolites are in very low concertation comparing to other type of urine making CU less or no toxic⁶.

So far, a few activities of CU have been revealed such as its capacity to inhibit the proliferation of some bacterial, fungal and viral strain due to its chemical composition^4,7. Also, CU has demonstrated anti-cancer properties⁸ by blocking the expression of some genes that activate cancer at the transcriptional and post-transcriptional stages⁹. Moreover, CU can play the role of an antiplatelet agent¹⁰ and in treating some diabetic complications¹¹; it was also found that camel urine has an efficiency against multi-drugs resistant Pseudomonas aeruginosa, isolated from effected burns, wounds and ears¹². However, though the potential capacities that CU may illustrate it didn’t take an important place in the traditional pharmacology researches unlike cow urine that took a wide place in literature researches^13,14.

The therapeutic forces exhibited by this urine may be directly related to the richness of Saharan plants species grazed by the dromedary camel with active ingredients such as second metabolites, where these components are believed to be the source of the biological activities of this liquid¹⁵, On the other hand, the uniqueness of this animal and its physiological structure can be the reason for the utility and capacities of its urine.

Thus, this study aims to evaluate the antioxidant activity of camel urine carried out under two breeding and feeding system domesticated and desertic.

MATERIALS AND METHODS:

Sampling:

The urine used in this work comes from non-lactating, disease-free, aged between 2-8years female camel (camel dromedaries) belong to the Sahrawi breed, from Ouargla, Algeria. Urine samples were collected from camels under two different breeding systems (five of each):

· Domesticated camels (DC), raised on a private farm where they had free access to water and food such as cereal grain, some plants cut and dried bread.

· Desertic (Saharan) camels (SC), desert living that fed only on the Saharan plants with the lack of water.

· CU was collected in the morning (4-5am) with experienced help in dark, sterile vials and immediately transported in coolers at 4°C to the laboratory to be lyophilized for the study assays.

Phenolic compounds determination:

The determination of the total phenolic compounds was carried out using the Folin-Ciocalteu reagent, applying the coulometric assay reported¹⁶.

A quantity of 100μl of each solution is introduced using a micropipette into test tubes, followed by the addition of 500μl of the Folin-Ciocalteu reagent (diluted 10 times). After incubation for 2min, 2ml of 20% sodium carbonates (Na₂CO₃) is added, then kept in the dark for 30min at room temperature. The absorbance of each solution was determined at 760nm against a blank prepared in the same manner except that it did not contain gallic acid. The standard calibration curve was obtained from gallic acid solutions of different concentrations (0.01-0.3mg/ml), where the optical density readings at 760nm of the solutions thus prepared allowed the calibration curve for gallic acid to be drawn ¹⁷.

Flavonoids Determination:

The determination of flavonoids was carried out using the aluminum trichloride (AlCl₃) method described by¹⁸. To a volume of 1ml of each urine 1ml of the AlCl₃ solution (2% in methanol) was added. After 10 minutes of incubation, the absorbance measuring was affected at 430nm. Using the rutin as a standard at different concentrations (0-10mg/L), for the calibration curve obtaining, following the same steps of the assay. The results were expressed in mg Rutin equivalent (RE) per liter of urine volume (mg RE/ LU)¹⁹.

Fourier-transform infrared spectroscopy (FTIR) characterization:

The equipment used for this study was the Bruker Tensor 27 spectrometer equipped with a DLA TGS detector (Bremen - Germany), and the data acquisition was accomplished using OPUS program version 7.2 for Windows from Bruker GMBH. The studied products were directly injected and compressed on the ATR crystal surface using distilled water as background, where the FTIR scanning was performed at a band interval of 4000-600 s-1, accumulating 16 scans per spectra.

Antioxidant capacity evaluation:

Four testes were chosen for the oxidation inhibition activity as following:

a) DPPH scavenging assay:

A volume of 0.05ml of different concentrations of each urine was added to 1.950ml of methanolic solution of DPPH (0.024 g/l). the 2ml solution was then incubated in the dark for 30min and at room temperature, the absorbance reading was taken at 515nm. The inhibition percentages were calculated by the following formula:

I% = ((Ab - AS)/ Ab) *100

Where: AS is the samples absorbance, Ab is the blank absorbance²⁰.

b) ABTS scavenging assay:

A 16h earlier prepared stock solution of ABTS was diluted with phosphate buffer (0.2 M, pH 7.4) to obtain an absorbance of 0.70 ±0.2 at 743 nm. From this solution a volume of 1.95 ml was mixed with 0.05 ml of urine at different concentration and kept for 6-7 min in obscurity at room temperature than the absorbance was recorded at the same wavelength mentioned before using phosphate buffer as blank²¹. Inhibition ration percentage was obtained using the following formula:

I% = ((Ab - AS)/ Ab) *100

Where: AS is the sample absorbance, Ab is the blank absorbance.

c) FRAP reduction test:

Applying the spectrophotometric assay reported by²² this test was accomplished. The absorbance of the final obtained solution is determined at 700nm, where the more the absorbance is high the more is the reducing power of the tested samples. This solution is obtained by mixing a volume of 1ml of urine at different concentrations with 2.5ml of PBS (0.2M, pH 6.6) and the same volume of potassium ferricyanide K₃Fe (CN)₆ solution (1% W/V). this mixer was put in a water bath of 50°C for 20 min. Another volume of 2.5ml of trichloroacetic acid (10% W/V) was added to end the reaction and the tubes were centrifuged at 3000rpm for 10min, a 2.5ml of the supernatant was combined with 2.5ml of distilled water and 0.5ml of aqueous FeCl3 solution 0.1% (W/V). similarly, prepared blank, replacing the urine with distilled water which makes it possible to calibrate the device (UV-VIS spectrophotometer)²³.

d) PPM (Phosphomolybdate) assay:

The method involves placing in a hemolysis tube 200μl of urine at difrent concentration mixed with 1800μl of a reagent composed of H₂SO₄ (0.6 M), NaH₂PO₄ (28 mM) and ammonium molybdate (4mM). The tube is then tightly closed and then incubated at 95°C. for 90 minutes in a water bath. After cooling the mixtures to room temperature, the absorbance is measured at 765 nm. the reducing power increasing is aligned with the absorbance increasing²¹. Reducing power was estimated using following formula:

Reducing power (%) = [(Ab - As)/Ab] * 100

Where: AS is the sample absorbance, Ab is the blank solution absorbance.

Statistical analysis:

Using the SPSS package program (IMB SPSS 25) Statistical analysis was carried out. Preforming a One-way analysis of variance (ANOVA) and the statisticaldifferences between the means were evaluated at the significance level of 0.05 using the Tukey test. The experimental analyses were performed in triplicate.

RESULT:

Phenolic and flavonoids content:

As presented in Table 1 phenolic compounds are reported in mg gallic acid equivalent per liter of urine. The urine of the SC illustrated a content of phenolic compounds significantly higher (p<0.05) comparing to the urine of the DC with an average of 0.84±0.01mg GAE/LU that was 0.45±0.04mg GAE/ LU in the (Table 1).

The quantification of flavonoids was carried out by a standard solution (Rutin) at different concentrations. The results obtained are shown in Table 1, where the flavonoid content in the urine of camels carried out in the desert was 0.014±0.003mg RE/LU, while the second type of urine contained a concentration of 0,005±0.001

Table 1. Total phenols (mg GAE/UL) and total flavonoids (mg RE/U L) in both domesticated and desertic camel urine

Samples	Total phenolics (mg GAE/ UL)	Total flavonoids (mg RE/ UL)
SC1	0.87±0.03^a	0.011±0.001^B
SC2	0.89±0.03^a	0.013±0.001^B
SC3	0.77±0.04^b	0.016±0.001^A
SC4	0.75±0.03^b	0.017±0.002^A
SC5	0.90±0.04^a	0.016±0.001^A
DC1	0.45±0.01^cde	0.004±0.000^C
DC2	0.48±0.02^cd	0.006±0.000^C
DC3	0.51±0.02^c	0.005±0.001^C
DC4	0.42±0.02^de	0.004±0.000^C
DC5	0.41±0.02^e	0.006±0.001^C

The values are the mean of three determinations ± standard error.

a-e: Different letters in the column indicate a significant difference (P<0.05) between the results of different camel samples; A-C: Different letters in the line indicate a significant difference (P<0.05) between the results of same camel sample.

Fourier-transform infrared spectroscopy (FTIR) characterization:

From Figure 1 and 2, both FTIR scans gave practically the same clear peaks pattern of the functional groups that exist in the studied samples, yet with an interval of 2 to 3 cm-1 between the peaks profile of the two types of urine which account insignificant.

From the obtained graphs profile the following functional groups where revealed (Figure 1,2), starting with a large band referring to the symmetric and asymmetric elongation of the hydroxyl group “OH” at ∼3440 cm−1, followed by a peak at ∼1630 cm−1 representing the NH corresponds to the nitrogen molecules and the carbonyl C=O groups that of urines. Two small successive bands at ∼1399 cm−1 ∼1460 cm−1 relating to the N-H deformation and the C=C stretching cm−1 causing by the to the hypochromic action of the amid and carbonyl groups and at ∼1245 cm−1 ∼1300 cm−1 bands at corresponding to the C-O group, with a high peak that was at ∼1100 cm-1 relating to the C-O group and the C-N vibrations^22-24.

Figure 1: FTIR absorption spectrum of urine obtained from domesticated camels (DC).

Figure 2. FTIR absorption spectrum of urine obtained from desertic camels (SC).

Antioxidant capacity evaluation:

The antioxidant activity of camel urine was investigated using four tests, ABTS and DPPH radical scavenging tests, with the phosphomolybdate and FRAP test that allowed us to assess the antioxidant status of camel urine. It appears from the results of the four testes that the antioxidant activity of the studied product is very important compared with the standard (ascorbic acid).

From Figure 3, it can be seen that the effective concentration to inhibit 50% of free radical’s DPPH by the SCU, DCU, and AA was 19.133±0.402, 15.467±2.411 and 6.427±0.369µg/ml respectively. Where both urines showed a great oxidation inhibition capacity, yet the anti-free radical activity of the urine of camels carried out in the private farms was significantly higher (p<0.05) than the ability demonstrated by the urine of camels carried out in the desert. the same results were illustrated in the ABTS scavenging test where the half maximal effective concentration (EC 50) value of the DCU was almost twice lower than (p<0.05) the EC50 of the SCU with a value of 16,80±0,382 and 9,600 ±1,992 for the SCU and DCU, respectively (Figure 3).

According to Figure 3, the reducing powers of the samples in the PPM and the FRAP tests of both tested samples was interesting, yet the reducing power of both urine in the FRAP test was more important with a very low concentration than the PPM test where the EC50 of the urine from camels conducted in the domesticated farms was 85,467±5,084 and 102,596±1,853 for the urine of the desertic camels (p<0.05).

Figure 3. Half maximal effective concentration (EC50) values of DC and SC urine and the standard. ANOVA, with Tukey test with *p < 0.05 (n=5, ±SD).

DISCUSSION:

The variability of the contents of phenolics and flavonoids in the two types of urine analyzed is linked to the physiological state of the animal and to the phenolic composition of the food ingested, part of which is eliminated mainly in the urine^28,²⁹. Indeed, where the urine of the SC showed a higher concentration of those compounds due to the richness of plants or the nutrients ingested in these compounds^30,³¹. Unlike the urine of the domesticated camels that showed a very low level of both phenolics and flavonoids due to the lack of their ingested food of those elements where their diet is mainly based on fodder that is exclusively cereal grain, some plants cut and dried bread, the data analysis reveals that the feeding system is related to the concentration of phenolics and flavonoids in both urine type.

The FTIR characterization showed that both of the tested urine demonstrated the same peak pattern which means the composition of the DC and SC urine is almost the same despite the feeding system difference, concluding that no matter what the feeding and breeding method is the camel kidneys will exhibit the same recycling and filtration processes.

The antioxidant capacity demonstrated by both type of urine can be related to the presence of the “OH” functional group as shown in the FTIR profiles of the SCU and DCU (Figure 3) which is widely known with its high capacity as a reducing and scavenging group³². Moreover, the oxidation inhibition ability can be also related to the presence of phenolic compounds in the two studied urine that is classified as the most powerful agents to trap the free radicals gents³³, yet the very low concentration of this compounds is insufficient to explain this important activity, especially that the concentration of those elements was lower in the DCU yet this urine showed a higher antioxidant capacity comparing to the SCU.

Moreover, it appears from the obtained results by all applied tests, that the significant anti-radical activity and reducing power of urine of camels conducted in the private farms was higher than the urine of camels conducted in the desert (Figure 3), which was not proportional to the phenolic compounds content in both studied urines that was higher in the urine of the desertic camels, suggesting that this activity is note related to the phenolic compounds concentration. Moreover, from the obtained absorbance of the FTIR spectrum, it appears that the concentration of the functional groups in the urine of DC is higher than the urine of the SC due to the free access of food and water unlike the other camels, where the more this animal is fed the more the secretion of the molecules with antioxidant ability is high which denied the theory that refers the CU value to the richness of the desertic plants consumed by this animal with active elements, which means that the oxidation inhibition ability demonstrated by CU is mainly related to the physiological, biological and metabolic characteristics of the Camelus dromedaries which can influence the pathways of the biosynthesis of this biological fluid by producing molecules responsible of activity in this urine³⁴.

The consumption of camel urine with high antioxidant power can therefore be applied for the prevention of diseases associated with oxidative stress such as cancer, digestive disorders, and cardiovascular diseases^33,³⁵.

CONCLUSION:

The results of the executed study demonstrate the existence of a small amount of phenolics and flavonoids where the concentration was proportional to the camel feeding system, it was also revealed from this work that the urine of the Camelus dromedaries exhibit a very imported antioxidant capacity no matter what the breeding method and the feeding system was desertic or domesticated, therefore the consumption and usage of camel urine with high antioxidant activity can contribute to the prevention of diseases and health disorders associated with oxidative stress.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 24.02.2022 Modified on 15.05.2022

Asian J. Pharm. Res. 2022; 12(4):261-266.

DOI: 10.52711/2231-5691.2022.00042