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Vol. 5(1), pp. 1-8, 2021
Copyright ©2021, the copyright of this article is retained by the author(s)
Comparing day 0 and day 28, Hematological and Biochemical parameter changes, on Male white Rattus norvegicus exposed to Urtica dioica leaves & stem Ethanolic Extracts; a herb used in the management of Diabetes mellitus by the Tugen Community-Kenya
Kipbichii Chebor1*, Osano Odipo3, Isaboke Job3, and Ng’wena Magak2
1. Department of Community Health and Extension (C.H.E.), School of Nursing, Midwifery and Paramedical Sciences (SONMAPS), Masinde Muliro University of Science and Technology (MMUST), P.O. Box 190-50100 Kakamega.
2. Department of Medical Physiology, School of Medicine (S.O.M.), Maseno University (M.U.), P.O. Box 333, Maseno.
3. Department of Environmental Biology and Health (E.B.H.), School of Environmental Studies (S.E.S.), University of Eldoret (UoE), P.O. Box 1125-30100.
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E-mail: senvebhm00119@ uoeld.ac.ke; fillmanjob@ gmail. com
Objective: To compare day 0 and day 28 hematological and biochemical parameters changes on male white Rattus norvegicus , exposed to Urtica dioica, stems & leaves mixtures, ethanolic extracts.
Materials and methods: Experimental design was employed. About 2kg of fresh Urtica dioica (U.D.) mixture of leaves &stems, obtained from Baringo County, Eldama-Ravine forest, Kenya, were cleaned with distilled water at the site to remove debris and transported to the University of Eldoret, where its identity was taxonomically ascertained by the University taxonomists. Later, the herb was dried at room temperature and crushed into a powder form. Five hundred (500g) grams was used in [ethanolic] the extraction [duration was 72 hours], after which the percentage yield, phytochemicals, quantity (done using HPLC), and later the dosages were determined, after exposing six rats, to different concentrations of the herb. The rats used, were obtained from the University of Eldoret department of biological sciences. Rats’ acclimatization was done for 7 days to make them adapt to the lab environment. Cage 1, which had six (6) rats were used for dosage optimization; cage 2, which had five rats, was used in the actual experiment (duration- 28 days). Cage 3 were the controls -given distilled water (controls). Blood samples for hematological; (hemoglobin [Hb.), red blood cells [R.B.C.], and white blood cells [WBC]) and biochemical (Urea, creatinine, Alanine Transaminase [A.L.T.s], Aspartate Aminotransferase [A.S.T.s]) tests, were drawn from the rats’ tail end, at day 0 and day 28. Day 0 lab tests results were compared to day 28 lab test results. Analysis was done using ADVIA 220i for hematology and Chemistry analyzer Cobas C311 for biochemicals. The outcomes were descriptively analyzed and presented in tabular form and figures. All the tests were 2-tailed, where applicable, and were considered significant at a p=0.05. Ethical approval to conduct this study was sought from, Animal Research and Ethics Committee (HAREC) of the University of Eastern Africa Baraton (UEAB)- Kenya (R.E.C.: UEAB/3/1/2018).
Results: With regards to hematological parameters, all the changes were within the normal ranges. WBC especially recorded a decrease in all the parameters on the 28 days. Although biochemical parameters recorded some changes (Urea recorded a significant increase from day 0 to day 28, p-value of 0.001), these changes were also within normal ranges.
Conclusions and Recommendations: Urtica dioica leaves and stems ethanolic extracts showed some elevation in urea levels, though they were within the normal ranges and a decrease in WBC, which may signify that U.D. possesses, some anti-microbial or anti-inflammatory activities. Nevertheless, U.D. is generally safe (all changes were within the normal ranges); however, more studies are needed.
With an increase in the use of herbal medications used today, so to the increasing concerns about their safety/toxicity. These concerns may be either general or herb-specific, hence the need to study them. A toxicity test is essential for developing new drugs and for the extension of the therapeutic potential of existing drugs. This toxicity/safety of medicinal plants may be related to the mixtures of active compounds that they contain; their interactions with other herbs and drugs, contaminants, adulterants; or their inherent toxicity
(Rodriguez-Fragoso et al., 2008).
More than 20,000 medicinal plants are used to manage numerous pathologies in Africa today, but less than 1% of them have been scientifically investigated for safety (Taika et al., 2018). An herb might be effective but not safe. Therefore, it’s imperative to study its safety/toxicity. Animals have been used since time immemorial as surrogates when examining the safety and efficacy of medicinal herbs. One such animal is the use of white Rattus norvegicus. One among many outcomes, tested/ studied parameters is hematological (the red blood cell [R.B.C.], white blood cells [WBC], hemoglobin [H.b.]content), and biochemical (Urea, creatinine, electrolytes, enzymes such as A.S.T.s and A.L.T.s) changes. Deviation from the regular usually signifies something (Arika et al., 2016).
Therefore, this study wanted to compare day 0 and day 28 hematological and biochemical parameter on white Rattus norvegicus, exposed to ethanolic extracts of Urtica dioica (stinging nettle) leaves & stems, a herb that comes from the Urticaceae family(Patel et al., 2018). Traditionally, used as a blood purifier, diuretic, a treatment for rheumatism, eczema, anemia, nephritis, hematuria, jaundice, menorrhagia, diabetes mellitus, hypertension, and diarrhea. It is widely distributed throughout the temperate and tropical areas around Africa, Asia, and the world (Chebor K, 2020; Joshi et al., 2014).
MATERIALS AND METHODS
Study area: The study was carried out in the University of Eldoret, Biotechnology Centre laboratory.
Study design: Experimental
Collection of the herbs, preparation, and extraction:
Collection, identification, and extraction
2 Kgs. of Urtica dioica plant materials were collected from Baringo County-Kenya and taxonomically verified based on their morphological characteristics (Beattie et al., 2005) by the University of Eldoret’s-Kenya, Department of Botany, taxonomists. They were cleaned at the place of origin (Eldama-ravine forest) with distilled water to remove external debris attached to them, then transported to the University of Eldoret (Kenya), Biotechnology Laboratory, where they were air-dried at room temperature to complete dryness before being crushed with a grinder- OHMS OCG-200, into a powder form in readiness for extraction.
[98% concentration-analytical reagent purchased from Sigma-Aldrich-Kenya]
The herb was extracted using the maceration extraction process. Five hundred grams (500 g) of the herbs were soaked in two liters of ethanol for 72 hours (Azwanida, 2015)at room temperature, after which the resultant mixtures were filtered using Whatman filter paper (No.1) and the filtrate concentrated to dryness using a vacuum-rotary evaporator machine- BUCHI Rotavapor R-3000 at a temperature range of between 400C -500C. Fifty milliliters (50mls) of distilled water were then added to the container containing the concentrated dried substance, and then, using a stirring rod, the contents of the container were stirred to dissolve the dried substance much as possible.
Determination of extraction yield
After extraction, U.D., percentage (%) yield was determined based on the formula described by (Qaid, 2020). Lyophilization was done with the help of ‘Harvest Right freeze drier (U.S.A)’ for 24 hours (ethanol extract).
The resultant yield powder was dissolved in 200mls of distilled water.
5mls was used in phytochemical screening (screening done as per the description of (Muralidharan, 2015)
1ml used in HPLC (determining the quantities) The screening was done using Shimadzu HPLC SYSTEM Machine from shimadzu cooperation Kyoto-japan. The column was: Silica 250 x 4 nm, ten μm. Injection.
50mls was used in optimization
144mls used in the actual experiment
N/B for the sake of optimization, the 50mls formed was classified as 100%. Further dilutions were made using distilled water—75%, 50%, 25% and 12.5%, and 0% and given to 6 rats via oral gavage at 9 am daily for seven days. First rat (1) rat given 100%, 1 rat 75%…and the last (1) rat given 0% (pure-distilled water). The aim was to determine the optimal concentration to be given to rats during the actual experiment. Physical and physiological parameters, e.g., temperature, behavior, etc. checked at seven days as described by (Hawkins et al., 2011). Findings analyzed and optimal dose with no or minimal effect, picked.
During the actual experiment, the rats were given the determined dose via oral gavage every morning for the duration of the experiment (28 days).
The feeding protocol was done in reference to the Piero et al. study of 2011 (Piero et al., 2011).
Collection, preparation, and protocol of feeding the rats (cases and controls)
Sixteen (16) white males Rattus norvegicus, between the ages of 4 and 6 months, weighing in the ranges of 200-230g, were recruited. The animals were obtained from the University of Eldoret-Kenya department of biological sciences. The sample size was selected based on systematic published peer-reviewed studies (Ranasinghe et al., 2012; Yeh et al., 2003).
Preparation (Acclimatization of the rats to the lab environment)
The rats were cared for under the laboratory procedure, and feeding was done using pellets from UNGA limited-Eldoret Kenya, morning and evening, and given water ad-libitum.
N/B Before the experiment commenced, the rats had to be acclimatized in the lab environment for seven days.
Before the experiment, blood samples were taken for baseline hematological (Hemoglobin, R.B.C., WBC) and biochemical tests (Urea, creatinine and A.L.T.s, and ASTS). The same was also done at the end of the experiment. Blood samples were collected through the tail end and put on heparinized sample bottles, where applicable, in readiness for the tests (the collection was done on days 0 and 28). Analysis was done using ADVIA 220i for hematology and Chemistry analyzer Cobas C311 for biochemicals.
Cage 1 was used for optimization, cage 2 (cases) rats were given ethanolic extracts of U.D. via oral gavage. In cage 3, the controls were fed with only distilled water and pellets from Unga Kenya ad-libitum.
The feeding of the cases was done once daily at 0.5mls U.D./100mg of a rat, orally at 0900 hours while continuing with the other regular feeding (i.e., the pellets and water, every morning and evening for the 28 days).
N/B the handling of the animals was as described by National Academies(Council, 2010).
Approval of the research was done by the Human and Animal Research and Ethics Committee (HAREC) of the University of Eastern Africa Baraton (UEAB)- Kenya (R.E.C.: UEAB/3/1/2018)
The resultant data were entered into excel sheet office 19 and analyzed using SPSS software version 21. Descriptive statistics were used to describe the data and summarized in tabular form. All the tests were 2-tailed, where applicable, and were considered significant at a p=0.05.
After collecting the Urtica dioica plant samples from Baringo County-where they are commonly used (Chebor K, 2020), the plants were extracted, and the extraction yield was determined as shown in table 1. Different quantities were then subjected to qualitative phytochemical screening, HPLC Analysis Hematological and biochemical responses using male white Rattus norvegicus rats. The hematological and biochemical responses were monitored for 28 days. The status at 0 day was compared to the status at 28 day.
N/B, Day 0 status with reference to the normal ranges was either low, normal, or high, at day 28th, and was described as either; increase-within normal ranges, increase above the normal ranges, decrease within the normal ranges, or a decrease below the normal ranges. [Normal ranges was based on the works of (Giknis & Clifford, 2008; Sharp & La Regina, 1998)]
The results are as shown below;
The percentage yield of Urtica dioica after extraction.
Table 1: Yield outcomes of extraction yields
The rate of extraction using the maceration method yielded 1,376 mg of the herb for 72 hours. This is approximately 0.28% extract obtained from the plants. After extraction, the extracts were subjected to qualitative phytochemical screening.
Phytochemical screening of ethanol extract
Table 2: Qualitative analysis of phytochemicals of Urtica dioica ethanolic extract
Fig 1: HPLC Chromatogram Analysis of Urtica dioica ethanol extract
Fourteen phytochemicals were tested qualitatively, and 50% of the tested compounds were present. Saponins were found to be of high quantity with the domination of “+++” while alkaloids, Flavonoids, and sterols were moderately present. Quinones, Glycosides, and coumarins were seen in trace amounts “+.” After a qualitative phytochemical screen of the extract, the outcome was further tested in HPLC to confirm the presence of the phytochemicals.
The HPLC analysis chromatogram of the plant produced ten peaks with different retention times and different areas. This confirmed more than seven (7) different compounds that can be extracted by ethanolic solvents. The extract was then tested for a hematological and biochemical response on the white Rattus norvegicus male rats.
Weight averages of the rats
Table 3: day 0 and 28 average weights
Dosages given to the rats
Based on the results of optimization, there was no notifiable effect on the six rats after exposing to various concentrations of Urtica dioica; hence the dosages were given using the 100% concentration. The dosages given to rats was, therefore, 0.5ml/100g of rat, which is equivalent to 3.44mg/100g of a rat once daily for 28 days.
Hematological parameters changes
Fig 2: Surface plots for Hematological responses of male white Rattus norvegicus rats exposed to Urtica dioica ethanol extracts: the test of red blood cells, white blood cells, and hemoglobin.
There was a significant increase of both R.B.C. and WBC in the controls but a decrease in hemoglobin (P<0.05). The hemoglobin of the rats administered with the extract (cases) significantly increased by 12.1% (p<0.05) from day 0 to day 28, while that of control decreased by 0.7%. WBC in cases generally decreased.
Different biochemical parameters were also tested on the responses from exposures of Urtica dioica, namely Urea, creatinine, ALTS, and ASTL.
Fig 3: Surface plots for Biochemical responses of white male Rattus norvegicus exposed to Urtica dioica ethanol extracts: the test of Urea and Creatinine.
Fig 4: Surface plots for Biochemical responses of white male Rattus norvegicus rats exposed to Urtica dioica ethanol extracts: the test of ALTS and ASTL.
There was a significant increase in Urea from day 0 to day 28, p-value of 0.001 for both the control and the cases. With regards to creatinine, there was an increase in the control but a decrease in the cases.
Both ALTS and ASTL increased significantly on the rats exposed orally to the herb extract while there was no significant increase (A.L.T.s) and decrease (A.S.T.) in controls (p>0.05)
This study aimed to determine the phytochemical’s constituents of Urtica dioica and their hematological and biochemical effects on exposure to white male Rattus norvegicus rats after 28 days.
Regarding phytochemicals, out of the fourteen tested, seven were present: alkaloids, flavonoids, saponins, quinones, glycosides, coumarins, sterols, and terpenoids. Based on the confirmation of phytochemicals by the HPLC analysis, there were more than seven (7) different peaks, with variations in retention times and area. This variation confirms different phytochemicals as confirmed in literature (Bourgeois et al., 2016; Dar et al., 2013; Grauso et al., 2020).
Concerning hematological changes, Hb, in cases increased significantly (p<0.05), indicating that this herb might be hemo-protective and hepato-protective, a finding that is in congruence with the findings (Juma et al., 2015), while a decrease was seen in WBC, signifying that this herb might be possessing an anti-microbial or autoinflammatory tendency. A finding that concurs with the studies of (Dar et al., 2012). R.B.C. parameter, also reduced as compared to the controls, nevertheless the levels were within the normal range
Regarding biochemical parameters, all urea levels recorded a higher level on day 28 than day 0, a finding suggesting that this herb might affect the Kidneys. The same increasing scenario was also seen with regards to creatinine levels. ALTS and A.S.T.s, though there were some changes, all the changes were within normal ranges—a finding concurrent with the findings of (Mukundi et al., 2017).
CONCLUSIONS AND RECOMMENDATIONS
With regards to hematological parameters, all the changes were within the normal ranges. WBC especially recorded a more of a decrease in all the parameters on the 28 days. Concerning biochemical parameters, though all the others recorded a change within normal ranges, Urea recorded some changes, higher than the normal. Nevertheless, U.D. is generally safe. However, much more studies are needed.
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