The Effect of Molasses in Reducing the risk of Chloramphenicol-induced Reticulocytopenia in White mice ( Mus musculus)Marika Allego Edzelle Marie Andalahao Anacleto Clent Banaay jr.

Inroduction Oxidative damage is implicated in the etiology of cancer, cardiovascular disease, blood dyscrasia and other degenerative disorders the production of Free radicals and Reactive oxygen species is prevented by antioxidant systems and enzymes

Review of Related literatureMolasses The Quality of Molasses is dependent on the process of Production, The method of Production, the soil it was grown.

Curtin, 1973

Review of Related literature Sugar Molasses where identified to be significant in the inhibition of DNA oxidative damage The Antioxidants found in molasses: syringic acid, p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde and ferulic acid They are significant in the inhibition of DNA oxidative damage

Guimaraes, 2004

Review of Related literature Molasses has more antioxidant than refined sugar Dark and blackstrap molasses had the highest FRAP ferric-reducing ability of plasma (FRAP) assay was used to estimate total antioxidant capacity. Calsen, 2009

Review of Related literatureCHLORAMPHENICOL Increased Dosage and Duration of Therapy are the two main factors why it can cause Bone marrow damage and dysfunction by Mitochondrial Leakage of the Marrow cells which includes the Erythroid and the myeloid populations McCurdy, 1963

Review of Related literature Chloramphenicol is given at 45 to 60 mg per kg of body weight every eight hours for Humans Maximum dose of 100 mg/kg and the duration ranges from 8 to 10 days

McCurdy, 1963

Review of Related literature Reticulocyte count is a semiquantitative indicator of bone marrow erythropoietic activity and is estimated by evaluating the presence of immature RBC's in the peripheral blood. The reticulocyte count is most useful for assessing the marrow response to anemia McPherson, 2007

Review of Related literature By increasing the dose and prolonging the duration of Chloramphenicol therapy, bone marrow suppression will develop as indicated by Reticulocyte count and Bone Marrow study

McCurdy, 1963

The exposure of the bone marrow to chloramphenicol is determined by the Plasma concentration of Chloramphenicol, the length of therapy and the possible concentration of drug in the Bone Marrow

McCurdy, 1963

Review of Related literature Mus musculus Linn is the common house mouse and the most commonly used Laboratory mouse the reticulocyte ranges from 1 to 3 % of the RBC count. The Retiulocyte estimate is from 100x106 /L to 300 x106 /L. Staats, 1979

Objective of the Study General objective To determine the effect of molasses in reducing the risk of chloramphenicolinduced Reticulocytopenia in White Mice ( Mus musculus).

Specific objective1. To determine the Baseline Reticulocyte count in the following groups: 1.1 Test group (Chloramphenicol and Molasses)Control group (Chloramphenicol)

2. To determine the Post- treatment Reticulocyte count of the two groups after 10 days.

. To determine the Mean Baseline Reticulocyte count and the Mean Posttreatment retiuclocyte count of the two groups. 4. To determine the significant difference between the Mean Baseline Reticulocyte counts of the Test group and the control group. 5. To determine the significant difference of the Mean change between the Test Group and the Control Group.

6. To determine the significant difference of the Post Treatment Reticulocyte between the Test Group and the Control Group.

Statement of Alternative Hypothesis There is a significant effect of Molasses in reducing the risk of chloramphenicolinduced Reticulocytopenia in White mice (Mus musculus). Therefore the Null hypothesis (Ho) is rejected.

Significance of the Study1. Patients undergoing Chloramphenicol therapy 2. Patients who are Potential victims of Aplastic anemia 3. The Medical practitioners

Methodology Resign designTrue experimental Research design Research environmentThe study was conducted at the Matias H. Aznar Memorial College of Medicine- Department of Pharmacology, 6th Floor MHAM Building, Southwestern University, Urgello Street, Cebu City Reticulocyte count was done at Vicente Sotto Memorial Medical Center, Department of Pathology, B. Rodriguez Street, Cebu city

Research subjects Twenty White Mice (Mus musculus) utilized in the study based on sample size determination Sample size formula:

Research ProcedureRandom assignment of the 20 mice

Treatment Group ( Group A)

Control Group (Group B)

Baseline Reticulocyte count determination

Test Group

Control group

Reticulocytes observed before the treatment period

Test Group

Control group

Molasses for 10 days

Chloramphenicol for 10 days Chloramphenicol for 10 days

On the Eleventh day

Post Treatment Reticulocyte count determination

Test Group

Control group

Testing the Null hypothesis if there is a significant difference between the Test group and the control group

Treatment of Data Independent T Test was utilized

NCSS Statistical software was used to Analyze to the Data

Definition of terms Molasses Molasses is a dark, viscous liquid that is a by-product of sugar production made from either sugar beets or sugar cane. It is produced when sugar syrup is boiled to get to crystalline form. It is the substance that is employed in the study to determine its effect in the prevention of Bone Marrow Suppression.

Definition of terms Chloramphenicol- it is a bacteriostatic antimicrobial which is considered to be a broad-spectrum antibiotic. It is associated with bone marrow toxicity, which may occur in two distinct forms: bone marrow suppression, which is a direct toxic effect of the drug and is usually reversible, and aplastic anemia, which is idiosyncratic (rare, unpredictable, and unrelated to dose) and generally fatal.

Definition of terms Bone Marrow suppression- is a myelotoxic condition usually resulted from a serious side-effect of chemotherapy and certain drugs affecting the immune system.

Definition of terms Baseline Reticulocyte countReticulocyte count is the index of Bone marrow production of Mature Red blood cells. The Baseline Reticulocyte count is performed on the First Day of the 15-day period of testing. The Mean Baseline Reticulocyte count is computed and compared with the Mean Post Treatment Reticulocyte count.

Definition of terms Post treatment Reticulocyte- The test is performed after a 15- day period of Chloramphenicol and molasses administration to determine if there is a change when compared to the Baseline Reticulocyte count. The Mean is also computed to determine the significant difference between the test group and the control group.

Results and DiscussionTable1. Mean Reticulocyte count of the Treatment Group and the Control group Group Baseline Post-treatment Mean change of Reticulocyte Reticulocyte Reticulocyte Count count count (X106 /L) (X106/L) 195.2 122.3 72.9

Treatment Group Control Group

157.1

81.4

75.7

The Treatment group has a higher Mean Baseline reticulocyte count with a value of 195.2x 106/L compared to the control group with only 157.1x106/L. The treatment group has a higher Mean Post treatment Reticulocyte count with a value of 122.3X 106/L while the Control group has only 81.4x 106/L.

Figure 1.Comparison of the Mean Baseline Reticulocyte and the Mean Post treatment Reticulocyte of both Groups.

Table 2.The significant difference between the Baseline Reticulocyte count of the Test Group and the Control Group. MEAN PROB. POWER DECISIO VARIABLE SD DF T N LVL (ALPHA VALU =0.05) (5 %) E TEST GROUP 195.2 56.3181 18 1.5519 0.06904 0.4397 53.4383 18 ACCEPT H0

(MOLASSES+ CHLORAMPHE NICOL) CONTROL 157.1 GROUP

Tests of Assumptions Section Value Probability Skewness Normality (treatment=molasses) -0.1080 Kurtosis Normality (treatment=molasses) Omnibus Normality (treatment=molasses) Skewness Normality (treatment=placebo) Kurtosis Normality (treatment=placebo) 1.2114 Omnibus Normality (treatment=placebo) 5.2831 Variance-Ratio Equal-Variance Test Modified-Levene Equal-Variance Test 1.1107 0.0448 -0.3565 0.1388 1.9534 0.225758 0.071253 0.878299 0.834698

Decision (5%) 0.914035 Cannot reject normality 0.721456 Cannot reject normality 0.932974 Cannot reject normality 0.050775 Cannot reject normality Cannot reject normality Cannot reject normality Cannot reject equal variances Cannot reject equal variances

Table 2 shows that there is no significant difference between the Test group and the Control group The tests for the assumptions were also determined to validate normality in each group, to signify that the variables are normally distributed and to prove equal variances among the groups

Table 3. The Mean change between the Test group and the Control group

Variable

Count

Mean

SD

DF

MD

T value

Prob. Level

P (Alpha=0 .05)

Decision

Test group Molasses+ chloramphenic ol

10

72.9

28.14032

18

2.8 0.1937 0.424367 0.07231 Accept Ho

Control group 10 Chloramphenic ol

75.7

36.0340

18

2.8

Legend: SD=Standard deviation; DF=Degree of Freedom; MD= Mean Difference; T=Test statistics; P=Power

Table 3 reveals that there are no significant differences in the Mean Change of the Test group and the Control group It implies that there are no significant differences in the Mean change and the decision is to accept Ho

Table 4. The Significant difference of the Post-Treatment Reticulocyte count between the Test group and the Control group. Variable Count Mean SD DF MD T value Prob. Level P (Alpha =0.05) Decision

Test group Molasses+ chlorampheni col Control group Chlorampheni col

10

122.3

38.32623

18

40.9 2.7498 0.006589 0.8411 Reject Ho

10

81.4

27.26496

18

40.9

Legend: SD=Standard deviation; DF=Degree of Freedom; MD= Mean Difference; T=Test statistics; P=Power

Table 4 shows that there is a significant difference of the Post treatment Reticulocyte count between the Test group and the Control group Since the P value falls within the significant limit of 95% (Power alpha= 0.05); the decision is to reject Ho

Discussion As shown in table 1, the Mean Baseline Reticulocyte of the Test group is comparatively higher compared to that of the control table 2, the results reveal that there is no significant difference between the two groups, thus, Null hypothesis is true

Tests for the assumptions show no disparity between the two groups, which validates normality and that each group were normally distributed. Nevertheless, there is no significant difference in the Mean Reticulocyte counts between the two groups statistically. All samples are Normal based on the Normal reticulocyte value.

Based on the results in table 3, there is no significant difference in the Mean change between the Test group and the Control group after determining the differences of the Baseline reticulocyte count and the Post treatment reticulocyte count of the said groups The decreased in the Mean Post treatment reticulocyte count of both groups proves that chloramphenicol can cause Bone marrow suppression by preventing erythropoiesis as indicated by reticulocytopenia (McPherson, 2007)

by increasing the dose of chloramphenicol and prolonging the duration of therapy, there would be significant Bone marrow suppression which can be verified by a fall in the reticulocyte count (McCurdy, 1962) It was verified in this study by giving 100 mg/kg/day to the subjects, the amount would be sufficient enough to cause reticulocytopenia among white mice

It was pointed out in table 4 that there is a significant difference of the Post treatment reticulocyte count between the Test group and the Control group The rejection of the Null hypothesis implies that Molasses has a promising effect of reducing the risk of bone marrow suppression perhaps because of the Antioxidants found in the Molasses (Guimaraes, 2004)

The effect of Molasses may not be that sufficient to prevent bone marrow suppression duly because of multifactorial causes which includes the processing of Molasses, its production, the type used, the soil where the sugar cane was grown that determines the nutritional value obtained from it which were cited by Curtin in his analysis on molasses. (Curtin, 1973)

statistically there is an existing difference when compared to the control group, suggesting that the presence of molasses in the Test group have reduced the risk of a very low reticulocyte count which was seen in the Control group.

CONCLUSION The demonstration of low Reticulocyte counts in both groups attests the damaging effect of Chloramphenicol on Bone marrow when its used is prolonged and its dose is increased, while Molasses has a potential effect in reducing the damage caused by chloramphenicol, thus, it can be used as an adjunct to Chloramphenicol therapy to reduce its side effect.

RECOMMENDATION Another Group of animals such as Rabbits should be used in further study obtain bone marrow smears to correlate with Reticulocyte count in order to determine the effect of Chloramphenicol on the marrow cells. A larger sample size should be used to increase the probability that the study represents the entire population. Reduce the variances to improve the Power, in this may the probability of detecting significant differences would increase.

Other parameters should be obtained like Complete blood Count (CBC) instead Reticulocyte count only, to determine the effect of chloramphenicol on the Leukocyte population, Platelets and Red cells. Determine the Therapeutic effect of Molasses by increasing its dosage. In this method, the therapeutic dose may be established, which could prevent Bone marrow suppression.

Serial determination of the different parameters (such as CBC and reticulocyte count) should be done to observe the effect of Chloramphenicol and molasses treatment as time progresses. In this manner, the exact duration where Molasses is ideal for the prevention of bone marrow can be achieved.

use Molasses as part of Chloramphenicol therapy to protect the Patients from having Bone marrow suppression. Lastly, since Molasses can reduce Bone marrow suppression by preventing oxidative damage, it is advised to determine the curative effect of molasses on Chloramphenicol-induced reticulocytopenia.

The end

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