Peripheral Blood Profiles of CCl

-Induced Wistar Rats after Date

Seed Steeping Treatment

Saryono

, Atikah Proverawati

, Hernayanti Hernayanti

and Ferry Effendi

School of Nursing, Health Sciences of Faculty, University of Jenderal Soedirman

Toxicology Department, Faculty of Biology, University of Jenderal Soedirman

Faculty of Nursing Universitas Airlangga, Kampus C Mulyorejo, Surabaya, Indonesia

Keywords: Monocytes, Neutrophils, Lymphocytes, Date Seeds, Anti-inflammatory

Abstract: A various household appliances that containing CCl

can cause an inflammation. Utilization of date seed as

an anti-inflammatory agent has never been done. The aim of the study was to explore the peripheral blood

profiles of CCl

-induced wistar rat after steeped date seed treatment. An experimental study, post-test only

with control group design was used. Rats were assigned into 6 groups. There are negative control, positive

control, healthy control and treatment groups (dose 1; 3; 5 g/kg). Data collected were neutrophils,

lymphocytes and monocytes. The data was compared between the groups with the ANOVA test and was

followed by post hoc test. Induction of CCl

causes an increase of monocytes, neutrophils and decreased

lymphocytes number. Giving of dates seeds can reduce the number of monocytes and neutrophils but

increase of lymphocytes number. A dose of 3 g/kg of dates seed has the same effect with a control group

that given dexamethasone. The study showed for the first time anti-inflammatory of activity of steeped date

seed can change the peripheral blood profile. Steeping of date seeds can repair tissue damage due to CCl

exposure to be marked by accelerating the inflammatory process.

1 BACKGROUND

Exposure to toxic compounds, microorganisms,

tumors and trauma will lead to the onset of

inflammatory process. The inflammatory process

involves the role of peripheral blood cells to

minimize damage. From the site of infection,

chronic irritation and inflammation can also develop

into various types of cancer. Lymphocytes and

monocytes play an important role as predictor

survival of several diseases (Li et al., 2014). The

high levels of monocyte and/or neutrophil

infiltration may be accompanied by cytotoxicity,

angiostasis and tumor regression. The neutrophil-

lymph node ratio (NLR) is an index indicating

systemic inflammation (Unal et al., 2013).

Lymphocyte-monocyte ratio (LMR) is a useful

predictive factor for various cancers (Huang & Feng,

2015). The amount of eosinophil also correlates with

inflammation in bronchial asthma (Hef et al., 2017;

Katz et al., 2014). It suggests that the peripheral

blood profile is sensitive to identify the status of

systemic inflammation that occurs.

The peripheral blood features include eosinophil,

neutrophil, basophil, lymphocytes and monocytes.

Neutrophil acts as a bacterial phagocytosis and cell

destruction with lysosome enzymes. Basophil plays

an important role in hypersensitivity reactions.

While eosinophil produces antihistamines and plays

a role to prevent hypersensitivity reactions. Systemic

inflammation responses play an important role in the

progress of some diseases. Round blood examination

is easy to do, accurate and low cost. Inflammation

modifies the quality of the immune response.

Lymphocytes and monocytes are the major immune

cells in the inflammation response. The platelet-

lymphocyte ratio is a critical sign and associated

with a poor prognosis in some oncologic disorders

(Gary et al., 2013).

Recently, several cross sectional and cohort

studies have shown that the number of leukocytes is

positively associated with the prevalence and

incidence of metabolic syndrome (Sun et al., 2014).

The use of drugs as anti-inflammation cause various

side effects. One of the herb plants that allegedly act

as immunomodulators is date seeds. The date seeds

contain polyphenols (Ardekani et al., 2010) which

Saryono, ., Proverawati, A., Hernayanti, H. and Effendi, F.

Peripheral Blood Proﬁles of CCl4-Induced Wistar Rats after Date Seed Steeping Treatment.

DOI: 10.5220/0008320100380043

In Proceedings of the 9th International Nursing Conference (INC 2018), pages 38-43

ISBN: 978-989-758-336-0

can function as antioxidants and useful for lowering

blood cholesterol (Saryono, Eliyan, et al., 2017;

Saryono, Rahmawati, et al., 2017) and glucose (El-

Fouhil et al., 2011; El-Fouhil et al., 2010). However,

there have been no studies that explain the

peripheral blood parameters in inflammation due to

CCl

toxicity given the steeping of date palms

(Phoenix Dactylifera L.). Thus, this study aimed to

determine the profile of peripheral blood in mice

exposed to CCl

after being given the steeping of

date seeds.

2 METHODS

2.1 Preparation of Dates

The matured seeds of Phoenix dactylifera were

purchased from local market. Date palm seed of

Deglet Nour was selected and washed with tap

water, left to dry under the sun for one day. The

dried date palm seed was roasted and ground with

blender. Filtered the flour to obtain a fine powder.

2.2 Experimental Animal

Animal try in the form of white rat wistar grown

male, age about 2-3 months with the weight range

150-200 grams. Rats were adapted for one week and

placed in separate cages according to the group. All

animals were given food and drink according to their

needs (add libitum), and kept on a light dark cycle

12/12 hours. They were conducted based on

standardized protocols for experimental animal

studies. This study followed the principles of

laboratory established by Ethic Committee of

Medical School, University of Sebelas Maret,

Surakarta, Indonesia.

2.3 Research Design

The research was conducted using laboratory

experimental, pre and post-test with control group

design. The white rats were grouped into 6 groups,

healthy control (HC), negative control (NC),

positive control (PC), treatment dose 1 g/kg (T1),

treatment dose 3 g/kg (T3), and treatment dose 5

g/kg (T5). Prior to treatment, all groups were

induced with CCl

with a single dose of 0.2 ml/100g

except group E6. The treatment of giving the date

seeds was given for 14 days. Positive control using

dexamethasone dose 0.5 mg/kg.

2.4 Statistical Analysis

The measured data were peripheral blood profiles

including the absolute number of monocytes,

eosinophil, neutrophil segments and stems, as well

as lymphocytes with GIEMSA staining. The data

obtained will be compared between groups with one

way ANOVA test. The test results are said to be

significant if p <0.05. If there is a significant

difference, the data are analysed further by the post

hoc Least Significant Difference (LSD) test.

3 RESULTS

3.1 Phytochemical Test

Based on phytochemical test, steeping of date seed

powder contain more flavonoid and tannin.

Quantitative test results show that the steamed of

date seeds powder contain flavonoids that is 20.68

mg/mL (Table 1).

3.2 Peripheral Blood Profile

The mean score of monocytes in the healthy control

group was 1.8 ± 1.09 cells/mm

while in the CCl

induced group 2 ± 0.76 cells/mm

. In the CCl

induced group, the number of monocytes increased

but did not differ significantly with the healthy

group. After the treatment period, the group given 5

mg/kg of date seeds had the greatest decrease and

significant compared to the other groups. The lowest

monocyte rate was 1.8 ± 0.44 in the treatment group

with 5 mg/kg of date seeds (Table 2). Based on the

Anova test, it was found that there was a difference

of monocyte level between groups (p = 0.000). In

the negative control group, the number of monocytes

is still high. When compared with the positive

control group, administering the seeds of dates can

lower the number of monocytes larger. Based on the

LSD post hoc test, treatment group given 3 g/kg and

5 g/kg duck seeds were not significantly different

(Table 3), but generally the steeping of date palm

seeds decreased the number of monocytes.

Peripheral Blood Proﬁles of CCl4-Induced Wistar Rats after Date Seed Steeping Treatment

Table 1: Qualitative and quantitative test of phytochemical

compound

Indicator

Level

Qualitative test

Flavonoid

+++

Tannin

+++

Saponine

Alkaloid

Quantitative test

Polyphenol level

8.44 mg/mL

Flavonoid level

20.68 mg/mL

The mean number of lymphocytes in the healthy

group was 40.8 ± 2.58 cells/mm

, whereas in the

induction group CCl

was 37.04 ± 2.75 cells/mm

There was a significant difference in the number of

lymphocytes between the healthy group and the

CCl

induced group (p<0.009). After the treatment

period, the average number of lymphocytes

increased (Table 4). Anova test results showed that

there were significant differences in the number of

lymphocytes between groups (p = 0.000). The

greatest increase occurred in the positive control

group given dexamethasone, and did not differ

significantly with the group given dose 3 g/kg

(Table 4). Based on the LSD post hoc test, the group

given doses of 1 and 5 g/kg date seeds were not

significantly different.

Table 2: Number of monocytes, lymphocytes and neutrophils before and after administration of dates seed steeping

Groups

Monocytes

Lymphocytes

Neutrophils

Pre test

Post test

Pre test

Post test

Pre test

Post test

Mean

1.80

1.09

1.40

0.54

40.80

2.58

36.40

2.30

57.2

2.38

60.8

3.27

1.40

0.54

6.00

38.40

2.70

23.80

3.03

58.4

2.88

59.2

3.42

2.20

0.83

2.80

0.83

38.60

2.40

40.60

2.60

57.6

2.96

52.6

2.96

2.20

0.44

3.00

37.40

2.96

36.00

1.22

58.6

2.70

57.6

2.07

1.80

0.83

2.40

0.54

35.40

1.51

37.00

4.00

61.6

1.94

55.4

3.64

2.40

0.89

1.80

0.44

35.40

2.96

34.60

2.70

59.8

1.92

58.8

2.28

Anova test

p = 0.000

p = 0.03

Table 3: LSD test results of monocyte count after treatment

*: Significant (p<0.05)

Table 4: LSD Test results on the number of lymphocytes after treatment

Group

0.000

0.015

0.051

0.002

0.025

0.000

0.015

0.574

0.432

0.821

0.000

0.051

0.574

0.184

0.735

0.000

0.002

0.432

0.184

0.315

0.000

0.025

0.821

0.735

0.315

*: Significant (p<0.05)

In the early stages of inflammation, neutrophils

have increased, while subsequently replaced by

monocytes. After induction of CCl

, neutrophils had

a slight increase with mean of 59.20 ± 2.70

cells/mm

, while in the healthy group 57.2 ± 2.38

cells/mm

. There was no difference in the number of

neutrophils between the CCl

-induced group and the

healthy control group (p>0.05). After the treatment

Group

0.000

0.683

0.416

0.049

0.008

0.000

0.683

0.226

0.020

0.003

0.000

0.416

0.226

0.049

0.000

0.049

0.020

0.226

0.416

0.000

0.008

0.003

0.049

0.416

INC 2018 - The 9th International Nursing Conference: Nurses at The Forefront Transforming Care, Science and Research

period, there was an average difference between

mature neutrophil levels between groups (p = 0.03).

Based on the LSD post hoc test, the positive control

group differed significantly with the treatment

group, but between treatment groups treated with

dates did not differ (Table 5).

Table 5: LSD Test result on the number of neutrophil after treatment

Group

0.002

0.407

0.057

0.835

0.407

0.002

0.014

0.153

0.003

0.000

0.407

0.014

0.258

0.533

0.105

0.057

0.153

0.258

0.086

0.009

0.835

0.003

0.533

0.086

0.302

0.407

0.000

0.105

0.009

0.302

*: Significant (p<0.05)

Based on the analysis of the overall data, giving

positive control (dexamethasone) showed no

difference with the giving of dill seeds dose 3 g/kg.

This means that the steeping dose of 3 g/kg doses is

proportional to the effects of dexamethasone drugs

as anti-inflammatory.

4 DISCUSSION

Epidemiological studies have shown that peripheral

blood profiles play a role in inflammation. CCl

toxicity can cause inflammation, but the mechanism

of using dates as anti-inflammatory in CCl

toxicity

is unclear. Giving CCl

effect on the occurrence of

acute inflammation, especially liver and kidney

organ. The number of monocytes and neutrophils

increased, while the number of lymphocytes

decreased after CCl

induction. These occur because

the inflammation caused spur the secretion of

monocytes and neutrophils to fight inflammation.

The presence of trauma to the cells will trigger an

inflammatory response, but some factors can boost

immunity so that it can reduce inflammation (Viana

et al., 2014).

In chronic inflammation, an increase in

monocytes in peripheral blood. After 24 hours, the

monocyte goes to the tissues and forms

macrophages. Macrophages will be activated by

TNF-α and activate iNOS (inducible nitrite oxide

synthase) and produce NO. The CCl

compound that

enters the liver will be converted to CCl

. CCl

bonding with NO will lead to new, more toxic

compounds, peroxinitrite (ONOO

). The ONOO

compound will cause endothelial poisoning,

resulting in decreased endothelial NO production.

This causes vasoconstriction so that the

inflammation gets worse. Giving dates flavonoids

(Yasin et al., 2015; Mohamed et al., 2014), can act

as antioxidants, by donating H+ to CCl

to become

neutral. This is what causes the number of

monocytes to return to normal.

Neutrophils are innate immunity components

that are abundant in the circulation. Neutrophils play

a role in the fight against invasive pathogens,

although it can also increase certain inflammatory

diseases. At the time of acute inflammation there is

an increase in the number of neutrophils that will

degrade the phospholipid membrane and form

arachidonic acid. With the help of the enzyme

cyclooxygenase-2 (COX2), arachidonic acid is

converted to prostaglandin. An increase in the

number of neutrophils is activated by IL-1β pro-

inflammatory cytokines. Along with cell repair by

the body's immune system, the number of

neutrophils will decrease. Some types of diseases

accompanied by high neutrophils have more severe

clinical outcome problems than controls such as

pain, bleeding, infarction and death (Zhang et al.,

2016). The results showed that neutrophil

recruitment to the site of infection was enhanced by

serotonin delivered by platelets in innate immune

reactions (Duerschmied et al., 2017). Several studies

have clarified the association of neutrophils with

prognostic diseases (Hashimoto et al., 2013; Li et

al., 2014; Jiang et al., 2015).

Increasing the number of lymphocytes at a time

of chronic inflammation is due to the immune

system trying to form antibodies as a defense against

CCl

radicals. But after the seeding of the date palm,

the free radicals CCl

decreases and the

lymphocytes return to normal. Baldness dates palm

seeds contain many flavonoids that can act as

antioxidants (Tang et al., 2013). This is evident from

previous research that the steeping of date palm

seeds can reduce malondialdehyde as a free radical

oxidation product and increase antioxidant enzymes

(Saryono, Hisni, et al., 2017). High leukocyte

content, neutrophil count and high neutrophil-

Peripheral Blood Proﬁles of CCl4-Induced Wistar Rats after Date Seed Steeping Treatment

lymphocyte ratios correlated with failure of ST

elevation myocardial infarction (STEMI). This

indicator is a good failure predictor of the case

(Ghaffari et al., 2014). Other studies have shown

that leukocytes play a role in the generation of

oxidative stress and inflammation in diabetic

mellitus patients (Shurtz-Swirski et al., 2001).

5 CONCLUSIONS

CCl

toxicity can lead to visible inflammation in the

presence of an increase in the number of monocytes,

neutrophils and decreased lymphocytes. Giving of

dates seeds can reduce the number of monocytes,

neutrophils and increase lymphocytes. Steeped of

palm seeds can repair tissue damage due to CCl

exposure in animals that to be marked by

accelerating the inflammatory process. The date seed

steeped of 3 g/kg have an comparable to the effects

of the drug dexamethasone as anti-inflammatory.

Implication: The use of date palm seed as anti-

inflammatory can be given in the form of steeping.

The first study to look at the effects of date seed as

anti-inflammatory based on peripheral blood profiles

can be used as an early reference for further

research.

ACKNOWLEDGEMENTS

Thanks to the Institute for Research and Community

Service, University of Jenderal Soedirman that has

provided research grants under contract number

4869 / UN23.14 / PN / 2017.

REFERENCES

Ardekani, M.R.S. et al., (2010). Comparison of

antioxidant activity and total phenol contents of some

date seed varieties from Iran. Iranian Journal of

Pharmaceutical Research, 9(2), pp.141–146.

Duerschmied, D. et al., (2017). Platelet serotonin promotes

the recruitment of neutrophils to sites of acute

inflammation in mice. Blood, 121(6), pp.1008–1015.

El-Fouhil, A.F. et al., (2011). An extract from date seeds

having a hypoglycemic effect. Saudi Med J 2011;,

2925(March), pp.791–796.

El-Fouhil, A.F., Ahmed, A.M. & Darwish, H.H., (2010).

Hypoglycemic effect of an extract from date seeds on

diabetic rats. Saudi Med J, 31(7), pp.747–751.

Gary, T. et al., (2013). Platelet-to-Lymphocyte Ratio : A

Novel Marker for Critical Limb Ischemia in Peripheral

Arterial Occlusive Disease Patients. PLOS ONE, 8(7),

pp.1–5.

Ghaffari, S. et al., (2014). The Predictive Value of Total

Neutrophil Count and Neutrophil / Lymphocyte Ratio

in Predicting In-hospital Mortality and Complications

after STEMI. J Cardiovasc Thorac Res, 6(1), pp.35–

41.

Hashimoto, T., Ohno, Y. & Nakashima, J., (2013).

Clinical significance of preoperative peripheral blood

neutrophil count in patients with non-metastatic upper

urinary tract carcinoma. World J Urol, 31, pp.953–

958.

Hef, E. et al., (2017). Point-of-care blood eosinophil count

in a severe asthma clinic setting. Ann Allergy Astma

Immunol, 119, pp.16–20.

Huang, Y. & Feng, J., (2015). Low preoperative

lymphocyte to monocyte ratio predicts poor cancer-

specific survival in patients with esophageal squamous

cell carcinoma. Onco target and Therapy, 8, pp.137–

145.

Jiang, L. et al., (2015). Prognostic value of monocyte and

neutrophils to lymphocytes ratio in patients with

metastatic soft tissue sarcoma. Oncotarget, 6 (11),

pp.9542–9549.

Katz, L.E. et al., (2014). Blood Eosinophil Count Is a

Useful Biomarker to Identify Patients with Severe

Eosinophilic Asthma. Ann Am Thorac Soc, 11(4),

pp.531–536.

Li, Y. et al., (2014). Peripheral blood lymphocyte /

monocyte ratio predicts outcome for patients with

diffuse large B cell lymphoma after standard first-line

regimens. Ann Hematol, 93, pp.617–626.

Mohamed, R.M.A. et al., (2014). Chemical composition ,

antioxidant capacity , and mineral extractability of

Sudanese date palm ( Phoenix dactylifera L .) fruits.

Food Science & Nutrition, 2(5), pp.478–489.

Saryono, Eliyan, J., et al., (2017). Anti-atherogenic

properties of Deglet Noor Date seeds ( Phoenix

dactylifera ) Methanol extract on Diet-Induced

Hypercholesterolemic Rats . IOP Conf. Ser.: Mater.

Sci. Eng., 172(12046).

Saryono, Rahmawati, E., et al., (2017). Effect of

Antioxidant Status and Oxidative Stress Products in

Pre-menopausal Women after Treatment with Date

Seed Powder ( Phoenix dactylifera L .): A Study on

Women in Indonesia. Pak. J. Nutr., 16(4), pp.477–

481.

Saryono, Hisni, D., et al., (2017). Plasma

Malondialdehyde and Vitamin E Levels after Date

Palm Seeds (Phoenix dactylifera) Steeping

Administration. Asian Journal of Clinical Nutrition,

9(3), pp.131–136.

Shurtz-Swirski, R. et al., (2001). Involvement of

Peripheral Polymorphonuclear Leukocytes in

Oxidative Stress and Inflammation in Type 2 Diabetic

Patients. Diabetes Care, 24(1), pp.104–110.

Sun, S. et al., (2014). Subnormal Peripheral Blood

Leukocyte Counts Are Related to the Lowest

Prevalence and Incidence of Metabolic Syndrome :

Tianjin Chronic Low-Grade Systemic Inflammation

and Health Cohort Study. Mediators of Inflammation,

INC 2018 - The 9th International Nursing Conference: Nurses at The Forefront Transforming Care, Science and Research

2014, pp.1–12.

Tang, Z., Shi, L. & Aleid, S.M., (2013). Date Fruit :

Chemical Compositions , Nutritional and Medicinal

Values , Products. , doi: 10.10, pp.1–51.

Unal, D. et al., (2013). Are Neutrophil / Lymphocyte and

Platelet / Lymphocyte Rates in Patients with Non-

Small Cell Lung Cancer Associated with Treatment

Response and Prognosis ? Asian Pac J Cancer Prev,

14(9), pp.5237–5242.

Viana, J.L. et al., (2014). Evidence for Anti-Inflammatory

Effects of Exercise in CKD. J Am Soc Nephrol, 25,

pp.2121–2130.

Yasin, B.R., El-Fawal, H.A.N. & Mousa, S.A., (2015).

Date (Phoenix dactylifera) polyphenolics and other

bioactive compounds: A traditional islamic remedy’s

potential in prevention of cell damage, cancer

therapeutics and beyond. International Journal of

Molecular Sciences, 16 (12), pp.30075–30090.

Zhang, D. et al., (2016). Neutrophils, platelets, and

inflammatory pathways at the nexus of sickle cell

disease pathophysiology. Blood, 127(7), pp.801–809.

Peripheral Blood Proﬁles of CCl4-Induced Wistar Rats after Date Seed Steeping Treatment