View of The Effects of Methotrexate and Matricaria Chamomilla Extract on Some Immunological and Hematological Parameters in Male Albino Rats

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The Effects of Methotrexate and Matricaria Chamomilla Extract on Some Immunological and Hematological Parameters in Male Albino

Rats

Afaf K. Shwaikh^1a, Alaa Jawad Hassan^2b, Kareem Hameed Rashid²

1Department of Biology, College of Science , University of Kerbala ,Iraq

2Department of Biology, College of Science , University of Babylon, Hilla-Iraq a) Corresponding author: [email protected]

b) Another author: [email protected]

Abstract

Matricaria chamomilla, is a popular herb that exhibits various pharmacological activities. The current study was conducted to investigate the relationship between methotrexate(MTX) and chamomile extract and to estimate their impact on some immunological and hematological parameters in rats. Thirty-six male rats (Rattus rattus) were divided into six groups (6 rats for each), G1(control), G2(MTXtreatment), G3(chamomile flowers methanolic extract), G4(chamomile flowers methanolic extract with MTX), G5

(

chamomile flowers aqueous extract), G6(chamomile flowers aqueous extract and MTX treatment). All the groups were treated for 30 days and then sacrificed on day 31. The results indicated that the blood parameters were significantly influenced by the type of group. The total number of WBCs in G4 and G6 were significantly lower (p<0.001) compared to other groups, while WBCs in other groups not significantly changed compared to the control (p>0.05). The number of lymphocytes in both G2 and G4 were significantly lower (p<0.001), while the changes in other groups were not statistically observed compared to the control (p>0.05). The number of monocytes was significantly decreased in G4 compared to the G1, G3, and G5, while this decrease was statistically disappeared compared to the G2 and G6. de among the above groups. In addition, number of GRA was only the one which was not significantly affected by the type of group. Furthermore, the level of IL-2 cytokine in treated groups did not show any significant differences compared to the control group and also among groups (P>0.05), while concentration of IL-10 cytokine in G6 group was significantly lower compared to the control group, on the other hand such changes were disappeared among other groups in comparison to the control group. In conclusion, according to the results, the immunosuppressant drug methotrexate

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influenced via the type of group and showed synergistic effect with Matricaria chamomilla extract on some immunological and hematological parameters.

Keywords

:

Methotrexate, Matricaria chamomilla flowers, Anti-inflammatory, Immunosuppressant.

Introduction

Methotrexate (MTX) is an effective therapy to manage immune-mediated inflammatory diseases , particularly rheumatoid arthritis (RA), when used at a low dose [1, 2], and is often used on its own or in combination with biologics or steroids [3].

MTX, a folic acid antagonist, suppresses dehydrofolate reductase, blocking the transition of dihydrofolate to tetrahydrofolate at massive doses, purine and pyrimidine biosynthesis are inhibited as a result, affecting with DNA synthesis, part of the anti- inflammatory impact of MTX at low dosage comes from the synthesis of adenosine, which inhibits neutrophil activity, in which this drug raised adenosine levels and inhibited leukocyte formation in inflammatory exudates in an in vivo model of inflammation[4]. Locally cell-cell interconnections are crucial in sustaining the chronicity of inflammation in diseases treated with MTX, such as psoriasis or RA, at the inflammatory location, interactions between infiltrated immune system cells and locally mesenchymal cells result in elevated production of pro-inflammatory cytokines notably IL-6, TNF, and IL-17 [5]. Plants which were abundant in natural polyphenolic compounds intensively researched in recent years, because of their strong anti- carcinogens, vitamins, and immunomodulants property ,one from the oldest medicinal herbs known to mankind is chamomile, which it belongs to the Asteraceae / compositae family and is represented by two species ,chamomile - Roman (Chamaemelum nobile) and Chamomile - German (Matricaria chamomilla) (Chamomilla recutita),the beneficial of dried chamomile flowers (Matricaria chamomilla) effects are linked to the presence of many flavonoids that contribute to the therapeutic potential [6]. The most common of which herbal phenolic compounds and the central structure it either flavonone (apigenin and luteoline) or flavonol derivatives (patuletine and quercetin),also certain major constituents are essential oils these include terpenoids, α- bisabolol and its oxides, and azulenes such derivatives of chalmuzene and acetylene[7].

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Materials and Methods

1.Chemicals

MTX injection 50 mg/5ml manufactured by KOCAK FARMA -Turkey. Methyl alcohol (99.8%) was obtained from Central Drug House, New Delhi.

2.Preparation and extraction of the chamomile plant materials A. Preparation of Methanolic Extract

Dried chamomile (Matricaria chamomilla) flowers seeds was purchased from Cairo, Egypt. Eighty grams of dried chamomile flowers (ground) were transported into 1 L Erlenmeyer flasks, then 800 mL of 80% (80 : 20, methanol: water) were added to the samples, extraction was carried out using an shaker at a room temperature for 8 hours, they were filtrated through filter paper ( What man No.1), then the extract were evaporated at 45℃by using an oven and stored at -4℃ until use [8].

B. Preparation of Aqueous Extracts

Dried chamomile flowers were weighed and crushed to powder with a marble pestle and mortar, and a 5% (w/v) suspension was prepared in a flask by adding hot boiled water. The flask was then placed on a shaker (200 rpm) for 4 h, and the temperature was maintained at 37 °C, after being shaken, the flask was brought to room temperature, and the suspension was filtered through a series of Whatman filters and finally passed through a 0.22 μm filter (Millipore, Billerica, MA), the filtered aqueous extract was freeze-dried and stored at -20 °C until it was used [9].

3. Preparation of MTX Concentration

The concentration of MTX that used for priming the experimental rats was 10 mg / kg (1000 mg) .

4. Laboratory Animals

The current study was conducted on thirty six male rats (Rattus rattus) their weight ranged from 200–300gm. Rats were housed in controlled condition of temperature (25+ 3C) and 12 hours (light – dark cycle) and fed commercial pellets and tap water / ad libitum maintained in the animal house of College of Science, University of Babylon .The rats were divided into six groups (6 rats for each), including ; Group I(G1): six rats received oral daily dose of distilled water (2 ml/ kg) for 30 successive days this group served as negative control. Group II(G2): six rats received single oral daily dose of methanolic extract of chamomile flowers (200 mg / kg) dissolved in 1 ml distilled water given by oral gavage. Group III(G3): six rats received single oral daily

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dose of aqueous extract of chamomile flowers (200 mg / kg) dissolved in 1 ml distilled water given by oral gavage. Group IV(G4): six rats received single oral daily dose of methanolic extract of chamomile flowers (200 mg / kg) dissolved in 1 ml distilled water given by oral gavage and on day 28 the animals given single IP dose of methotrexate (10mg / kg ). Group V(G5)0: six rats received single oral daily dose of aqueous extract of chamomile flowers (200mg/kg) dissolved in 1 ml distilled water given by oral gavage and on day 28 the animals given single IP dose of methotrexate (10mg/ kg). Group VI(G6): six rats received single oral daily dose of 1 ml distilled water given by oral gavage and on day 28 the animals given single IP dose of methotrexate (10mg/ kg ).

Then all the animals were anesthetized by chloroform and sacrificed on day 31.

5. Samples and Hematological Assay

The animals were anesthetized after 31 days by chloroform and sacrificed. About 7 ml of the blood was collected directly by heart puncture with sterile syringes, then about 2 ml of blood were put in EDTA tubes for hematological parameters analysis and about 5 ml were put in gel tubes for 30 minutes at room temperature to allowing clotting . The clotted blood was centrifuged at 3000 ^rpm for 10 minutes, and serum was collected for the estimating the immunological parameters (IL - 2, IL - 10). A complete blood cell count (CBC) and a 3-part white blood cell count (WBC) differential assessment via the Mythic 18 VET is a fully automated hematology bench-top analyzer using impedance technology [10].

6. Evaluation the levels of IL-2 and IL-10.

The sera levels of IL-2 and IL-10 cytokines were estimated by ELISA kits according to the manufacturer’s instructions (MyBioSource, USA).

7.Statistical Analysis

Statistical analysis were conducting using paired Minitab for Windows (version 17;

Minitab Inc) p≤ 0.05 were considered as significant[11].

Results

The finding from the current study revealed that the blood parameters were significantly influenced via type of group. Total number of WBCs in G4 and G6 groups were significantly decreased (p<0.001) compared to other groups, while WBCs in other groups not significantly altered compared to the control group (p>0.05), the number of lymphocytes in both G2 and G4 were significantly lowered (p<0.001), while changes in other groups were not statistically observed compared to the control group (p>0.05).

The number of monocytes was significantly lower in G4 compared to the G1, G3, and

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G5 , while this lower was statistically not observed compared to the G2 and G6. The concentration of IL10 in G6 was significantly lower compared to the control group, while such changes were disappeared among other groups comparing to the control group other hand , the concentration of IL2 did not show any significantly changes compared to the control group and also among groups (P>0.05), as shown in table (1) and figures 1,2,3,4,5,6, and 7.

Table 1: The statistical outcomes of blood and immunological parameters among treated and control groups.

Blood Parameters

G1, Mean±SE

G2, Mean±SE

G3, Mean±SE

G4, Mean±SE

G5, Mean±SE

G6, Mean±SE

Group's Effect

Sample size N=6 N=6 N=6 N=5 N=6 N=5

WBC 7.78âb±0.91 3.78^bc±0.27 8.97â±1.59 2.38^c±0.62 10.02â±2.17 3.54^c±0.83 F5,28=10.51, P<0.001 RBC 5.76âb±0.25 6.87â±0.37 6.41âb±0.40 4.52^b±0.84 5.89âb±0.12 6.54â±0.40 F5,28=3.54, P=0.013 LYM 5.58âb±0.56 2.25^c±0.26 5.65âb±0.92 1.64^c±0.432 6.8â±1.04 2.60^bc±0.74 F5,28=8.84, P<0.001 MON 1.53âb±0.24 0.85âbc±0.17 1.91âb±0.60 0.28^c±0.03 2.36â±0.83 0.52^bc±0.13 F5,28=6.16 , P<0.001 GRA 0.66â±0.15 0.7â±0.203 1.41â±0.35 0.5â±0.30 0.85â±0.33 0.42â±0.13 F5,28=2.44 , P= 0.059

IL2 (pg ̸ ml)

170.9â ±11.7 170.0â ±15.7 157.14â±9.34 191.2â ±10.9 158.6â±11.5 170.48â±8.84 F5,28= 1.11 , P>0.05

IL10 (pg ̸ ml)

1015.4â±96.4 1033.8â±50.8 985.5âb±82.5 1127.4â±51.0 969.2âb±71.4 527^b±128 F5,28=3.98 P=0.008

WBC= White Blood Cells , RBC=Red Blood Cells , LYM= Lymphocyte , MON = Monocyte , GRA=

Granulocyte G1=Control, G2= MTX Treatment , G3 = Methanolic Extract , G4= Methanolic Extract and MTX Treatment, G5= Aqueous Extract , G6=Aqueous Extract and MTX Treatment

.

Means that do not share a letter are significantly different at p<0.05.

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Control MTX treatment Methanolic Extract

Methanolic Extract &MTX

Aqueous Extract Aqueous Extract &MTX

Figure (1): The total number of white blood cells in treatments and control groups.

Aqueous Extract Aqueous Extract

Figure (2): The total number of red blood corpuscles in treatments and control bc

c a

a

c

ab

a ab a

ab b

G1 G2 G3

G4 G5 G6

G1 G2 G3

G4 G5 G6

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Figure (3): The total number of lymphocytes in treatments and control groups .

Methanolic Extract & MTX

Figure (4): The total number of monocytes in treatment and control groups.

ab

c

ab

c

bc a

ab

c abc

ab

bc

G1 G2 G3

G4 G5 G6

G1 G2 G3

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Figure (5) : The total number of granulocytes in treatment and control groups

Aqueous Extract Aqueous Extract&MTX

Figure (6): The level of IL-2 in treatment and control groups of rats.

G4 G5 G6

G1 G2 G3

a

G4 G5 G6

G1 G2 G3

a a

a

a a

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Aqueous Extract Aqueous Extract&MTX

Figure (7): The level of IL-10 in control and treatment groups of rats.

Note : Small letters represent a significant difference between treatment groups and control group .

Discussion

MTX alone had a modest inhibitory effect on WBCs while large anti- inflammatory effect was shown in groups received chamomile extract, the different results obtained between MTX and chamomile extract in these experiments could come from the different modes of action of the MTX drug and plant extract, one key mechanism for action of the MTX is the induction of adenosine signaling, the finding of present study revealed that combinations of chamomile extracts either aqueous or methanolic resulted in synergistic effects with MTX drug in reducing the total number of white blood cells, these effects may be contribute to the anti-inflammatory activity for chamomile plant components. Herbs, in natural, may still have drug interactions, very few interactions between chamomile and over-the-counter or prescription drugs have actually been documented, however the most significant are linked to the use of this herb with Coumadin (warfarin), a typical blood thinner, and cyclosporine, a

G4 G5 G6

G1 G2 G3

a ab

a

ab

b

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treatment that used for prevent rejection following organ transplantation, due to chamomile biochemistry, theoretical interactions also can occur, chamomile does have compounds which suppress drug-metabolizing enzymes in human from cytochrome family P450 (CYP1A2, CYP2C9, CYP2D6, and CYP3A4 CYP2C9, CYP2D6, and CYP3A4), as a result, when chamomile is consumed , blood levels may be higher than expected for various drugs, no findings of this occurrence, other than warfarin and cyclosporine, have been documented[12]. Furthermore, a single dose from low concentration of methotrexate was very well tolerated, with a low incidence of suppression for bone marrow[13]. Also , the reduction of lymphocyte count occurred may because of the influence of methotrexate on normal lymphocyte transformation and mitosis, the antimitotic activity of MTX dependent upon the concentration, lymphocyte contact period, and also the developmental stage for lymphocytes at the time of treatment and the using of folinic acid abolished the antimitotic effect of MTX drug [14]. The results showed that the level of IL-10 was significantly lowered in G5 compared to the control group. In vitro model to determine the relationships between synoviocytes and stimulated immune cells for mimic locally in vivo inflammatory condition ,the effects of MTX were first studied with a response-dose ,for each evaluated the cytokine IL-17, IL-6, IL—1β, IFN-ɤ, and IL-10, no strong dose-response was noticed and alone MTX had a moderate inhibitory effect on some cytokine production[15]. Other study refers to that MTX was shown to decrease IL-10 level , but was less reliable indicators of response to therapy[16]. Meantime, study on patients undergoing treatment with methotrexate suggest that therapeutic strategies simultaneously affect the formation of the pro-inflammatory and anti-inflammatory cytokines such as IL-10 [17]. The study revealed that non- significant alteration in IL-2 level among studying groups. Previous study revealed that methotrexate reduced cytokine production in long-term T-cell culture, but low dose methotrexate had no effect in primary culture, while others demonstrated that in WB cultures for RA patients after their primary oral intake for methotrexate. MTX levels in plasma are sufficient to suppress cytokine secretion, even when diluting the blood for four times, their showed that suppression for purine and pyrimidine formation is the basic mechanism via which cytokine secretion is inhibited in the cultures of methotrexate [18]. In conclusion , the result of current study shown the immunosuppressant drug methotrexate influenced by the type of group and showed synergistic effect with Matricaria chamomilla extract on some blood parameters , while, the level of IL10 in G6 group was significantly lower

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compared to the control group , in addition, the level of IL-2 did not show any significant changes compared to the control group and also among groups.

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