1. Introduction

Issues of morbidity/mortality in pregnancy have been endemic in developing world particularly sub-Saharan Africa with associated causes often multifaceted ^{[1, 2, 3, 4]}. One often reported cause especially in rural communities are unverified herbal products use by pregnant women ^{[5, 6]}. Ricinus communis oil (RCO) despite being reported contra-indicated in pregnancy by few studies; other studies still reported on its preferred use as labour inducer by some traditional birth attendants ^{[7, 8, 9]}. In Nigeria where this study was carried out, traditional birth attendants still rely on the use of local herbs and concoction like RCO during pregnancy. Apart from exposure through traditional birth attendants, pregnant women are also inadvertently exposed to RCO during different stages of pregnancy due to its accessibility and availability oftentimes as over the counter drug. We have previously reported on the effects of RCO on biochemical and hormonal parameters in pregnant rats ^[10]. Furthermore, we also reported on the generational reproductive implication of maternal exposure to RCO in offspring of exposed mothers ^{[11, 12]}. However, literature search shows specifically nothing on effects of RCO on haematological parameters of pregnant rats during different gestation periods. This study investigates the gestational haematological effect after RCO consumption in Wistar rats.

2. Materials and Methods

Animals used were sourced from the Lagos state University College of Medicine and allowed access to standard rat pellets (Ladokun feeds) and water ad libitum. Guidelines for animal care and use were strictly adhered to and ethical approval given by the college animal house committee (LASUCOM/EAC/27). Thirty mature nulliparous female (220-250 g) albino rats (12 weeks) with normal estrous cycle were used. Positive mating was confirmed by the presence of a sperm positive vaginal smear or copulatory plug and day after which either of this was found was taken as gestational day 0 as we have previously reported ^[12]. 950 mg/kg BW of RCO (recommended therapeutic dose in humans, (Drug store.com, Inc. 2004) ^[13] was administered orally to pregnant wistar rats (6 per group) at gestation days 1-7, 7-14, 14-21 and 1-21 respectively (groups 2-5). Control group received distilled water (group 1). The treatments to groups were chosen to target all the critical periods of gestation (i.e. early, mid, late and entire gestation periods).

Ricinus communis seeds were air dried to a constant weight and then pulverized. The pulverized seeds (2 kg) were extracted by cold extraction using 5 litres of methanol. RCO obtained by filtration was evaporated of the solvent in a rotatory evaporator at 37°C ^[10].

For haematological studies, blood samples (2ml) were collected in heparinized bottles via the orbital sinus under penobarbitone anesthesia. Blood samples were subsequently analyzed using automated blood analyzer H1Z Hema-screen 13 by Hospitex Diagnostic, San Pierro Quaracchi, Firenze, Italy. Red blood cell count (rbc) hemoglobin concentration (Hb), packed cell volume (pcv), mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch), mean corpuscular hemoglobin concentration (mchc) and differential leucocytes counts (lymphocytes, neutrophils, eosinophils and monocytes) were all determined and recorded.

Mean values and standard error of mean (Mean ± SEM) were calculated. The test of significance between two groups was by Students T test and for more than two groups by the analysis of variance (ANOVA) using prism graph pad statistical software version 5.

3. Results

There were significant increases in PCV, Hb and RBC in GD 14-21 treated rats as compared to control, while MCV and MCH decreased significantly in GD 14-21 as compared to control (Table 1). As also shown in Table 1, pregnant rats treated at gestation days 1-7 showed a reduction in packed cell volume when compared to control, while pregnant rats treated on gestation days 7-14, 14-21 and 1-21 showed an increase in PCV. These variations in values were however not significantly different from the control.

Table 1. Effects of RCO (950 mg/kg B.W.) administered to pregnant female rats at different gestation period on PCV, HB, RBC, MCV, MCH, and MCHC

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Percentage variations/changes in leucocyte cells values were all not statistically different from control values (Table 2).

Table 2. Effects of RCO (950 mg/kg B.W.) administered to pregnant female rats at different gestation period on leucocyte values

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4. Discussion

An earlier study ^[14] (NTP, 1992) reported that there were no haematological abnormality except for statistically significant decrease in reticulocyte count after exposure of female rats to RCO. However, the study design was different from this study because 10 % RCO was added to diet and fed non pregnant rats for 13 weeks in the study. In this study, RCO was given orally to pregnant rats at different gestation periods.

There are several implications for marked variations in haematological parameters particularly during pregnancy. PCV may be altered and increased falsely as a result of dehydration while its decrease might indicate the presence of anemia as a result of blood loss or deficiency. Reduced MCV may be precipitated by iron deficiencies and thalasemia while increased MCV may follow Vitamins B12 and folate deficiencies. Pregnant rats given RCO at GD 14-21 were observed to show significant increase in PCV, Hb concentration and RBC when compared to control. The actual mechanism by which RCO influenced these is not known. However, enhanced erythropoiesis is known to be predicated on the duo of a highly functional endometrium at this stage of gestation and uteroferrin secreted by the endometrium. Efficient erythropoiesis is reported to require iron, folate, Vitamin B12 and erythropoietin ^[15]. Jepson, ^[16] also attributed increase in erythropoiesis late in pregnancy to erythropoietin transfer to the mother and that erythropoietic processes starts later in pregnancy and not early as fetal processes joins that of the mother. Furthermore, apart from erythropoietin, prolactin is known to increase as gestation days increase peaking at parturition ^[17]. Suggestion that prolactin may function as an erythropoietic stimulatory component in pregnant mouse has earlier been postulated by Jepsin and Lowenstein ^[18]. Jepsin and Lowenstein ^[19] further asserted that major increase in red blood volume occurs normally in late pregnancy as reticulocytes and plasma iron progressively decrease. It is therefore safe to suggest for now that RCO may have positively influenced any of these processes that have been identified to enhance erythropoiesis late at pregnancy.

On the other hand, the reduced erythropoiesis (though not statistically significant) observed in GD 1-7 treated rats may suggest that RCO use may be greatly contraindicated at early gestation period. In addition, the inhibiting effect on erythropoiesis induced by estrogen (levels of which are usually elevated at early gestation period) cannot be ruled out as it has been previously reported ^[17]. This usual inhibiting effect of estrogen can be further exacerbated by the estrogenic propensity of RCO which has been richly reported in literature ^{[10, 11, 20, 21]}. Our previous study Salami and Raji, ^[10] gives credence to this line of thought by reporting higher estrogen levels in pregnant rats exposed to RCO at early gestation periods compared to late gestation period.

There were no significant differences in the differential white cells count of treated pregnant rats when compared to control. This suggests that RCO possibly has no overriding immuno-modulatory effects. Monocytes, Eosinophils and Basophils levels are known to increase during infection stress leukemia and inflammation while reduced neutrophils and lymphocytes are usually recorded during intake of some medications, autoimmune and bone marrow diseases and during severe infections ^[22].

Conclusively, RCO exposure at different gestation periods has no significant effect on differential leucocytes cells count. However, RCO exposure seems to militate and enhance erythropoiesis at early and late gestation period respectively.

References

[1]	Tinker A. Safe motherhood: from advocacy to action. Finance Dev.1991 28.4: 18-20.
	In article
[2]	Donnay F. Maternal survival in developing countries. What has been done, what can be achieved in the next decade? Int J. of Gynaecol and Obst, 2000 70: 89-97.
	In article		View Article
[3]	Wilder-Smith A. Currents status of essential obstetric care activities internationally: a literature review. Trop Doct., 2003. 33: 135-138.
	In article		View Article  PubMed
[4]	WHO (2015). Trends in maternal mortality: 1991-2015. Estimates by WHO, UNICEF, UNFPA, World Bank Groups & UN population division. www.unicef.org. July 26, 2016.
	In article
[5]	Wang CL, Li L, Tang LY, Leung PC. Safety evaluation of commonly used Chinese herbal medicines during pregnancy in mice. Human Reprod., 2012. 27:2448-56.
	In article		View Article  PubMed
[6]	Bothren MA, Hunter EC, Munthe-Kass HM, Souza JP, Vogel JP, Gulmezoglu AM. Facilitators and barriers to facility based delivary in low income and middle income countries: a quantitative evidence synthesis. Reprod. Health., 2014. 11: 71.
	In article		View Article  PubMed
[7]	Steingrub, JS., Lopez, T., Teres, D. and Steingart, R. Amniotic fluid embolism associated with castor oil ingestion. Crit Care Med, 1988, 16: 642-643.
	In article		View Article  PubMed
[8]	Cosmetic Ingredient Review Expert Panel CIREP. Final report on the safety assessment of Ricinus communis (Castor) seed oil, hydrogenated castor oil, ricinoleic acid, potassium ricinoleate, sodium ricinoleate, zink, and octyldodecyl ricinoleate. Glyceryl ricinoleate, methyl ricinoleate, cetyl ricinoleate, ethyl ricinoleate, glycol ricinoleate, isopropyl ricinoleate. Int J of Toxicol, 2007, 26: 31-77.
	In article		View Article  PubMed
[9]	Boel, ME., Lee, SJ.,Rijken, MJ., Paw, MK., Pimanpanarak, M., Tan, SO., Singhasivanon, P., Nosten, F. and Mcgready, R. Castor oil for induction of labor: not harmful, not helpful. Obst & Gynecol Survey, 2010, 65: 77-78.
	In article		View Article
[10]	Salami SA.,Raji, Y. Oral Ricinus communis oil exposure at different stages of pregnancy impaired hormonal, lipid profile and histopathology of reproductive organs in Wistar rats. J of Med Plant Res, 2014. 8: 1289-1298.
	In article
[11]	Salami SA.,Raji, Y. Generational reproductive outcomes in Wistar rats maternally exposed to Ricinus communis oil at different stages of gestation. J of Dev Orig of Healt and Dis, 2015 6: 443-453.
	In article		View Article  PubMed
[12]	Salami, S. A., Omidiran, J., Balogun, M.and Raji, Y. Testicular and epididymal histomorphometric assessment in F1 male Wistar rats after gestational Ricinus communis oil exposure J. Morphol. Sci., 2015. 32: 187-191.
	In article		View Article
[13]	Drugstore.com. Rite aid castor oil, stimulant laxative U.S.P. Package details: directions. Bellevue, 2004. Available from: <https:// www.drugstore.com>. Access in: 1 dec. 2004.
	In article
[14]	National Toxicology Program (NTP). 1992. NTP technical report on the toxicity studies of castor oil (CAS No. 8001-79-4) in F344/N rats and B6C3F1 mice (dosed feed studies). NTIS Report No. PB93151439.
	In article
[15]	Vallet JL. Fetal erythropoiesis and other factors which influence uterine capacity in swine J appl. Anim. Res.2000. 17: 1-26.
	In article		View Article
[16]	Jepson JH. Endocrine control of maternal and fetal erythropoiesis. Canad. Med. Ass J, 1968. 98: 844-849.
	In article		PubMed  PubMed
[17]	Rodney AR, Bell DR. Medical Physiology: Principles for Clinical Medicine, 3rd edn, 2009. Lippincott Williams and Wilkins: Baltimore, MD. pp. 174, 710-714.
	In article
[18]	Jepson JH, Lowenstien L Effect of prolactin on erythropoiesis in the mouse. Blood, 1964. 24: 726-738.
	In article		PubMed
[19]	Joanne Jepson and Louis Lowenstein Hormonal control of erythropoiesis during pregnancy in the mouse. Brit J of Heamatol, 2008 14: 555-562.
	In article
[20]	Okwuasaba, FK.,Osunkwo, UA., Ekwenchi, MM., Ekpenyong, KI., Onwukeme, KE., Olayinka, AO., Uguru, MO. and Das, SC. Anticonceptive and estrogenic effects of a seed extract of Ricinus communis var. minor. J. of Ethnopharmacol, 1991. 34:141-145.
	In article		View Article
[21]	Onwuliri VA, Anekwe GE. Amino acids and other Biochemical components of Ricinuscommunis (Variety minor), an anticonceptive seed. Pak J Biol Sci. 2001; 4, 866-868.
	In article		View Article
[22]	Ganong’s review of medical physiology. 23rd edition. Kim E. Barret edited 2010 McGraw Hill New York. pg 424-425.
	In article