Phytochemical Composition of Ethanol Extract of 𝘉𝘳𝘺𝘰𝘱𝘩𝘺𝘭𝘭𝘶𝘮 𝘱𝘪𝘯𝘯𝘢𝘵𝘶𝘮 leaves (EEBP) with its Effects on Haematopoietic Indices and Bone Marrow Histology of Cadmium-intoxicated Rats
Subject Areas : Journal of Chemical Health Risks
Doris Akachukwu
1
,
Charles N. Chukwu
2
,
Philippa C. Ojimelukwe
3
,
Anthony C.C. Egbuonu
4
,
Inemesit C. Ubiom
5
,
Rosemary Izunwanne Uchegbu
6
1 - Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, Nigeria
2 - Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria
3 - Department of Food Science and Technology, College of Food Science and Technology, Michael Okpara University of Agriculture, Umudike, Nigeria
4 - Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria
5 - Department of Biochemistry, Faculty of Sciences, University of Uyo, Uyo, Nigeria
6 - Department of Chemistry, Alvan Ikoku Federal College of Education, Owerri, Imo State, Nigeria
Keywords: Bone marrow, 𝘉𝘳𝘺𝘰𝘱𝘩𝘺𝘭𝘭𝘶𝘮 𝘱𝘪𝘯𝘯𝘢𝘵𝘶𝘮, Cadmium-challenged, GC-MS, Haematology, Histology,
Abstract :
Cadmium intoxication may arise from environmental pollution and cause bone and nervous system disorders. The phytochemical composition of ethanol extract of Bryophyllum pinnatum leaves (EEBP) with its effects on haematopoietic indices and bone marrow histology of cadmium-intoxicated rats was investigated. Twenty-four male albino rats were grouped for four treatments: Group 1 - normal control, Group 2 - 5 mg kg-1 bodyweight CdCl2,Groups 3 and 5 - 200 and 400 mg kg-1 body weight EEBP, while Groups 4 and 6 received 5 mg kg-1 bodyweight CdCl2, and treated with 200 and 400 mg kg-1 bodyweight EEBP respectively for 14 days. Feed and water were given ad libitum. Six bioactive compounds were obtained with 2H-Benzocyclohepten-2-one, decahydro-9a-methyl-, trans-13-octadecanoic acid methyl ester (35.81%) being the most abundant. The extract-treated groups showed a significant increase in haemoglobin (from 10.61 to 10.78 mmol L-1), packed cell volume (41.75%), red (6.36-6.42 × 106 mm3), and total white blood cells counts (17.30-19.25 × 106 mm3). The cadmium-intoxicated groups treated with 200 and 400 mg kg-1 body weight of EEBP showed a mild reduction in progenitor cells in the bone marrow. The results suggest that EEBP possesses potent bioactive compounds and nutrients that could improve hematological properties and attenuate bone marrow degeneration in cadmium-intoxicated rats.
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Phytochemical composition of ethanol extract of Bryophyllum pinnatum leaves (EEBP) with its effects on haematopoietic indices and bone marrow histology of cadmium-intoxicated rats
Doris Akachukwu1*, Charles N. Chukwu1, Philippa C. Ojimelukwe2, Anthony C.C. Egbuonu1, Inemesit C. Ubiom3 and Rosemary I. Uchegbu4
1Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, Nigeria
2Department of Food Science and Technology, College of Food Science and Technology, Michael Okpara University of Agriculture, Umudike, Nigeria
3Department of Biochemistry, Faculty of Sciences, University of Uyo, Uyo, Nigeria
4Department of Chemistry, Alvan Ikoku Federal College of Education, Owerri, Imo State, Nigeria
*Corresponding author’s e-mail: dorisakachukwu@yahoo.com
ABSTRACT
Cadmium intoxication may arise from environmental pollution and causes bone and nervous system disorders. Phytochemical composition of ethanol extract of Bryophyllum pinnatum leaves (EEBP) with its effects on haematopoietic indices and bone marrow histology of cadmium-intoxicated rats were investigated. Twenty-four male albino rats were grouped for four treatment: Group 1 - normal control, Group 2 - 5 mg/kg bodyweight CdCl2, Groups 3 and 5 - 200 and 400 mg/kg bodyweight EEBP, while Groups 4 and 6 received 5 mg/kg bodyweight CdCl2 and treated with 200 and 400 mg/kg bodyweight EEBP respectively for 14 days. Feed and water were given ad libitum. Six bioactive compounds were obtained with 2H-Benzocyclohepten-2-one, decahydro-9a-methyl-, trans-13-Octadecanoic acid, methyl ester (35.81%) being the most abundant. There was significant (P<0.05) increase in haemoglobin (17.07-17.35 g/dl), packed cell volume (41.75%), red (6.36-6.42 × 106 mm3) and total white blood cells’ counts (17.30-19.25 × 106 mm3) of the extract-treated groups. There was mild reduction of progenitor cells of the bone marrow in the cadmium-intoxicated groups treated with 200 and 400 mg/kg bodyweight of EEBP. The results suggest that EEBP possess potent bioactive compounds and nutrients that could improve haematological properties and attenuate bone marrow degeneration in cadmium-intoxicated rats.
Keywords: Bone marrow, Bryophyllum pinnatum, cadmium-challenged, GC-MS, haematology, histology
INTRODUCTION
Cadmium (Cd) intoxication has been reported from several parts of the world as one of the global health challenges, which has deleterious effects on several organs and in some cases, can cause death [1]. The human system is vulnerable to harmful effects of toxicants including drugs and heavy metals [2]. Residents or workers near waste sites or industrial areas that emit cadmium into the air have been shown to develop impaired health, including injured lungs, diarrhoea, stomach aches and serious nausea, bone fracture, reproductive failure and probably even infertility, harm to the central nervous system, psychological disorder, DNA damage or cancer development [3]. Cadmium toxicity also affects the blood and bone systems [4]. The hematopoietic system comprises the bone marrow, spleen, tonsils, lymph nodes and cells; and it is responsible for continuous production of mature circulating blood cells including white blood cells, red blood cells and platelets. According to El-Boshy et al. [5] cadmium upsets the hematopoietic function by damaging genetic material of stromal and hematopoietic cells and altering cytokine homeostasis. Kocak [6] reported that cadmium exerts its effect on hematopoiesis by causing microcytic anemia in rats. Cadmium exerts deleterious cytotoxic and genotoxic effects on peripheral blood and bone marrow cells. Increased numbers of polychromatic erythrocytes and transient and rapid leukocytosis in the blood are examples of harmful effects of Cd [7]. Cadmium has been reported to reduce red-blood cells shelf-life and induce haemolysis that leads to anaemia in animals and man [8].
Bryophyllum pinnatum (Lam.) is a perennial therapeutic herb, which is native to Madagascar, but extensively distributed in some other areas, including the temperate regions of Asia, Africa, Australia and New Zealand [9]. In Nigeria, B. pinnatum is commonly called ‘Never Die’, Hausa – ‘Karan masallachi’ , Igbo – ‘Oda opue’ or ‘Alupu’ and Yoruba – ‘abamoda’. It has been found useful in the management of variety of conditions including, rheumatism, body pain, arthritis, heartburn, skin ulcers, peptic ulcer, diabetes mellitus, microbial infections, and hypertension [10]. In Nigeria, the plant is particularly known for its effective wound healing properties and detachment of the umbilicus of infants. In Nigeria, the plant is particularly known for its effective wound healing properties and detachment of the umbilicus of infants. Extract of B. pinnatum has been reported to increase white blood cell count, reduce neutrophil count, without affecting lymphocyte count and packed cell volume [9]. It has also been shown to possess therapeutic and biological effects including anti-inflammatory, antihypertensive, immunomodulatory, antitumor, antioxidant properties etc. [11,12,13,14,15]. Several phyto-constituents including alkaloids, triterpenes, glycosides, flavonoids, steroids [11,16,17] have been reported to be responsible for these activities. In a recent study, Kısadere et al. [18] reported the protective effect of melatonin in hematological characteristics of cadmium-induced rats. There is no available literature on the possible effect of ethanol extract of B. pinnatum leaves in rats under cadmium toxicity and this has necessitated this study. The study was therefore aimed at investigating the GC-MS composition of the ethanol extract of B. pinnatum leaves (EEBP) and its effect on haematological indices and bone marrow histology of cadmium-challenged rats.
METHODS
Collection and preparation of plant material
The fresh matured leaves of Bryophyllum pinnatum were collected from the compound of National Soil, Plant and Water Laboratory under the Federal Ministry of Agriculture and Rural Development, Umuahia North LGA, Abia State, Nigeria. The plant was identified and authenticated by the Taxonomic unit of Plant Science and Biotechnology Department, Michael Okpara University of Agriculture, Umudike. The plant leaves were sorted, dirt removed and washed in running tap water. Thereafter, they were kept in a container to drain off water and left to air-dry. A quantity of 626.13 g of Bryophyllum pinnatum leaves was weighed and ground with the aid of a hand milling machine.
Extraction of plant sample
The ground sample was soaked with 1 L of ethanol (99.8%) with periodic shaking for 48 hours for efficient extraction. It was thereafter filtered using Whatman No 1 filter paper and the filtrate was dried using a rotary evaporator and then placed in the oven to concentrate it to obtain the methanol extract of Bryophyllum pinnatum (MEBP), while the residue was discarded. The extract was weighed and stored in a small beaker and kept in the refrigerator until it was ready for use.
GC-MS
Gas chromatography-mass spectra (GC-MS) analysis was conducted using GC-MS (Agilent 7890A) equipped with a DB-5MS column (30 m × 0.25 mm i.d., 0.25 um film thickness, J & W Scientific, Folsom, CA). The initial oven temperature was 60 °C. Helium was used as the carrier gas at the rate of 1.0 mL/min. The eluent of the GC column was introduced directly into the source of the MS via a transfer line (250 °C). Ionization voltage was 70 eV and ion source temperature was 230 °C. Scan range was 41- 450 amu. The components were identified by comparing their retention times to the reference in the National Institute of Standards and Technology (NIST, ver. 2.0, 2008) mass spectral database.
Experimental animal
A total of 24 male albino rats (104-185 g) were procured from Animal Breeding House of the Department of Veterinary Medicine, University of Nigeria, Nsukka. The animals were transported to the Animal House of the College of Veterinary Medicine, Michael Okpara University of Agriculture, Umudike and they were acclimatized for 2 weeks in clean cages.
Experimental Design
The animals were randomly divided into six (6) groups of four (4) animals each. The animal groupings were as follow:
Group 1: Control (Feed +Water only)
Group 2: 5 mg/kg body weight b.w CdCl2)
Group 3: 200 mg/kg b.w EEBP only
Group 4: 5 mg/kg BW CdCl2 co-treated + 200 mg/kg b.w EEBP
Group 5: 400 mg/kg b.w EEBP
Group 6: 5 mg/kg BW CdCl2 co-treated + 400 mg/kg b.w EEBP
The animals had free access to feed and water. The experiment was for a period of 14 days, after which they were weighed and sacrificed through cervical dislocation on the 15th day. The blood was collected into EDTA (Ethylenediaminetetraacetic acid) bottles with capillary tubes from the optical plexus for hematological assays. The bone marrows of the rats were collected by cutting their femur bone and a smear of the bone marrow was made on labeled slides according to their respective groups. The bone marrow slides were stored with methanol solution.
Haematological Investigation
The red and white blood cells’ counts, packed cell volume and haemoglobin concentration were determined using standard protocols. Red blood cell (RBC) count was determined using Haematocrit method [19]. Total white blood cell (WBC) count and Packed cell volume (PCV) was determined by hematocytometry following the method described by Ochei and Kolhatkar [20]. Packed cell volume (PCV) was estimated as described by [20] while haemoglobin (Hb) concentration determination was done using Cyanmethaemoglobin method as described by [20]. The Hb concentration was obtained by multiplying the absorbance of the sample with a calibration factor (36.8) derived from the absorbance and concentration of the standard [21].
Histological investigation
This was done using paraffin technique as described by Bancroft and Stevens [22]. In this technique, the bone marrow tissues were fixed and embedded in paraffin wax. This makes the tissue hard and much easier to cut sections from. The sections were then stained and examined with the microscope. The samples were put into separate sample bottles with label, and fixed in 10% formal saline solution. Thereafter, the samples were dehydrated through ascending grades of alcohol viz: 70% (1 hour); 95% twice (1 hour each); absolute alcohol twice (1hour 30 minutes each); and another absolute alcohol for 2 hours; equal volumes of absolute alcohol and xylene, i.e. 50/50 (overnight); then cleared in two changes of xylene for one hour each.
Paraffin wax infiltration was carried out with two changes of paraffin wax at interval of one hour each, in an electric hot air oven at 60 oC. The samples were then embedded in paraffin wax, trimmed and mounted in a wooden chuck. Each sample was sectioned on the microtome at 5 µm and floated on the floatation bath. The floated sections were picked out with clean albumenized microscopic slides; the slides were stained using Haematoxylin and Eosin (H & E) protocol and cover-slipped with a Depex mountant. The slides were later viewed with a binocular Olympus microscope (Zeedo, 5MP internal camera, e-PLAN objective). nLabelling was done with Photoscape v3.7.
RESULTS AND DISCUSSION
Table 1 summarized the GC-MS analysis of ethanol extract of B. pinnatum (EEBP) while Figure 1 is the chromatogram showing 6 peaks. As presented on the table, six (6) compounds were detected namely: 2H-Benzocyclohepten-2-one, decahydro-9a-methyl-, trans- (35.81%), Heptadecanoic acid, 16-methyl-, methyl ester (16.22%), Bis (2-ethylhexyl) phthalate (14.31%), MDMA methylene homolog (24.28%) and thymol (9.39%). These constituents obtained from GC-MS analysis have several biological activities, which can be exploited for therapeutic purposes. 2H-Benzocyclohepten-2-one, decahydro-9a-methyl-, trans-13-Octadecenoic acid and methyl ester have been reported to possess anti-angiogenic and anti-tumor effects [23]. Heptadecanoic acid, 16-methyl-, methyl ester is a fatty acid methyl ester with anti-microbial properties. Bis (2-ethylhexyl) phthalate is a major bioactive metabolite with potent antibacterial, antimicrobial and cytotoxic activities [24-26]. Benzo[h]quinoline, 2,4-dimethyl- and other quinolone analogues and derivatives have been reported to possess numerous type of biological activity including anticancer, anti-inflammatory, anti-microbial, anti-convulsant, anti-malarial, anti-hypertensive, etc [27]. Several reports have shown that thymol possess various pharmacological properties including antioxidant, free radical scavenging, anti-inflammatory, analgesic, antispasmodic, antibacterial, antifungal, antiseptic and antitumor activities [28]. These therapeutic properties of thymol are possible due to the pharmacophore of the phenolic hydroxyl group in its chemical structure, which confers protection by mutual absorption or neutralization of free radicals or by supplementing endogenous antioxidants [29].
Figure 1. Chromatogram of ethanol extract of Bryophyllum pinnatum
Table 1: Chemical Composition (GC-MS) of the EEBP
Peak No. | Name of compound | RT (Mins) | Exact mass (g) | Conc. (%) | Formula | Structure |
1 | 2H-Benzocyclohepten-2-one, decahydro-9a-methyl-, trans-13-Octadecanoic acid, methyl ester
| 16.03 | 180.15 | 35.81 | C12H20O |
|
2 | Heptadecanoic acid, 16-methyl-, methyl ester | 16.30 | 298.29 | 16.22 | C19H30O2 |
|
3 | Bis (2-ethylhexyl) phthalate | 18.95 | 390.28 | 14.31 | C24H38O4 |
|
4 | Benzo[h]quinoline, 2,4-dimethyl- | 20.09 | 207.10 | 1.80 | C15H13N |
|
5 | MDMA methylene homolog | 20.32 | 207.13 | 24.28 | C12H17NO2 |
|
6 | Thymol, TMS | 20.67 | 150.10 | 9.39 | C10H14O |
|
![2,4-Dimethylbenzo[h]quinoline.png](data:image/png;base64,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)
EEBP - Ethanol Extract of Bryophyllum pinnatum leaves
Figure 2. Effect of EEBP on Haemoglobin Concentration of Cadmium-Challenged Rat
Bars are mean ± standard deviation (n=4). Bars with asterisk (*) are significantly (p < 0.05) different compared with CdCl2-treated group. BP = Bryophyllum pinnatum
Figure 3. Effect of EEBP on Red Blood Cell Concentration of Cadmium-Challenged Rat
Bars are mean ± standard deviation (n=4). Bars with asterisk (*) are significantly (p < 0.05) different compared with CdCl2-treated group. BP = Bryophyllum pinnatum
Figure 4. Effect of EEBP on Packed Cell Volume of Cadmium-Challenged Rat
Bars are mean ± standard deviation (n=4). Bars with asterisk (*) are significantly (p < 0.05) different compared with CdCl2-treated group. BP = Bryophyllum pinnatum
Result of the effect of ethanol extract of Bryophyllum pinnatum leaves on haemoglobin concentration, red blood cell count and packed cell volume of cadmium-challenged rats are shown in Figures 2, 3 and 4 respectively. As indicated in Figure 2, there was a significantly (p <0.05) higher haemoglobin concentration of the group treated with 400 mg/kg of EEBP only when compared to the control and cadmium-intoxicated groups. In addition, there was a significant elevation of Hb concentration in the groups intoxicated with cadmium and co-treated with EEBP (200 and 400 mg/kg) compared to the normal control and cadmium control groups. There was no significant (p > 0.05) difference between the Hb concentration of the extract control (200 mg/kg) and normal control groups. The same trend of the effect of EEBP on haemoglobin concentration was observed in PCV (Figure 4). As shown in Figure 3 There was a significantly (p <0.05) lower blood cell (RBC) count of the cadmium control group when compared to the normal control group and the groups intoxicated with cadmium and co-treated with EEBP (200 and 400 mg/kg). There was no significant (p >0.05) difference in the RBC count of the cadmium control group compared to the 200 mg/kg EEBP group.
Several reports have linked cadmium toxicity to several forms of anaemia via haemolysis caused by deformation of peripheral red blood cells (RBCs), iron deficiency and insufficient erythropoietin production [5,30]. This was evident in this study as shown by the significant suppression of Hb, RBC count and packed cell volume in the cadmium group. However, treatment with ethanol extract of B. pinnatum leaves (EEBP) significantly improved the hematological parameters (Hb, RBC count and PCV). This is in consonance with the report of Ufelle et al. [31] who reported increased Hb and PCV levels in rats administered crude methanol leaf extract of B. pinnatum. The observed marked elevation in Hb concentration and PCV could be due to the stimulatory effect of B. pinnatum on the bone marrow to produce more hemoglobin and red blood cells. Presence of important phytochemical constituents in B. pinnatum such as tannin, ascorbic acid and phenol could boost erythropoiesis [30-33]. Flavonoid, zinc, riboflavin and niacin contents of B. pinnatum, as earlier reported by [34,35] could be responsible for the improvement of the hematological parameters observed in this study. Also, benzo[h]quinoline, 2,4-dimethyl-, one of the constituents discovered from the GC-MS analysis was also present in the stem bark extract of Mangefera indica L. and showed significant anti-anaemic property in haemolytic anaemic rats [36]. Hence, B. pinnatum could serve as a haematinic product in terms of improving blood volume and erythropoietic processes.
Table 2. The Effect of Ethanol Extract of B. pinnatum Leaves on Total White Blood Cell (TWBC) Count and Differentials of Cadmium-Challenged Rats
GROUP | TWBC (x103/mm3) | Lymphocytes (%) | Neutrophils (%) | Monocytes (%) | Eosinophil (%) |
Normal Control | 9.73 ± 1.16 | 54.67 ± 1.76 | 37.67 ± 2.02 | 4.67 ± 0.67 | 3.00 ± 0.58 |
CdCl2 | 12.13 ± 0.26 | 54.25 ± 0.63 | 38.00 ± 0.58 | 5.25 ± 0.48 | 2.50 ± 0.65 |
200mg/kg BP | 19.25±1.06* | 60.00 ± 0.41* | 31.00 ± 0.91* | 5.75 ± 0.48 | 3.25 ± 0.48 |
400mg/kg BP | 14.70 ± 0.66 | 59.00 ±0.71* | 32.25 ± 1.25* | 5.50 ± 0.64 | 3.25 ± 0.48 |
Cd+200mg/kg BP | 14.20 ± 2.07 | 58.25 ± 0.85* | 33.50 ± 0.96* | 5.25 ± 0.48 | 3.00 ± 0.81 |
Cd+400mg/kg BP | 17.30 ± 0.14* | 57.33 ± 11.45* | 33.33 ± 1.86* | 5.67 ± 0.67 | 3.67 ± 0.67 |
Values are mean ± standard deviation. Values with asterisk (*) are significantly (P < 0.05) different compared with CdCl2-treated group. TWBC = Total white blood cells; BP = Bryophyllum pinnatum
As shown in Table 2, the cadmium control group had the least TWBC count when compared to the rest of the groups – showing statistical significant (P<0.05) with reference to extract-treated (200 mg/kg EEBP) and cadmium-extract co-treated (5 mg/kg Cd + 400 mg/kg EEBP) groups. The percentage lymphocyte and neutrophil concentrations of the extract-only and cadmium-extract co-treated groups (Groups 3 – 6) were significantly (P<0.05) elevated compared to those of the normal and cadmium control groups. No significant (P > 0.05) difference in the percentages of monocytes and eosinophil was observed.
Total white blood cell (TWBC) count and its differentials are
quantifiable parameters of the blood that are commonly used to appraise hematopoietic function [37]. White blood cells (WBC) are vital in conferring protection to living organisms against foreign invaders and rise in their concentration could denote an immunological challenge. On the other hand, neutrophils are essential phagocytic cells generally elevated in the early inflammatory response [38,39] whereas lymphocytes are subtypes of WBC critically vital for providing cell-facilitated protection. The observed elevation in total white blood cell (TWBC) count in the EEBP-treated groups of the rats is in tandem with earlier findings [31,40] which is suggestive of the fact that B. pinnatum possesses antimicrobial activity. The observed significant elevation of TWBC count and lymphocytes by the extract as seen in this study (Table 2) suggests that B. pinnatum could serve as potent antimicrobial agent and immune booster against cadmium-induced toxicity. These effects could have been enhanced by the presence of the notable antimicrobial constituents (heptadecanoic acid, 16-methyl-, bis (2-ethylhexyl) phthalate, benzo[h]quinoline, 2,4-dimethyl- and thymol) shown by the GC-MS analysis (Table 1).
Furthermore, the significant reduction of neutrophil (Table 2) as seen in this study tallies with earlier report of Ufelle et al. [31]. This strongly suggests that the plant has the ability of weakening primary immune response, including inflammation. Previous reports showed that aqueous extract of B. pinnatum possess anti-inflammatory [41,42] and immunosuppressive activities [38] which could in part be connected with observed neutrophilic effect.
Result of Histological Investigation
Plates 1 – 6 show the photomicrographs of bone marrow of the animals in the study with a range of normal erythroid and granulocytic progenitors at different stages of differentiation including abundant metamyelocytes (MM), erythroblasts (EB), lymphoblasts (LM), megakaryocyte (MK), occasional macrophages (M) and band forms of neutrophils (NB) among others. Occasional mitotic figures (MF) are also seen. The group induced with CdCl2 showed moderate to severe reduction of the different progenitor cells compared to the normal control group. However, there was mild reduction of the different progenitor cells in the cadmium-challenged groups treated with 200 and 400 mg/kg b.w of B. pinnatum compared to the normal control. There were no pathological alterations in the groups treated with only the ethanol extract (200 and 400 mg/kg b.w) of B. pinnatum compared to the normal control.
Administration of the extract only did not cause any form of alteration in the histology of the bone marrow. This is suggestive of the fact that B. pinnatum was not harmful against the bone marrow, and hence, may not affect haemopoitic process. Cadmium toxicity has been reported to inhibit the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts, and directly causes BMSC apoptosis. Cadmium toxicity can affect the skeletal system via direct interaction with bone cells thereby causing demineralization [43]. Cadmium induces osteoblast injury and oxidative stress – giving rise to DNA damage, dysfunctional mitochondria, and endoplasmic reticulum stress, resulting in apoptosis [44]. This was evident in this study as shown by the moderate to severe reduction of the different progenitor cells of the cadmium-intoxicated group relative to the normal control group. This effect could have also possibly contributed to the significantly lower red and white blood cells’ counts and PCV in the cadmium group (Plate 2). This is because the bone marrow is the main haematopoietic organ responsible for the production of red blood cells, white blood cells (including its differentials) and platelets [45]. The mild reduction in the different progenitor cells by the B. pinnatum extract suggests that it could ameliorate the degeneration of the progenitor cells. Earlier study by Cruz et al. [46] also highlighted the potential of B. pinnatum which inhibited degranulation and cytokine production of bone marrow-derived mast cells following IgE/FcRI crosslinking [46].
Plate 1: Photomicrograph of the bone marrow of normal control group
Plate 2: Photomicrograph of the bone marrow of CdCl2-challenged group
Plate 3: Photomicrograph of the bone marrow of B. pinnatum extract group (200 mg/kg b.w)
Plate 4. Photomicrograph of the bone marrow of B. pinnatum extract group (400 mg/kg b.w)
Plate 5: Photomicrograph of the bone marrow of CdCl2-challenged rats co-treated with 200 mg/kg b.w of B. pinnatum extract
Plate 6: Photomicrograph of the bone marrow of CdCl2-challenged rats co-treated with 400 mg/kg b.w of B. pinnatum extract
Acknowledgements
The authors appreciate the Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine for providing the laboratory space.
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