Effect of Chysophyllum albidum Ethanol Leaf Extract on Haematological and Platelet Indices of Apparently Healthy Wistar Rat

Juliet N. Ozioko1, Obinna V. Ayogu2, Benjamin O. Ezema1, Dilibe C. Uramah3 and Kingsley O. Omeje*,1

1Department of Biochemistry, University of Nigeria

2Department of Zoology and Environmental Biology, University of Nigeria

3Department of Plant Science and Biotechnology, University of Nigeria

ABSTRACT

Chysophyllum albidum ethanol leaf extract was studied to ascertain it potentials on hematological indices and hepatocyte of wistar albino rat. Twenty-five rats that weighed 98-168g were separated into five groups. Phytochemicals were quantified using standard methods. Alkaloids, flavonoids, tannins, saponnins and terpernoids were detected. The leaf extract had 0.415 % of alkaloids, 0.102 mg/TAEq of tannins, 0.211 mg/QEq of flavonoids and 0.360 mg/GAEq of phenolics. Phytol, oleic acids, hexadecanoic acids and octadecanoic acids were detected in the leaf extract by Gas Chomatography-Mass Spectroscopy. White blood cells, hemoglobin and platelets were some of the hematological indices increased after the oral administration of the extract. Red cell distribution width (RDW), a platelet index implicated in cardiovascular ailments was lowered after consumption of the leaf extract of C. albidum. The liver marker enzymes aspartate transaminase and alanine transaminase showed no significant changes in their serum activity. Similarly, superoxide dismutase and catalase showed no significant increase after the period of study.

Article Information

Received 03 October 2020

Revised 18 November 2020

Accepted 04 December 2020

Available online 07 May 2021

(early access)

Published 14 February 2022

Authors’ Contribution

EBO, OKO designed research. OJN, AOV and OKO carried out laboratory work, wrote manuscript. EBO, UDC and OKO analyzed results and wrote manuscript.

Key words

Platelet indices, Hematological indices, Phytomedicine, Chysophyllum albidum, Enzymes

DOI: https://dx.doi.org/10.17582/journal.pjz/20201003141005

* Corresponding author: kingsley.omeje@unn.edu.ng

0030-9923/2022/0003-1095 $ 9.00/0

Copyright 2022 Zoological Society of Pakistan

INTRODUCTION

Chysophyllum albidum is common tree found in Africa, known for its edible fruits and medicinal values (Amusa et al., 2003). It is predominant in rainforests, which can reach 25-37m when matured (Orwa et al., 2009). It is known commonly Nigeria as Agbalumo and Udara among the Yorubas and Igbos. It is rich in phytochemicals such as alkaloids, phenols and tannins (Okoli and Okere, 2010), alkaloids and tannins (Ibrahim et al., 2017). Imaga and Urua (2013) and Egharevba et al. (2015) reported that the plant is rich in phytochemicals.

The plant is used to cure yellow fever, malaria, treatment of infections and and sin eruptions (Idowu et al., 2006). Similarly, the stem, seeds and root extracts of C. albidum exhibit antimicrobial, anti-inflammatory, anti-diarrheal and anti-haemorrhoidal properties (Okoli and Okere, 2010). These pharmacological activities are elicited by phytochemicals they contain (Omeje et al., 2014), it is a remedy for fever due to alkaloids present and the flavonoid is responsible for anti-allergic, anti-microbial and anti-cancer activity (Adisa, 2000). There are several side effects associated with the usage of phytomedicine in the treatment of ailments, such as deleterious effect on blood (Esonu et al., 2001), liver and kidney (Stickel et al., 2005).

Different parts of Chysophyllum albidium plant (leaf, stem-bark and root extracts) have been employed in ethno-medicine as antimicrobial agent (Okoli and Okere, 2010), fertility enhancer (Oigbochie et al., 2019) and antimalarial agent (Odediran et al., 2020). Though, the choice of preparation method depends on the type of ailment targeted, plant extract could be taken as a tonic (Morton, 1987), boiled or using local gin.

Despite all the medicinal benefits attributed to the different parts of C. albidum plant, some individual reports on dizziness and other allergic conditions after ingesting the trado-medicine have appeared. For these reasons it becomes important to evaluate the ethanol C. albidum leaf extract on the biochemical indices of Wistar rats.

MATERIALS AND METHODS

C. albidum leaf was harvested from its natural habitat Edem-ani, Nsukka LGA, Enugu State of Nigeria in the month of March, 2019. After fourteen days drying, it was pulverized into fine powder using electric blender. The ground leaf material (773.760 g) was extracted by macerating in 1.2L of 70% ethanol for 48 h and concentrate, yielding 18.48 g of the extract stored for further use.

Qualitative and quantitative phytochemical analysis

Qualitative analysis of the phytochemicals was determined using AOAC (2010) method with little modification. The phytochemicals screened were alkaloids, flavonoids, tannins, saponin, terpenoid and steroids.

The phenolic content was determined using the method described by Nwidu et al. (2017). The total flavonoid, alkaloid and tannin content of the leaf extract were determined according to Senguttuvan et al. (2014), using quercetin as the reference compound.

Gas chomatography-mass spectroscopy (GC-MS) analysis of the ethanol extract

GC-MS analysis was done using GC system comprising a Gas Chomatograph interfaced to a Mass Spectrometer (Schimadzu GCMS-QP2010).

Experimental animal

Twenty-five (25) adult Wistar albino rats (98-168 g) were used for the study. They were housed Biochemistry Department Animal House, and fed with commercial rat chow. The animals were divided into 5 groups, wach of five rats. They received the extract orally for twenty-eight days as follows: Group I: 2 ml of distilled water/kg body weight per day; Group II: 50 mg/kg b.w. of ethanol extract per day; Group III: 100 mg/kg b.w. of the ethanol extract per day; Group IV: 150 mg/kg b.w. of the ethanol extract per day; Group V: 200 mg/kg b.w. of the ethanol extract per day.

On day 28, blood samples were drawn by cardiac puncture; chloroform was used as a sedative agent. Each container was thoroughly mixed with anticoagulant (3.8% Tri-sodium-citrate) to prevent coagulation of the blood.

Haematological indices

The blood samples were drawn from the animals (control) at the start of experiment and then on the 28th day for determination of hematological parameters such as hemoglobin, white blood cells, haemocrit, red blood cells, platelet concentration, mean cell volume and lymphocyte concentration, platelet indices (red cell distribution width standard deviation, red cell distribution width coefficient of variation, platelet distribution width, mean platelet volume, platelet large cell ratio) by an Automated Hematology Sysmex Analyzer (Coulter Electronics, Bedfordshire, England).

Biochemical components of blood

Superoxide dismutase activity was determined using Martin et al. (1987) method. Alanine aminotransferase and aspartate aminotransferase activities were measured as described by Reitman and Frankel (1957). Catalase activity was determined as described by Aebi (1984) by monitoring the ultraviolet absorption of the sample at 240 nm as hydrogen peroxide is decomposed in by catalase.

Statistical analysis

The statistical packaged used was the statistical package for social sciences (SPSS), version 1. Differences were considered significant at p < 0.05.

RESULTS AND DISCUSSION

C. albidum was harvested from Edem-ani community, its natural habitat and macerated for 48 h. The extract yield was 18.48 g. Alkaloids, tannins, saponins, terpernoids and flavonoids were detected, while steroids was not detected. The result agreed with the report of Oputah et al. (2016), who reported saponins and flavonoids, while tannins, alkaloids, terpenoids, saponins, reducing sugars, steroids and flavonoids were reported by Akinpelu et al. (2016) in their study on C. albidum stem bark extract. It is evident that the plant parts of C. albidum are rich source of phytochemicals, which could be responsible for the reported pharmacological activities. Similarly, Oguntoyinbo et al. (2015) reported flavonoids, tannins, terpernoids and cardiac glycoside in the leaf of C. albidum.

Table I shows the concentration of alkaloids (0.415%), tannins were (0.102 g/TAEq), total flavonoids (0.211 mg/QEq) and total phenolic (0.360 mg/GAEq). Total phenolic and flavonoid content were high when compared to 68.3 μg GAE/g and total flavonoid of 3.71 mg reported by Asare et al. (2015). Also, low total phenol content and total flavonoid content of 0.47mg/gQE were reported by Oguntoyinbo et al. (2015). The total flavonoid content of C. albidum leaf extract was low, when compared to 1.79 mg/gQE reported for Euphorbia nerufolia leaf extract (Pracheta et al., 2010). The antimicrobial property may be attributed to the presence of phenolic compounds. This result indicates that the leaf extract could possess antioxidant activity due to the presence of polyphenols. Some pharmacological activities attributed to alkaloids include antimicrobial, cytotoxic, antioxidant, antimutagenic, and hallucinogenic properties (Patel et al., 2012). Tannin is important because of its ability to mop up free radicals (Deng et al., 2019), that can bind and precipitate or shink proteins (Ashok and Upadhyaya, 2012).

Figure 1 shows Gas Chomatography-Mass Spectrometry analysis (GC-MS) (Shibula and Velavan, 2015) of the ethanol extract of C. albidum which shows eight peaks. The corresponding compounds identified are shown in Table II. n-Hexadecanoic acid is an important biocidal agent. It has hypo-cholesterolemic and antioxidant properties (Komansilan et al., 2012). Phytol has been reported to significantly reduce the parasitic load of human schistosomiasis (Moraes et al., 2014). GC-MS was employed in studying the bioactive components of Physalis minima leaves (Karpagasundari and Kulothungan, 2014).

 

Table I. Quantitative phytochemical composition of ethanolic leaf extract C. albidum.

Phytochemicals	Composition
Alkaloid content	0.415 %
Tannin	0.102 mg/TAEq
Total Flavonoid content	0.211 mg/QEq
Total phenolic content	0.360 mg/GAEq

 

Table II. Compounds identified by GC-MS from ethanol extract of C. albidum.

S. No.	Name of compound	Conc. (Area %)	Retent. time (s)	Mol. formular
1	Pentadecanoic acid	1.72	19.32	C17H34O2
2	n-hexadecanoic acid	19.86	19.97	C16H32O2
3	11, 14, 17 eicosatrienoic acid	1.33	21.79	C21H36O2
4	Trans-phytol	2.92	22.08	C20H40O
5	Oleic acid	56.40	22.77	C18H34O2
6	Octadecanoic acid	9.42	23.15	C18H36O2
7	Hexadecanoic acid	1.70	25.16	C19H38O4
8	9-Octadecenal	6.65	27.44	C18H34O

 

Table III shows that the oral administration of the extract had no significant impact on the red blood cell count, while the white blood cell increased significantly when compared with the control. At 200 mg/kg bwt of the extract, the hemoglobin concentration increased significantly when compared to the control. Also, PCV and MCHC reduced significantly when compared to the control. The values of lymphocytes and neutrophil did not show any significant change when compared with the control. This suggests that, the leaf extract of C. albidum contain some important pharmacological agents responsible for impairment in the production of blood cells (PCV and MCHC) (Adebayo et al., 2011). The changes observed in some of the indices are an indication that some of the phytochemicals present have impact on blood indices synthesis and morphology. Tannin and alkaloids have been reported to cause changes in blood parameters (Deng et al., 2019). Similarly, the mechanism of antimicrobial property of C. albidum leaf extract could by increasing the concentration of the white blood cells, thereby mobilizing them to the site of infection. Omeje et al. (2014) had reported the alteration of blood indices by the leaf extract of C. olitorus. The significant increase in the values of platelet at all doses suggests that C. albidum may be of great importance to an individual who is to under cesarean section in order to help boost platelet aggregation, thereby leading to wound healing.

 

Table IV shows the effect of leaf extract on platelet indices. There was significant decrease of RDW-CV and PDW when compared to control group. A dose dependent decrease of the RDW-CV concentration was observed. The concentrations of MPV and P-LCR and RDW-SD increased significantly when compared with the control group. This is in accordance with the findings of Ofem et al. (2012) where doses of plant extract caused decrease in MPV, P–LCR and PDW of albino rats. This suggests the ethanol extract of C. albidum does not contain bioactive compound that affects the tested parameters. One of the indicators of cardiovascular mortality is increased red cell distribution width (RDW) (Söderholm, et al., 2015). This suggests the consumption of C. albidum leaf extract may not be predisposing a consumer to any cardiovascular illness. According to Tonelli et al. (2008), increased RDW is implicated in long-term mortality of patients suffering from coronary artery disease. Hence, the decrease observed, shows that the ethanol extract of C. albidum leaf may not cause or induce cardiovascular diseases by platelet index increase. Similarly, iron deficiency anemia has been linked with increased RDW (Khan et al., 2014). The non-significant increase in RDW shows that the extract may not have deleterious effect or inhibit red blood cell synthesis. Presence of tannin in plant extract has been implicated in the alteration of platelet indices (Muriithi et al., 2015).

 

Table III. Effect of C. albidum leaf extract effect on haematological indices of albino rats.

Hematological parameter	Control	50 mg/kg bwt	100 mg/kg bwt	150 mg/kg bwt	200 mg/kg bwt
White blood cell (×109/L)	5.28±1.29	5.35±0.14	7.41±1.62	7.21±0.22	9.05±0.13
Red blood cell (×1012/L)	5.11±1.77	5.20±1.31	6.45±0.31	5.34±0.40	6.09±0.62
Hemoglob. (g/dl)	6.72±0.43	6.53±0.83	8.64±0.92	6.07±0.11	8.35±1.03
HCT (%)	32.17±0.38	23.91±1.65	31.60±0.52	24.31±1.46	33.24±0.29
PCV(fl)	66.24±2.46	45.72±2.52	48.14±3.87	49.74±2.38	44.04±4.23
MCHC(pg)	22.75±0.18	21.92±0.59	19.20±0.53	19.73±1.26	19.60±0.22
Platelet (×109/L)	131.73±63.34	147.72±11.07	129.66±21.40	301.80±0.74	344.46±4.23
Lymphocyte (%)	68.29±0.56	69.85±0.62	66.39±2.81	74.27±3.70	70.04±0.28
Neutrophils (%)	25.42±1.37	24.72±0.56	30.44±0.41	33.63±1.64	23.89±3.22

 

Table IV. Effect of oral administration of ethanol extract of C. albidum on platelet indices of wistar rat

Platelet indices	Control	50 mg/kg bwt	100 mg/kg bwt	150 mg/kg bwt	200 mg/kg bwt
RDW-CV (%)	20.73±1.92	16.22±1.50	14.38±0.80	13.85±0.62	13.42±0.00
PDW (fL)	9.94±0.41	5.42±0.46	3.76±0.63	5.11±1.28	7.93±2.06
MPV (fL)	-9.27±0.77	5.29±0.31	-6.83±0.40	-9.61±.0.43	-8.63±0.10
P-LCR (%)	4.65±0.43	9.42±0.38	5.61±0.92	7.52±0.10	7.16±0.53
RDW-SD (fL)	27.59±0.28	27.65±0.90	25.40±0.65	22.65±0.00	19.34±0.44

 

RDW.CV, red cell distribution width coefficient of variation; PDW, platelet distribution width; MPV, mean platelet volume; P-LCR, platelet large cellratio; RDW-SD, red cell distribution standard deviation.

 

Table V. Effect of oral administration of ethanol extract of C. albidum on representative liver function and oxidative stress related enzymes in wistar rats.

Biochemical indices	Control	50 mg/kg bwt	100 mg/kg bwt	150 mg/kg bwt	200 mg/kg bwt
Alanine transaminase (IU/L)	116.05±0.43	113.92±0.30	109.20±0.42	132.09±0.18	193.22±0.12
Aspartate transaminase (IU/L)	184.76±0.26	184.45±0.18	139.28±0.30	170.58±0.07	186.41±0.25
Catalase (IU/L)	194.50±0.49	207.53±0.32	171.06±0.61	193.41±0.70	176.39±0.10
Superoxidismutase (IU/L)	202.70±0.90	200.65±0.25	186.14±1.30	185.29±0.05	213.12±0.29

 

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are some enzymes considered as indicators of the health of the liver (Trampuz et al., 2003). Table V shows effect of varying concentrations (50, 100, 150 and 200mg/kg) of extract of C. albidum. Increase in serum ALT is common in hepatic necrosis (Duncan et al., 2006). At concentrations of 150 and 200 mg/kg body weight, there was significant increase in the serum ALT. The observed increase suggests the low concentration of free radicals in the biological system, which could be attributed to the ability of some bio active agents to mop up these free radicals or maintaining the integrity of the hepatocytes to produce adequate enzymes needed to play the antioxidant role. Omeje et al. (2016) reported hepatoprotective potential of ethanol extract of C. olitorus. AST increase in serum signifies liver damage (Dasofunjo et al., 2013). The oral administration of the extract showed a similar result to Buchholzia coriacea extract on Plasmodium berghei infected mice as reported by Enechi et al. (2016). Therefore, the significant decrease obtained suggests the extract has the ability to protect the integrity of the animal liver.

Superoxide dismutase (SOD) is an enzyme responsible for scavenging free radicals for physiological defense strategies in living organisms (Stephanine et al., 2020). Varying concentration of C. albidum ethanol extract has shown a significant decrease when compared to control group. The results show a dose dependent decrease in the activity of SOD except for 200 mg/kg group. This is suggestive that high concentration of free radical was generated in vivo, hence the decreasing concentration of the enzyme responsible its mop up. This report is in line with the study by Owolabi et al. (2008). Similarly, Nwobodo et al. (2018) reported decreased SOD in their work on Azadirachta indica. Catalase is one of the antioxidant enzymes that plays essential role in the antioxidant protection biological systems (Ighodaro and Akinloye, 2018). Among the concentrations studied, there was no significant difference when compared to the control. Suggesting that, the rate of generation of free radical was in equilibrium with the rate of its clearance. This could be attributed to the presence of polyphenolic compounds in the ethanol extract. Sani et al. (2012) reported increased concentration of catalase and superoxide dismutase in their work on thee medicinal plants. There is synergistic relation between catalase and superoxide dismutase, since they are the first line of body defense against oxidative stress (Ighodaro and Akinloye, 2018).

CONCLUSION

In conclusion, the oral administration of C. albidum leaf extract did not cause significant alteration in the hematological and platelet indices (RDW, PDW, MPV and P–LCR) at the doses studied after 28 day consumption. One of the indicators of cardiovascular mortality is increased red cell distribution width (RDW), which was reduced by the ethanol extract. This suggests that the consumption of C. albidum leaf extract may not be predisposing a consumer to any cardiovascular illness. This suggests therefore, there is no harmful effect associated with the consumption of C. albidum, however, having established the preservatory property of the C. albidum extract on healthy models, further studies could be carried out on diseased conditions to ascertain if the extract is curative.

Statement of conflict of interest

The authors have declared no conflict of interest.

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