Introduction

Ginkgo biloba has existed for millions of years. The plant belongs to the family Gink- goaceae and it originated from Asia (Nuhu, 2014). The standardised extract of Ginkgo biloba (EGb 761®) with well defined com- ponents used in this study is one of the most accepted and  widely  used  herbal  medici-  nal products in the world (Xie et al., 2014). The extract is considered relatively  non-tox- ic and safe for consumption (Abdulrazak et al., 2018). The major constituents of the ex- tract are flavonoids (Nuhu et al., 2017) and terpenoids (Van beek, 2002). The flavonoid fraction is composed of quercetin, kaemp- ferol, isorhamnetin and glycosides, while the terpenoid fraction contains the ginkgolides A, B, C and J, as well as bilobalide. These com- pounds in  EGb  761®  are  actively  involved in the regulation of cellular metabolism, cell protective effect against toxins, and assist in balancing physiological status under oxida- tive stress (Yoshikawa et al., 1999), unlike most synthesized drugs with only one target function in their mechanism of action (He et al., 2009). The mechanisms of action of the extract are related to its antioxidant effect, and anti-platelet activating factor (Anti-PAF) ac- tivity (Smith and Luo, 2004). He et al. (2009) investigated the effects of EGb 761 on the properties of human red blood cells in the presence and absence of amyloid peptide (A   β 25-35), peroxide and hypotonic stress. The findings reported that the protective effect of EGb 761® on red blood cells was dependent on the type of external stress present. Accord- ing to Chan et al. (2007), antioxidant protec- tive ability of EGb 761® is related to its ability to inhibit nitric oxide-stimulated protein ki- nase C (PKC) activity.

Chlorpyrifos is widely used today espe- cially  in  northern  Nigeria,  as  a   pesticide,

and it has been known to induce oxidative stress (Nuhu et al., 2017). Exposure in both humans and animals is commonly via food products. It elicits many adverse effects, some of which are: haemotoxicity, genotox- icity, neurochemical and neurobehavioural changes (Uchendu et al., 2012). United States Environmental Protection Agency restricted some of its domestic use in 2000 based on human health risk (Uchendu et al., 2012). Some of the symptoms of chlorpyrifos poi- soning in human and animals include head- ache, dizziness, salivation, unconsciousness, convulsion and death (Akhtar et al.,  2009).

The underlying principle behind the ther- apeutic action of the Ginkgo leaf extract on chronic ailments (such as oxidative stress, poisoning, neurodegenerative and cardiovas- cular diseases) has focused on its antioxidant effects (Xie et al., 2014), which are elicited via two mechanisms of action; inhibition of free radical formation, and free radical scav- enging activity. It scavenges reactive oxy- gen species (ROS) such as hydroxyl radicals (OH˙), peroxyl radical  (ROO˙),  superox-  ide anion radical (O2−˙), nitric oxide radical (NO˙), hydrogen peroxide (H2O2), and ferryl

ion species (Mahadevan and Park, 2007). The

extract can also enhance activities of antioxi- dant enzymes such as superoxide distmutase (SOD), glutathione peroxidase, catalase, and/or heme oxygenase-1, thereby indirectly contributing as an antioxidant (Mahadevan and Park, 2007). It has been suggested that Ginkgo leaf extract increases expression of mitochondrial enzymes, NADH dehydroge- nases, which can influence  ROS generation in the mitochondria.

This study aimed to evaluate the phyto- chemical constituents of EGb 761®, effect of the extract on haematological parameters and

erythrocyte osmotic fragility in rats exper- imentally exposed to chlorpyrifos-induced oxidative stress, and also to determine the effect of the extract on erythrocyte osmotic fragility under hypotonic  stress.

Materials and Methods

Phytochemical Screening of the Extract Phytochemical screening of the stan- dardised extract of Ginkgo biloba leaf extract (EGb 761®) (Nutra Green Biotechnology Co. Ltd, United Kingdom) was carried out using standard methods (Silva et al., 1998;   Trease

and Evans 2002).

Experimental Animals and Management Albino rats, weighing between 180 - 200 g, were acclimatised for 2 weeks prior to the experiments; the rats were given access to pellets of feed prepared from grower’s mash, maize bran and groundnut cake at the ratio of 4:2:1 and water was provided ad libitum. The feed was produced in the Animal section of the Department of Veterinary Physiology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria. All animal experi- mentation was done in accordance with Ah- madu Bello University Animal Use and Care Guidelines. Ethical clearance was obtained with approval number ABUCAUC/2016/015 from  Committee  on Animal  Use  and Care,

Ahmadu Bello University,  Zaria.

Experimental Design

The method of Ambali et al. (2012) was used with modifications to  accommodate both the pre-treatment and post-treatment activity of EGb 761® vis-à-vis the treated control. The dosages for Vitamin E (100 mg/ ml), Ginkgo biloba leaf extract (100 mg/ml), and chlorpyrifos (10.6 mg/ml) used for this study were 100 mg/kg, 100 mg/kg, and 10.6 mg/kg, respectively. Timing and the choice of  treatment  doses  for  the  study  were  in-

formed by the acute toxicity study (L.D50 > 5000 mg/kg) and several  trial  experiments, as supported by previous studies (Sarikçio- glu et al., 2004, Biddlestone et al., 2007 and Ambali et al. 2012) .

For the effects of the extract on haemato- logical parameters and on erythrocyte osmot- ic fragility in chlorpyrifos-exposed rats, thir- ty-five albino rats were divided into seven groups of five rats each; Group A (Distilled water, 5 ml/kg) served as untreated control and administered only distilled water. Group B (VE+CPF) was pre-treated with vitamin E and then administered chlorpyrifos 30 min later, Group C (CPF+VE) was administered with chlorpyrifos and then vitamin E 30 min later, Group D (Extract+CPF) was pre-treat- ed with the extract, and then with chlorpy- rifos 30 min later, Group E (CPF+Extract) was administered with chlorpyrifos and the extract 30 min later, Group F (Extract only) was administered with the extract only and Group G (CPF only) was administered with chlorpyrifos only. The regimens were ad- ministered orally once daily for two weeks. The administered doses in ml of vitamin E, Ginkgo biloba leaf extract and chlorpyrifos used for this study were obtained by the for- mula;

Consequently, each animal received   0.18-

0.20 ml dose of the respective treatment type per day depending on their individual body weight (180-200 g).

In the erythrocyte osmotic fragility  study of non-chlorpyrifos-exposed rats, fifteen rats were divided into 3 groups of five animals each. Groups A, B, and C were administered with  Ginkgo  biloba leaf extract, Vitamin  E,

and isotonic saline at a dosage of 100 mg/ kg, 100 mg/kg, and 5 ml/kg respectively. The regimens were administered once daily by oral gavage for a period of 2 weeks.

The animals were sacrificed by jugular venesection at the completion of the ex- periments.  Blood  samples  were  collected  in clean plastic centrifuge tubes containing heparin as anticoagulant for haematological assay and erythrocyte osmotic fragility  test.

Determination of blood cellular components

Determination of packed cell volume (PCV) was done as described by Weiss and Wardrop (2010), with increased centrifuga- tion time as a slight modification. Erythro- cytes and leucocytes counts were done using a haemocytometer. Haemoglobin meter (XF- 1C-China) was used to determine haemoglo- bin concentration (Hb), and the mean cor- puscular volume (MCV), mean corpuscular hae‌moglobin (MCH) and mean corpuscu- lar haemoglo‌bin concentrations (MCHC) were then calculated.

Determination of Erythrocyte Osmotic Fragility

Erythrocyte osmotic fragility was deter- mined according to the method as described by Buhari et al. (2014). Exactly 5 ml of varying concentrations of sodium chloride (NaCl) solutions with phosphate buffer (3.22 g/L)  at a pH of 7.4 (0.0,  0.1,  0.3,  0.5,   0.7,

and 0.9%) were prepared in sets of 6 centri- fuge tubes each.  Blood  (20 µL) was added to each concentration of the test solution in each tube. The contents were mixed and in- cubated at room temperature for 30 min and then centrifuged at 3000 × g for 10 min. The concentration of haemoglobin in the super- natant solution of each tube was measured   at 540 nm using a spectrophotometer (Spec- tronic-20, Philip  Harris  Limited, Shenstone,

England) by reading the absorbance. By assuming that the haemolysis of haemoglo- bin in 0.0% NaCl solution was 100%, per- centage haemolyses of the various treatment groups were then calculated, as described by Deriyeli et al. (2004).

Data Analysis

Data were expressed as mean ± standard error of mean (SEM) and then analysed by one-way analysis of variance (ANOVA) fol- lowed by Tukey’s post-hoc test. The analy- ses were done using Graphpad Prism version

5. Values of P < 0 .05 were considered sig- nificant.

Results

Phytochemical Screening of the Extract The result of phytochemical screening is presented in Table 1. Phytochemical screen- ing of EGb 761® revealed the presence of carbohydrates, glycosides, saponins, triter- penes, tannins, flavonoids, and alkaloids. However, the result shows that the extract did not contain anthraquinones and steroids. Effect of Gingko biloba Leaf Extract  on

Haematological Parameters

The results of haematological parameters are presented in Tables 2 and 3. Packed cell volume and haemoglobin were significantly (P˂0.05) higher in Group D when compared to Groups A, B, and C. Similarly, total red blood cell count was significantly (P˂0.05) higher in Group D when compared to Groups A, B, C, and F. Total protein was significant- ly (P˂0.05) higher in Group D and E, com- pared to Group A. There was no significant (P˂0.05) effect of EGb 761® administration on total and differential leucocyte  counts.

Table 1. Phytochemical Screening of Ginkgo biloba Leaf Extract (EGb 761)

                 
Table 2. Effect of Ginkgo biloba Leaf Extract on Hematological Parameters (Mean ± Standard Error of Mean)

RED BLOOD INDICES

Tukey's test: Means having different superscript (a,b) letters are significantly (P˂0.05) different

PCV= Packed cell volume, HGB=haemoglobin, TP=total protein, TRBC=total red blood cell count, MCV= mean corpuscular volume, MCH= mean corpuscular haemoglobin, MCHC= mean corpuscular haemoglobin concentration

NOTE:

• Group A= (Distilled water)
• Group B= (Vitamin E+ Chlorpyrifos)
• Group C= (Chlorpyrifos + Vitamin E)
• Group D= (Extract+ Chlorpyrifos)
• Group E= (Chlorpyrifos +Extract)
• Group F= (Extract only)
• Group G= (Chlorpyrifos only)

Table 3. Effect of Ginkgo biloba Leaf Extract on White Blood Cells and Differential Count Parameters (Mean ± Standard Error of Mean)

Means within the same column are not statistically different at 95% CL (α=0.05)

TWBC= Total white blood cell count, Lymp= lymphocytes, Neutro= Neutrophils, Eosino=Eosinophils, Mono= Mono- cytes, Band=Band cells.

•

NOTE:

• Group A= (Distilled water)
• Group B= (Vitamin E+ Chlorpyrifos)
• Group C= (Chlorpyrifos + Vitamin E)
• Group D= (Extract+ Chlorpyrifos)
• Group E= (Chlorpyrifos +Extract)
• Group F= (Extract only)
• Group G= (Chlorpyrifos only)

Effect of Gingko biloba Leaf Extract Erythrocyte Osmotic Fragility

The result of the effect of EGb 761 on erythrocyte osmotic fragility in chlorpyrifos induced oxidative stress in rats is presented in Table 4. This result indicated that the dif- ferences in percentage haemolysis were not statistically significant (P>0.05) between  all compared groups. All groups have the same minimum erythrocyte osmotic fragil- ity level at 0.9% NaCl concentration which is isotonic, with Group F having the low- est percentage haemolysis vis-à-vis other groups. Group G has the highest erythro- cyte osmotic fragility level at 0.1% NaCl concentration while the other groups have the   highest   erythrocyte   osmotic fragility

level at 0.0% NaCl concentration, both of which are hypotonic.

Table 5 shows the results of the effect of Ginkgo biloba leaf extract on erythrocyte osmotic fragility. Results of this study in- dicated that Groups A and B have lowest percentage haemolysis at 0.9% NaCl con- centration which was isotonic and served as reference in this study, with Group A having the overall lowest value compared to oth-   er groups. Also, Group A has significantly (P˂0.05) lower percentage  of  haemolysis in hypotonic medium at 0.5, 0.3, and 0.1% NaCl concentration respectively, when compared to Group C. Group B has sig- nificantly (P˂0.05) lower percentage hae- molysis at 0.5% NaCl concentration    when

compared to Group C. Group A has signifi-

cantly (P˂0.05) lower percentage  haemoly-

sis at 0.7, 0.3, and 0.1% NaCl concentration respectively when compared to Group  B.

Table 4. Effect of Ginkgo biloba Leaf Extract on Erythrocyte Osmotic Fragility of Chlorpyrifos-Ex- posed Albino Rats, Measured as Percent Haemolysis (Mean ± Standard Error of Mean)

Means within the same column are not statistically different at 95% CL (α=0.05)

NOTE:

• Group A= (Distilled water)
• Group B= (Vitamin E+ Chlorpyrifos)
• Group C= (Chlorpyrifos + Vitamin E)
• Group D= (Extract+ Chlorpyrifos)
• Group E= (Chlorpyrifos +Extract)
• Group F= (Extract only)
• Group G= (Chlorpyrifos only)

Table 5. Effect of Ginkgo biloba Leaf Extract on Erythrocyte Osmotic Fragility of Non- Chlorpyri- fos-Exposed Albino Rats, Measured as Percentage Haemolysis (Mean ± Standard Error of Mean).

Tukey's test: Means having different superscript (a,b) letters are significantly different (P˂0.05)

NOTE:

• Group A= (Ginkgo biloba leaf extract)
• Group B= (Vitamin E)
• Group C= (Isotonic saline)

Discussion

Phytochemical Screening of the Extract Phytochemical screening of Ginkgo bi- loba leaf extract in this study showed the presence of carbohydrates, glycosides, sa- ponins, triterpenes, tannins, flavonoids, and alkaloids. This is also consistent with the findings of other investigators (Ibrahim and Nuhu, 2016; Goh and Barlow 2002; DeFeu- dis and Drieu 2000). The medicinal values of Ginkgo biloba lie in the presence of these ac- tive compounds which have been reported to have protective effect against ailments such as cardiovascular diseases, diabetes, and ox- idative stress (Abdulrazak et al., 2018;  Chan

et al., 2007; Saw et al., 2006).

Effect of Gingko biloba Leaf Extract on Haematological Parameters

In this study, Ginkgo biloba leaf extract significantly (P˂0.05) increased total red blood cell counts (8.35±0.29 ×1012/L), haemoglobin concentration (16.23±0.63 g/ dL) and packed cell volume (49.25±1.70 %) when compared with group pre-treated with Vitamin E (39.50±2.59 %, 13.13±0.86  g/dL,

6.65±0.37 ×1012/L respectively) and control (39.60±0.92 %, 13.16±0.31 g/dL,  6.62±0.15

×1012/L respectively). This, therefore, sug- gests that EGb 761® has more prophylactic protective effect on  erythrocyte  parameters in rats exposed to oxidative stress than vi- tamin E. Also, post treatment with EGb  761® significantly increased total protein (10.20±0.36 g/dL) when compared to con- trol (7.65±0.89 g/dL). These findings sug- gest that EGb 761® has more prophylactic than therapeutic protective effect on erythro- cyte parameters in rats exposed to oxidative stress. This effect may be due to accelerated erythropoiesis. However, further investiga- tion is required to confirm this. These find- ings  are  in  accordance  with  those reported

by Khafaga and Bayad, (2016), He et al., (2009), Abdel Baieth (2009) and He et al., (2008) that EGb 761® caused an increase in blood cellular components.

Effect of Gingko biloba Leaf Extract Erythrocyte  Osmotic Fragility

The prophylactic and therapeutic antiox- idant activities of EGb 761 on erythrocyte osmotic fragility in chlorpyrifos-induced oxidative stress were not significant when compared to vitamin E and  control.  Also, the prophylactic antioxidant activity of EGb 761 was not statistically significant vis-a-vis the therapeutic antioxidant activity of the ex- tract. Therefore, this study suggests that the percentage of haemolysis was not statistical- ly significant between all the groups.

However, the osmotic fragility of non-chlorpyrifos-exposed rats  showed  that rats treated with Ginkgo biloba leaf extract markedly afforded red blood cells an increased erythrocyte osmotic resistance against hypo- tonic stress (i.e. 0.7 %  NaCl  concentration has 3.30±0.79 % haemolysis, 0.5% NaCl concentration has 47.58±4.85% haemolysis, 0.3% NaCl concentration has 62.68±2.69 % haemolysis and 0.1% NaCl concentration has 58.84±10.22 % haemolysis ) when compared to the control group (4.91±0.50 %, 78.07±5.55

%, 92.80±7.20  %, 95.03±4.63  % haemolysis

at 0.7, 0.5, 0.3, and 0.1 % NaCl respectively) (Table 5). In the same vein, the extract also markedly increased red blood cell resistance  to hypotonic stress (i.e. at 0.7 % NaCl con- centration  was  3.30±0.79  % haemolysis, 0.3

% NaCl concentration was 62.68±2.69 % haemolysis and at 0.1 % NaCl concentration was 58.84±10.22 % haemolysis respectively) when compared to vitamin E (11.18±0.94, 87.54±5.33   and  97.57±2.34   % haemolysis

at 0.7, 0.3, and 0.1 % NaCl concentration re- spectively). Furthermore, vitamin E  increased