Food is thought about as a basic need for human. However, unhealthy food consumption degrades the quality of life instead of a balance diet. In this regard, Slavin and Lloyd (2012) stated that vegetables and fruits contain essential nutrients such as dietary fiber, carotenoids, vitamins, minerals, folate, plant sterols, and various phytochemicals are required for human daily diet. According to Wang et al. (2014); Crowe (2015) and Aune et al. (2017), these nutrients help to reduce mortality and prevent chronic diseases, including various cardiovascular diseases, cancers and even mental illnesses which is ascribed to the presence of different phytochemicals on those fruits and vegetables, functioning as anti-inflammatory agents, phytoestrogens, and antioxidants (Gouegni and Abubakar, 2013; Rodriguez-Casado 2016).

 

Enejoh et al. (2015) reported that the fruit juice of Citrus aurantifolia (Rutaceae), commonly known as lime, is added to sugar and palm oil or honey to relief cough and considered as tonic for libido and as antidote for poison. Aibinu et al. (2007) reported that the diluted form of the C. aurantifolia fruit juice is used for mouth wash to treat sore mouth and sore throat. The fruits of C. aurantium are sources of flavonoid-type compounds with diverse biological effects (Kang et al., 2011; Hamada et al., 2017; Liu et al., 2008) besides the essential oil and its components (Moraes et al., 2009; Barceloux, 2008). Additionally, Zhang et al. (2017) reported the isolation of flavonoid glycosides from the plant and the determination of biogenic amine and flavanone contents by Pellati et al. (2004) and Bagatela et al. (2015). Akhtar (2013) reported the use of the juice in treating irritation, diarrhoea and swelling due to mosquito bites, and is sometimes used as vermifuge when mixed with oil and also incorporated into weight management diet. Medicinal plants play important role in the management of diseases due to their rich antioxidants, phytochemicals, easy accessibility and affordability by the populace (Alaebo et al., 2020; Onochie et al., 2020; Iloanya et al., 2021).

 

Phytopharmaceuticals have shown an outstanding role in new drug discovery (Khan and Amin, 2016; Khan et al., 2016) in the last years and a large population globally are getting therapeutic benefits from them (Marya et al., 2018) both in the crude form as well as pure chemical entities. Kuding leaves known to be a medicinal plant has been observed in some studies to affect bodyweight and packed cell volume (Alisa et al., 2015; Ezeigwe et al., 2018). Some herbs may be beneficial in bodyweight reduction but may have adverse effect on the blood parameters. There is always needed to investigate the effect of medicinal plants on biochemical and haematological parameters before recommending them to those that need them for bodyweight reduction. One of our earlier studies investigated the effect of C. aurantifolia fruit juice on biochemical parameters of Wistar rats (Ezeigwe et al., 2022). There is paucity of information on the effects of C. aurantifolia on the blood parameters of Wistar rats, thus, the present study aimed to evaluate the effect of C. aurantifolia juice on bodyweight and hematological indices of rats.

Sample collection and identification

 

The C. aurantifolia fruits were purchased from Eke Market, Awka, Awka South Local Government Area, Anambra State, Nigeria. The sample was identified by a taxonomist in the Department of Botany, Nnamdi Azikiwe University, Awka. The voucher number as deposited in the herbarium of Nnamdi Azikiwe University, Awka is NAUH 196^A.

 

Test animals

 

A total of 20 Wistar albino rats of both sex weighing between 120-130 g were purchased from Chris Experimental Animal Farm and Research Laboratory, Awka, Anambra State and used for the experiment. They were maintained and housed in cages under standard environmental conditions (27±3°C, 12 h light/dark cycle) in the Department of Applied Biochemistry Laboratory, Nnamdi Azikiwe University, Awka. They were allowed to acclimatize with the environment for one week before use. The animals were fed vital grower’s mash pellets purchased from Vital Feed Distributor at Awka, Anambra state and fed ad libitum. At the end of the one-week acclimatization period, the animals were weighed, grouped, and labeled.

 

Study design

 

A total of 20 rats of both sex weighing between 120 and 130 g were randomized into 4 groups of five rats each and used. The C. aurantifolia fruit was freshly squeezed and the juice was administered to the test groups for a period of three months.

 

Group A: Normal Control

Group B: 2 ml/kg bodyweight of C. aurantifolia fruit juice

Group C: 4 ml/kg bodyweight of C. aurantifolia fruit juice

Group D: 8 ml/kg bodyweight of C. aurantifolia fruit juice

 

Determination of bodyweight

 

The weight of the experimental subjects was checked using an electronic weighing scale. The bodyweights of the rats were monitored before, during, and after the experiment to know whether the continuous administration of lime juice caused a noticeable increase or decrease in bodyweight. Percentage bodyweight was also calculated using the formular below:

 

 

Haematological analysis

 

Haematological parameters were determined using automated haematology analyzer (Mindray-BC-5300). The haematological parameters that were analysed include haemoglobin (HGB), packed cell volume (PCV), red blood cells (RBC), platelets (PLT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MON), eosinophils (EOS), basophils (BAS).

 

Statistical analysis of results

 

Data obtained from the experiments were analyzed using the Statistical Package for Social Sciences software for windows version 23 (SPSS Inc., Chicago, Illinois, USA). All the data collected were expressed as Mean ± SEM. Statistical analysis of the results obtained were performed by using ANOVA Tests to determine if significant difference exists between the mean of the test and control groups. The limit of significance was set at p<0.05.

 

Haematological analysis

 

Table 3 showed the Hemoglobin (HGB) concentration of rats administered with C. aurantifolia. HGB of the subjects from Group A (Normal control) and Group B decreased from their baselines after 1st month, followed by a further decrease in the 2nd month but increased in the 3rd month. Group C showed a decrease from its baseline after the 1st month, peaked in the 2nd month and even higher in the3rd month. Group D followed the same pattern as in Group C with a decrease in the 3^rd month. Table 4 showed the Hematocrit concentration of rats administered with C. aurantifolia. HCT of the subject from Group A (Normal control) decreased from its baseline after 1^st month, followed by a further decrease in the 2^nd month but increased in the 3^rd month. HCT of the subject from Group B increased from its baseline after 1^st month, followed by a sudden decrease in the 2^nd month but shot up in the 3^rd month. Group C increased from its baseline after 1^st month, which steadily increased in the 2^nd month, up till the 3^rd month. Group D showed a decreased and increased pattern after 1^st month, 2^nd month and 3^rd month, respectively.

 

 

MCV results were shown in Table 5. Although there was a decrease in all the groups in the 3^rd month of administration from their baselines, no significant differences (p>0.05) were observed throughout the three months of administration with Lime Juice. MCH results were shown in Table 6. There were no significant differences (p>0.05) observed throughout the three months of administration with lime Juice as the same level was maintained in all the groups. The results in Table 7 showed the MCHC of rats administered with different doses of C. aurantifolia fruit juice. There were no significant differences (p>0.05) observed throughout the three months of administration with lime Juice.

 

 

Red blood cell count (RBC) of rats administered with C. aurantifolia is shown in Table 8. RBC of the subject from Group A (Normal control) decreased from its baseline after 1^st month, followed by a further decrease in the 2^nd month but peaked in the 3^rd month. Group B showed a decreased and increased pattern after 1^st month, 2^nd month and 3^rd month, respectively. Group C and D decreased from their baselines after 1^st month, which steadily increased in the 2^nd month, up till the 3^rd month.

 

 

Platelet count (PLT) of rats administered with C. aurantifolia is shown in Table 9. PLT of the subject from Group A (Normal control) decreased from its baseline after 1^st month, followed by a sharp increase in the 2^nd month up till the 3^rd month. Group B increased from its baseline after 1^st month, which steadily increased in the 2^nd month, up till the 3^rd month. Group C increased from its baseline after 1^st month, followed by a sharp decline in the 2^nd month, which further decreased in the 3^rd month. Group D showed a decreased and increased pattern after 1^st month, 2^nd month and 3^rd month, respectively. All platelet counts in both Normal control and experimented groups are within the normal range (NV=500-1300 × 10⁹/L) according to Barrientos et al. (2020).

 

 

White blood cell count (WBC) of rats administered with C. aurantifolia is shown in Table 10. WBC of the subject from Group A (Normal control) decreased from its baseline after 1^st month, followed by a further decrease in the 2^nd month and peaked in the 3^rd month. Group B displayed an increased and decreased pattern after 1^st month, 2^nd month and 3^rd month, respectively. Group C decreased from its baseline after 1^st month, followed by a sudden increase in the 2^nd month, up till the 3^rd month. Group D increased steadily from its baseline after the 1^st month and continued till the 3^rd month. All white blood cell counts in both Normal control and experimented groups are within the normal range (NV=6-18 × 10⁹/L) according to Barrientos et al. (2020).

 

 

Neutrophils of rats administered with C. aurantifolia were shown in Table 11. Group A (Normal control) and Group B displayed a decreased and increased pattern after the 1^st month, 2^nd month and 3^rd month. Groups C and D increased from their baselines in the 1^st month, followed by a further decrease in the 2^nd month and peaked in the 3^rd month.

 

 

Lymphocytes of rats administered with C. aurantifolia were shown in Table 12. Group A (Normal control) and Group B displayed an increased and decreased pattern after the 1^st month, 2^nd month and 3^rd month. Group C and Group D displayed a decreased and increased pattern after the 1^st month, 2^nd month and 3^rd month.

 

 

Monocytes of rats administered with C. aurantifolia were shown in Table 13. Group A (Normal control) revealed an absence of the cell throughout the testing time. Group B revealed an absence of the cell in its baseline, 1^st, and 3^rd month except in the 2^nd month. Group C revealed a decrease from its baseline after 1^st month, which peaked in the 2^nd month, with an absence of the cell in the 3^rd month. Group D revealed the presence of the cell after the 3^rd which increased significantly (p<0.05) from its baseline.

 

 

Eosinophils of rats administered with C. aurantifolia were shown in Table 14. A decreased pattern of the cell and an increased pattern of the cell throughout the experiment were observed in Group A (Normal control) and Group D, respectively. Group B decreased from its baseline after 1^st month, followed by a further decrease in the 2^nd month, and peaked in the 3^rd month. Group C showed a decreased and increased pattern after the 1^st month, 2^nd month and 3^rd month, respectively. There were no observed changes in the basophyls levels of the different groups of rats starting from the normal control to the experimental groups (Table 15). Table 15 revealed the absence of basophyls in rats adiministered with different doses of C. aurantifolia fruit juice.

 

 

A reduction in bodyweight and food consumption was observed following the intake of C. aurantifolia fruit juice in rats after 12 weeks (Table 1). However, this decrease was only observed in the group of rats administered 4 and 8 ml/kg bodyweight of C. aurantifolia after 12 weeks of administration. This entails that lime juice can be a good regimen in the management of bodyweight at certain doses. However, some medicinal plants such as Justicia secunda Vahl leaves (Onochie et al., 2020) do not have the capacity to cause observable changes in the bodyweight of experimental animals. It is possible that phytoestrogens of C. aurantifolia inhibit serotonin re-uptake and thereby increase the levels of serotonin in synaptic clefts as in the case of C. cyminum L. which was reported by (Amin and Nagy, 2009), which in turn would result in enhanced satiety. Additionally, bodyweight may be reduced by limonene through increased lipolysis by a histaminergic response and reduced appetite (Shen et al., 2005).

 

The HGB concentration results obtained and represented in Table 3 were all within the normal range (NV=11-19.2 g/dL) according to Barrientos et al. (2020), and no significant difference (p > 0.005) was recorded throughout the three months of administration with lime juice. It suggests that administration with lime juice at different dosages used does not have any negative impact on HGB concentration and could be effective in the treatment of anaemia. Blood indices (MCV, MCH, and MCHC) results as shown in Tables 5, 6 and 7 respectively showed no significant differences (p>0.05) throughout the three months of administration with lime Juice. There was no significant difference (p>0.05) observed in RBC counts of both normal control and groups administered with lime juice (Table 8), and the values were within the normal range (NV= 6.76-9.75 × 10¹²/L) according to Barrientos et al. (2020). The steady values of RBC could be attributed to the rich milieu of antioxidants that protects the RBC from any oxidative damage.

 

Platelets are small, colorless cell fragments in our blood that form clots and stop or prevent bleeding. The result in table 9 revealed a significant increase (p<0.05) in group B relative to other administered groups and Normal control (Group A) after 3^rd month of lime juice administration. The lowest value was found in group D after 3^rd month of lime juice administration. The results although either showing a decreasing or increasing patterns in the different groups administered with lime juice were all with the normal range (NV=500-1300 × 10⁹/L) according to Barrientos et al. (2020), hence neither thrombocytosis nor thrombocytopenia can be implied.

 

Eyong et al. (2004) reported that rapid increase in WBC counts following a foreign attack on the system by pathogens gives rise to a boost in the body’s defense mechanisms as the normal physiological response. After 3^rd month of administration of lime juice, it was observed that the administered groups showed significant (p<0.05) increases relative to their baselines (Table 10). This could imply that lime juice could serve as an immune booster. The effects of the prolonged administration of lime fruit juice on the white blood cells differentials were also investigated. Following the results obtained at monthly intervals, there was no significant difference (p<0.05) in the neutrophil and lymphocyte levels of the administered groups. However, monocytes and eosinophils revealed an inconsistent increase and decrease at different doses analysed at monthly intervals (Tables 13 and 14). The white blood cell and its differentials are vital for the protection of the body against foreign invaders and help to stimulate cytokine erythropoietin which subsequently stimulates blood cell synthesis (Oladejo and Osukoya, 2021).

The domestic and ethnomedical use of C. aurantifolia cannot be overemphasized. The observations from this study revealed that fruit juice of C. aurantifolia not only could serve as immune booster but can also be used in bodyweight management. There should be a move from pre-clinical screening of the plant’s fruit juice to the isolation of active compounds and the actual development of useful drugs from the plant.

The authors have not declared any conflict of interests.