Modulatory Effects of Lawsonia inermis, Waltheria indica, Moringa oleifera and Nigella sativa on Haematology, Blood Pressure and Electrocardiographic Parameters of Wistar Rat Exposed to Acute Pain

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Pain is an obnoxious stimulus usually triggered by stressors that can affect homeostasis.The inability to sense pain is usually connected to abnormal fluctuations in normal body functions, leading to shortened life expectancy.
This study aimed to assess the effects of Lawsonia inermis, Waltheria indica, Moringa oleifera and Nigella sativa on hematology, electrocardiography, and blood pressure in Wistar rats experimentally exposed to acute pain.
The extracts showed non-significant increases in most hematological parameters, including Packed Cell Volume, Red Blood Cells, and White Blood Cells counts, compared to positive controls.The effects of the extracts on the electrocardiographic parameters were not significant.Although the extract relieved pain, the effect of acetic acid on the heart was not completely ameliorated.The results of this study showed high blood pressure in untreated rats, which could be linked to pain induction.Moringa oleifera and Waltheria indica exhibited minimal blood pressure-lowering effects.Waltheria indica and Lawsonia inermis showed transient blood pressure-lowering effects.
This study concluded that the extract had a modulatory effect on hematology and a lowering effect on blood pressure and electrocardiac parameters in all treatments.

INTRODUCTION
Pain is an obnoxious stimulus usually triggered by stressors that can a ect homeostasis, and the inability to sense pain is usually associated with shortened life expectancy (Swi , 2018).Internal medicine prioritizes the association between pain and changes in vital signs associated with hypertension and tachycardia (Dayoub and Jena, 2015).Reports have shown that acute pain is physiologically linked to stress responses involving increases in blood pressure, heart rate, pupil diameter, and plasma cortisol level (Rathmel and Fields, 2012).A Previous study examining the physiological response to pain in a small group of healthy patients found an increase in heart rate when individuals are exposed to noxious stimuli (Hilgard et al., 1974).e degree and interval of the stimulus in these spinal re exes causes activation of the sympathetic nervous system, which increases peripheral resistance, heart rate, and stroke volume (Silvani et al., 2016).
Medicinal plants are the primary source of drugs for the management of animal and human diseases (Aremu et al., 2022).e World Health Organization (WHO) estimates that approximately 80% of the world's population, particularly in developing countries, relies mainly on herbal plants for health care needs, primarily using the active principles in plant extracts (Aiyelero et al., 2009).ese plants have minimal side e ects and have been used to treat various diseases since ancient times (Aiyelero et al., 2009;Aremu et al., 2022).Kang-Hua Chen et al., (2012) reported the use of Moringa oleifera in the treatment of hypertension by lowering the pulmonary arterial blood pressure in monocrotaline-induced hypertensive rats.It has also been reported that the seed of Moringa oleifera shows heart-protective function in spontaneously hypertensive rats by raising diastolic cardiac function and lowering the nocturnal heart rate without altering the rat's blood pressure (Randriamboavonjy et al., 2016) the modulatory e ect of medicinal plants on hematology has been reported as shown by Waltheria indica increasing the Packed Cell Volume, Red Blood Cells, Hemoglobin Count, and Mean Corpuscular Hemoglobin in rats fed an iron-decient meal (Oladijin and Yakubu, 2005).Nigella sativa oil has been reported to increase hematocrit levels but reduce Mean Corpuscular Hemoglobin Concentration (MCHC) and Mean Corpuscular Volume (MCV) concentration (Kökdil et al., 2006) whereas Lawsonia inermis has no signi cant e ect on Packed Volume Cell (PVC), Red Blood Cells (RBC), and Hemoglobin concentrations, as reported by (Aremu et al., 2022).
Reports have shown that reduced hemoglobin levels, which are usually characterized by anemia, have been associated with bromyalgia and chronic fatigue syndrome, both of which are frequently accompanied by chronic pain (Yao et al., 2021).Additionally, by a ecting neuronal activity and tissue viability, insu cient oxygen transport to tissues as a result of low hemoglobin levels might worsen pain perception (Premont et al., 2020).Full blood counts and haematological parameters are useful, inexpensive, and widely accessible paraphernalia for the management and prognosis of patients

Plant Collection, Preparation and Authentication
e leaves of Lawsonia inermis and Moringa oleifera and the stem bark of Waltheria indica were harvested from di erent areas in Kwara State.e parts of these plants were used for this study because of their reported medicinal activities (Azeez et al., 2017;Aremu et al., 2022) the plants were authenticated at the Herbarium, Department of Botanical Sciences, University of Ilorin, Ilorin, with voucher specimen no.134289, which was deposited for reference.Nigella sativa oil (Hemani 125 mL) was obtained from a reputable store in Ilorin, Kwara State, Nigeria.e leaf samples of Lawsonia inermis, Moringa oleifera and the stem-bark of Waltheria indica were washed, dried, and crumpled into powder.Powdered particles (500g) were soaked in 99% methanol (ratio 1:3, w/v), and Waltheria indica was infused into 98% ethanol at room temperature for 48 h. is procedure was repeated twice for the second and third extraction processes.e ltrates were sieved through lter paper (Whatman No.1) and vaporized using a rotary evaporator.e dry residue was weighed and preserved in a refrigerator at 4 C until used.

Extraction of Plants Material
Experimental Design with coronary heart disease, heart failure, hypertension, arrhythmias, and stroke (Mozos, 2015).e present study aimed to assess the comparative and modulatory e ects of Lawsonia inermis, Waltheria indica, Moringa oleifera and Nigella sativa on the hematology, electrocardiography, and blood pressure of Wistar rats experimentally exposed to acute pain.
A total of 21 matured female Wistar rats (8 weeks old and non-gravid) were divided into seven groups, each consisting of three rats, and the extracts were administered through the oral route using a cannula.Each group was treated as follows: Group 1: (Negative control) was untreated and uninduced.Group 2: (Positive control) untreated induced.Group 3: Lawsonia inermis extract administered orally at 200 mg/kg daily for 14 days.Group 4: Waltheria indica extract was administered orally at 200 mg/kg daily for 14 days.Group 5: Nigella sativa oil was administered orally at 200 mg/kg daily for 14 days.Group 6: Moringa oleifera extract administered at 200 mg/kg daily for 14 days.Group 7: Diclofenac was orally administered at a dose of 10 mg/kg daily for 14 days.

Pain Induction Using Acetic Acid
Pain was induced using the writhing test.
e rats were treated with various extracts for 14 days and injected intraperitoneally with 0.6% acetic acid at 10 ml/kg to induce acute pain.Writhing activity characterized by hindlimb extension, abdominal muscle contraction, and arching of the back has been observed in animals.

Electrocardiograph and Blood Pressure Measurement
Electrocardiography was performed to assess cardiac function using an EDAN®-VE 1010 digital veterinary electrocardiographic machine, as described by (Azeez et al., 2017).Parameters such as HR (Heart Rate), P (atrial contraction), PR (delay at Atrioventricular bundle), QRS (ventricular contraction), QT (time from ventricular contraction to ventricular relaxation), and QTc (QT corrected) were measured.
e blood pressure of each rat was recorded.Electrocardiogram (ECG) and blood pressure were monitored on day 14.

Blood Collection and Analysis
At the end of the experiment, the rats were humanely euthanized (rats were anesthetized with light chloroform followed by cervical dislocation).Blood samples were collected via the intraocular route for hematology.e method adopted by Aremu et al., (2017) was used to insert a heparinized capillary tube into the ocular vein, and blood was drawn from the median canthus of the eye.1-2 ml of blood were drawn and placed in EDTA sample containers.

Statistical Analysis
Values are expressed as means ± standard deviation (mean ± SD). e di erences within the groups were compared using Dunnett's post hoc ANOVA with GraphPad Prism statistical package (San Diego, California, U.S.A; www.Graphpad.Com).

RESULTS Haematology
Table 1 shows the e ect of Rats treated with Lawsonia inermis, Waltheria indica, Moringa oleifera, and Nigella sativa on blood parameters a er 14 days of treatment following pain induction.ere were no signi cant (p>0.05)changes in any of the haematological parameters (PCV, RBC, HB, and WBC) when compared to the two controls.e ECG results of untreated rats showed a tall P, irregular RR interval, and widened T, which was higher than half of R, whereas rats treated with Lawsonia inermis presented a very short R and irregular RR interval.Waltheria indica showed tall P waves, and inversion of the P waves was observed in Nigella sativa.Moringa oleifera showed widening of P, PR, and T waves, while diclofenac showed very distressed R and Irregular RR intervals when compared to the negative control, which showed normal PQRS and T wave patterns (Figure I-VII).Table 2 shows the e ects of Lawsonia inermis, Waltheria indica, Moringa oleifera, and Nigella sativa on electrocardiographic parameters when administered orally for 14 days following the induction of pain.No signi cant (p>0.05)changes were observed in any of the treated groups when compared to the two controls.

Blood Pressure
Table 3 shows the e ects of Lawsonia inermis, Waltheria indica, Moringa oleifera, and Nigella sativa on blood pressure.
ere were no signi cant changes compared to the control group.

DISCUSSION
Reports have shown a complicated and multifaceted phenomenon linking haematological parameters and acute pain (Orsolini et al., 2020).e results obtained in this study showed that the extracts increased the values of RBC, PCV,  WBC, and other haematological parameters. is result agrees with (Günaydın and Günaydın, 2022) who reported that RBC distribution was signi cantly higher in patients exposed to subacute/chronic pain than in those exposed to acute pain. is result contradicts (Abdelgadir et al., 2010).reported a signi cant decrease in blood parameters in rats treated with an aqueous extract of Lawsonia inermis. is di erence could be due to variations in the doses administered and intensity of pain. is result also disagrees with (Ajugwo et al., 2017) who reported a signi cant increase in RBC, PCV, Hb, and WBC counts in rats treated with Moringa oleifera.is result agrees with (Basiru and Olayemi, 2014) who reported no signi cant changes in the haematological parameters of rats treated with 200 mg/kg of Waltheria indica.Reports have shown that Nigella sativa seed oil increases the total number of RBCs, hemoglobin levels, and hematocrit percentage (Fadeifard et al., 2018).e results obtained from this study followed the reports of (Liang et al., 2021) who showed that Nigella sativa has a potential stimulatory e ect on erythropoiesis.ese ndings also corroborate the reports that Nigella sativa enhances WBC function by increasing its phagocytosis and natural killer cell activities (Majdalawieh and Fayyad, 2015).Electrocardiography (ECG) is useful for indicating the perception of pain among all the physiological signals captured by wearable devices.
e ECG results from this study showed irregularities in heart function in the form of arrhythmias and myocardial infarction. is could be associated with the presence of cardiac glycosides in the extracts, which have positive ionotropic e ects and predispose to cardiotoxicity (Biobaku et al., 2021).
e e ects of the extracts on the electrocardiographic parameters were not signi cant.However, it could be deduced that acetic acid has strong negative e ects on cardiovascular function (Bao et al., 2017;Kalishwaralal et al., 2018) because the electrical conduction systems of the heart were a ected in all treated and untreated rats.Although the extract relieved pain, the e ect of acetic acid on the heart was not ameliorated.Reports have shown that pain activates the sympathetic nervous system, resulting in increased blood pressure, leading to increased stimulation of baroreceptors that consecutively activate the descending inhibitory pathways of pain, thereby restoring initial homeostasis.e relationship between pain and hypertension is potentially of great pathophysiological and clinical interest but is poorly understood (Saccò et al., 2013).e results of this study showed high blood pressure in untreated rats, which may be linked to acetic acid-induced pain.Moringa oleifera and Waltheria indica exhibited minimal blood pressure-lowering e ects.
Medicinal plants o er a diverse range of mechanisms through which they can e ectively lower blood pressure via vasodilation, diuretic e ects, ACE inhibition, and antioxidant and calcium blockade (Verma et al., 2021).Moringa oleifera and LLawsonia inermis leaves have been reported to inhibit angiotensin converting enzyme (ACE), and this e ect is attributable to various phytochemical compounds present in the extract (Mounika et al., 2022). is outcome conforms to the report of (Aekthammarat et al., 2019) stating that M. oleifera possesses traditional antihypertensive activities by alleviating vascular dysfunction and oxidative stress (Aekthammarat et al., 2019).
Reports have shown that supplementation of N. sativa in hypertensive patients signi cantly reduced both systolic and diastolic blood pressure compared with placebo (Rashidmayvan et al., 2022).
e results of this study also showed a decrease in blood pressure, which could be attributed to the active compounds found in N. sativa.Moreover, its antioxidant and anti-in ammatory properties may contribute signi cantly to its potential bene ts in lowering blood pressure (Maideen, 2020).However, reports on the antihypertensive e ect of W. indica and L. inermis are still scarce, even though there is a transient blood pressure-lowering e ect, as seen in this study.
Negative control (uninduced) showing a normal PQRS and T waves pattern.B. Positive control (induced but not treated) showing very tall P, irregular RR interval, and widened T which is higher than half of R suggestive of myocardial infarction.C. Lawsonia inermis 200 mg/kg indicating atrial and ventricular brillation, very short R, and irregular RR interval.D. Waltheria indica 200 mg/kg showing tall P waves which is indicative of inconsistent SA stimulation.E. Nigella sativa 200 mg/kg showing Inversion of the P waves.F. Moringa oleifera 200 mg/kg indicating ectopic stimulation and widening of P, PR, and T waves.G. Diclofenac 10 mg/kg showing atrial brillation and ventricular brillation, very distressed R and Irregular RR interval.

Note:
All values are expressed as Mean ± SD Note: All values are expressed as Mean ± SD

Table 2 .
E ect of Lawsonia inermis, Waltheria indica, Moringa oleifera, and Nigella sativa on the Electrocardiographic indices of pain induced Wistar rats

Table 3 .
E ect of Lawsonia inermis, Waltheria indica, Moringa oleifera, and Nigella sativa on the Electrocardiographic indices of pain induced Wistar rats