MATERIALS AND METHODS

Collection and authentication of plant: Acacia tortilis plant was plucked from Al-Abdiyah region, Makkah, KSA, in December, 2016 and verified by Dr. Ibrahim Abd-Elhady, Associate Professor, Department of Pharmacognosy, Faculty of Pharmacy, Umm Al-Qura University. A voucher specimen number PHG128 was deposited. Acacia tortilis leaves were separated and washed by using tap water.

Preparation of Acacia tortilis leave decoction (ATLD): For the preparation of ATLD, leaves (100 g) were grinded and macerated in distilled water for 1 h after that boiled in same distilled water as reported by Chinese medicinal herbs²⁶. At the end a bright green color extract was obtained and filtered. This filtrate was used for intravenous administration in rats.

Oral toxicity test (acute): Acute oral toxicity test was performed in healthy rats and mice, following OECD (Organization of Economic Cooperation and Development) Guideline 423. In rats and mice (3 each), 2000 mg kg^–1 (maximum dose) of ATLD was administered orally. Animals were monitored for the next 48 h²⁷ for any sign of toxicity and mortality.

Experimental animals: In the current study ten albino Sprague-dawley rats (200-250 g: either sex) were used for the recording of data. The rats were fed on a standard pellet diet and tap water ad libitum. The experimental protocol followed was permitted by the Animal Ethics Committee, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia (Ethical approval number HAPO-02-K-012-2015-11-125).

Measurement of normal blood pressure of rats (In vivo activity): Sprague-Dawley rats (normotensive: 200-250 g) were anaesthetized with thiopental sodium (40 mg kg^–1, i.p)²⁸. Once animal was anaesthetized, it was positioned on dissection bench (dorsal decubitus), under an overhead lamp to maintain body temperature. Afterward the trachea of a rat was intubated with polyethylene cannula to promote extempore respiration. A mid trachea longitudinal incision (20 mm) exposed the right jugular vein and both common carotid arteries. The right jugular vein was catheterized with a scalp vein set (BROMED) filled with a saline solution to administer drugs. The left carotid artery was catheterized and reusable blood pressure transducer (Millar Micro tip Pressure volume catheter SPR 838, Powerlab, AD Instruments) was inserted. Transducer was used for monitoring the systolic and diastolic pressure (mm Hg), as well as heart rate (beats min^–1, bpm) continuously during the experiment and calculated internally in the Powerlab using the Chart software (labChart Pro v8.1.5, Powerlab AD instrument). After surgery, rat and all parameters were equilibrated for at least 30 min before starting the tests. For control reading saline was administered (0.20-0.25 mL) through a scalp vein set introduced into the external jugular vein. After normal Data (Control), Ach (10‾⁶ M) and extract were administered one by one and their activity was recorded. These records were used for the calculation of arterial blood pressure (Systole and Diastole: mmHg), cardiac cycles (s) and Heart rate (BPM).

Statistical analysis: A software Minitab 17 was used for statistical analysis. A power analysis suggested that the sample size be set at N = 3 to achieve a power of 0.8 at an alpha of 0.05 calculated by G*Power²⁹. However, in the present study number of animals per group were N = 10. All hypotheses were tested using one way analysis of variance (ANOVA) tests. Alpha was set at (0.05). Significant effects were subjected to Tukey's multiple-range tests for comparison of treatment means with a control.

RESULTS

The blood pressure recording and parameters before (normal) and after the administration of ATLD are presented in Table 1. ATLD doses (10, 30 mg kg^–1) lowered the systolic, diastolic and the mean arterial pressure of normotensive rats significantly (p<0.05) and the effect of ATLD was more pronounced as compared to Ach in normotensive rats. Fall in Blood pressure was dose dependent. Greater effect was observed at 30 mg kg^–1 with reductions of 17.4±2.9 and 15.2±1.8% in systolic and diastolic blood pressures respectively. Decreases in various blood pressure parameters were statistically significant (p<0.005).

Effect of Acacia on heart rate and cardiac cycle: There was a significant decrease in the heart rate in rats following the administration of the Acacia extract. Dose of 10 mg kg^–1 caused significant reduction in heart rate (p<0.01), initially 361.72±26.13 beats/min, to 238.91±65.5 (Table 1). Intravenous administration of ATLD at 10 and 30 mg kg^–1 dose led to decrease in heart rate at (34 and 42%, respectively, p = 0.0001) and increase in the duration of cardiac cycle significantly (p<0.001) as shown in Table 1.

Table 1:	Effect of Acacia tortilis on various blood pressure parameters
n = 10, Each value represents the Mean±STDEV

Fig. 1:	Tracing from a typical experiment showing the ATLD for its effects on blood pressure (BP) in the anesthetized rats

Toxicological studies in mice and rats: Toxicological studies of ATLD were carried out in mice and rats. Administration of ATLD (P.O. 2000 mg kg^–1) did not show any significant change in physical behavior of mice and rats. None of the groups showed any significant change.

DISCUSSION

This present study is related to in vivo pharmacodynamics cardiovascular efficacy of KSA Acacia tortilis using rat model. Acacia tortilis have scientifically been screened for hypotensive activity. The results obtained from the acute oral toxicity study showed that ATLD demonstrated high safety margin when the animals tolerated ATLD up to 2000 mg kg^–1 b.wt., orally. Previously Mukhtar et al.³⁰ has also reported that the margin of ethanolic extract of Acacia tortilis leaves safety up to 2000 mg kg^–1.

In the present study the i.v. administration of ATLD caused a transient fall in blood pressure parameters (Systolic, Diastolic, MABP) and heart rate significantly as shown in Fig. 1. These hypotensive effects were brief returning to normal within 5 min. The significant hypotensive effect after ATLD administration depends on the Acacia tortilis components ratios. Previously another species (Acacia catechu) was reported to produce a dose dependent decrease in blood pressure in both anaesthetized dogs and rats³¹. Acacia Nilotica³² and Acacia Phenol³³ were also considered to exhibit anti-hypertensive effects.

It was suggested that serum sodium was dropped after the administration with gum arabic³³. This might be the cause of hypotensive activity and drop of blood pressure. The presence of N, NDimethyltryptamine (DMT) has been confirmed previously in Acacia leaves water extract³⁴ belong to tryptamine family (psychedelic compound), tend to reduced systolic blood pressure and heart rate³⁵. DMT (inhaled or injected), the effects last a short period of time (about 5-15 min), our results are in accordance with this study that after the i.v. administration of ATLD effect persist for only 5 min³⁶.

Crowch and Okello³⁷ demonstrated acetylcholinesterase antagonist activity of Acacia nilotica and Acacia auriculiformis in experimental models^37,38. Inhibition of acetylcholinesterase amplifies and prolongs the cholinergic response. Acetylcholine binds and stimulates the M2 receptor in heart cause bradycardia (negative chronotropic effect)³⁹. However the stimulation of M3 receptors causes vasodilation and decreased peripheral resistance which lowers blood pressure⁴⁰.

CONCLUSION ANS FUTURE RECOMMENDATION

Conclusively, ATLD exhibits profound hypotensive and heart rate lowering (Bradycardia) activities. It is proposed that ATLD might causes vasodilation and decreased peripheral resistance which lowers blood pressure. However, further studies are needed to be carried out for understanding the mechanism of action of ATLD.

SIGNIFICANCE STATEMENTS

In present study oral administration of ATLD (2000 mg kg^–1) was safe and did not cause any mortality or side effect in rats.

In this research work, an intravenous administration of different doses of ATLD produced hypotensive activity significantly comparable to Ach.

ATLD not only produced profound hypotensive effect in experimental rats, but also appeared to have a heart rate lowering (Bradycardia) activities.

It was a preliminary study, due to increase in the rate of hypertension in Saudi Arabia. This study will open the doors for further research and will provide an alternative low cost therapy for hypertensive patients.

For future it is recommended to carry out further studies on understanding the mechanism of action of ATLD.

ACKNOWLEDGMENT

The authors are thankful to the institute Deanship of Scientific Research for providing financial support through grant no.15-MED-3-1-0064, Umm Al Qura University, Makkah, KSA.