Jasminum sambac: A Potential Candidate for Drug Development to Cure Cardiovascular Ailments

Jasminum sambac (L.) is a South Asian folkloric medicinal plant that has traditionally been used to treat cardiovascular problems. The current investigation was meticulously organized to explore the pharmacological foundation for the medicinal uses of J. sambac pertaining to cardiovascular ailments and to investigate the core mechanisms. Mechanistic investigation revealed that crude leaf extract of J. sambac produced ex-vivo vasorelaxant effects in endotheliumintact aorta ring preparation and hypotensive effect was recorded via pressure and force transducers coupled to the Power Lab Data Acquisition System. Moreover; J. sambac showed cardioprotective effects against adrenaline -induced left ventricular hypertrophy in rabbits observed hemodynamic. CK-MB, LDH, troponin, CRP, ALT, AST, ALP levels were shown to be lower in the myocardial infarction model, as were necrosis, oedema, and inflammatory cell recruitment in comparison to control. J. sambac has shown good antioxidant potential as well as prolonged the noradrenaline induced platelet adhesion. The vasorelaxant and cardioprotective effects in both in vivo and ex vivo experiments, which are enabled by activation of muscarinic receptor and/or releasing the nitric oxide and by reducing the adrenaline, induced oxidative stress, justifying its usage in cardiovascular disorders.


Introduction
Myocardial infarction is the leading cause of death in humans in many developed countries [1]. Because of the changing lifestyle patterns in many developing countries such as Pakistan, India, Bangladesh, and Afghanistan are rapidly catching up with this epidemic at an alarming rate [1]. Though synthetic medicines are very useful in the treatment of cardiovascular ailments their use is restricted due to their harmful effects [2,3]. Myocardial infarction is a serious ischemic situation in which extreme necrosis of myocardial tissue takes place. It happens because of the imbalance between blood supply and oxygen requirement of the cardiac muscles [4,5].
Adrenaline (ADR) is a catecholamine synthesized in the adrenal medulla. Adrenaline is a non-selective agonist of all adrenergic receptors consisting of alpha, beta and their subtypes primarily located on the cardiovascular system [6]. It causes myocardial infarction above the therapeutic dose (2 mg/kg body weight) [6]. This method is applied to induce myocardial infarction in the experimental animal to evaluate the cardioprotective effect of test drugs. It causes myocardial infarction because of lipid peroxidation that leads to the depletion of cellular antioxidants. It causes the overproduction of nitrosative derivatives oxidative stress occurs due to the overproduction of ROS [7]. It increases the Ca ++ influx by opening the calcium channels of cardiomyocytes, increases oxidative stress by increasing the workload [8]. In a higher dose, it causes aortic and coronary vasoconstriction. It also promotes blood coagulation by increasing the level of clotting factor VIII, fibrinolysis as well as platelet count [9].
The importance of herbal medicines in the treatment of various ailments is invaluable globally. Herbal medicines are continuously providing important therapeutic agents not only in traditional but also in modern medicines and also because they are less toxic than synthetic medicines [3,5].

Priliminary Phytochemical Screening
Preliminary phytochemical screening of J. sambac (Leaf) revealed the presence of saponins, coumarins, phenols, and flavonoids while alkaloids, tannins and anthraquinones were not detected during the phytochemical investigation (Table 1).

HPLC Analysis
The analysis of High Power Liquid Chromatography (HPLC) revealed so many phytoconstituents in varing concentrations, in which the most prominent phytoconstituents are rutin and isoquercetin ( Figure 1).

DPPH Assay
In DPPH assay, the inhibitory concentration shown by the hydroalcoholic leaf extract of J. sambac was 120 µg/mL in comparison with ascorbic acid (Figure 2).

Acute Myocardial Infarction Study
ADR significantly increased the level of cardiac markers, CK-MB, LDH, troponin, CRP, ALT, AST, ALP (p < 0.05) concerning control. ADR group rabbits (group 2) had severe elevated cardiac markers, while the groups treated with the different doses of J. sambac showed resistance against the cardiac damage caused by the ADR in a dose-dependent fashion. In comparison, three groups receiving the leaf extract of J. sambac showed that the average amount of CK-MB, LDH, troponin, CRP, ALT, AST, ALP were reduced (p < 0.05) compared to those of the ADR group (Figures 3 and 4). and CK-MB (c) against ADR-induced myocardial infarction in rabbits. One-way ANOVA was performed for the statistical analysis, comparisons among different groups was carried out by Dunnett's multiple comparison test; ** p < 0.005 and *** p < 0.0001 (n = 5). Effect on Heart to Body Weight, and Weight of Heart to Tail Length Ratios ADR significantly increased the ratios as compared to the control group. All groups treated with J. sambac were found to reduce the abovementioned ratios less than the ADR treated group ( Figure 5).

Figure 5.
Cardioprotective effect ofdifferent doses of J. sambac on (a) cardiac weight to body weight ratio and (b) cardiac weight to tail length ratio in ADR-induced cardiac hypertrophied rabbits. One-way ANOVA was performed for the statistical analysis, comparisons among different groups was carried out by Dunnett's multiple comparison test; ** p < 0.005 and *** p < 0.0001 (n = 5).

Histopathology
Analysis of segments of heart tissues in the ADR-induced group showed a remarkable change in the cardiac cell structure. In ADR administrated group histological variations for example mononucleate cellular infiltration, interstitial oedema, disintegration and tear of muscular fibers, vacuolar disintegration, distention of capillaries, mottled staining, haemorrhage, and obstruction of the myocardium were observed. Severe necrotic lesions were noticed in all the rabbits while in J. sambac treated groups less myocardial deterioration was observed ( Figure 6).

Isolated Aortic Tissue Preparation and Vasorelaxant Activity
Phenylephrine (1 µM) caused a contraction in endothelium intact and denuded rabbit aorta. J. samac relaxed completely the endothelium intact aorta at a dose of 3 mg/mL, while at a similar dose, in endothelium-denuded aortic strip no vasodilation was recorded ( Figure 7a). Noradrenaline (10 µM) caused vasoconstriction in endothelium intact and denuded rabbit aorta, but J. sambac produced partial vasodilation in endothelium intact aorta as compared to PE induced contraction at a similar dose while no vasodilatory effect was observed in endothelium-denuded aorta similar to PE induced contractions ( Figure 7b). In L-nitro-arginine (1 × 10 −4 M) and atropine (1 × 10 −6 M) pretreated aortic strips, the vasodilator effect of J. sambac was disappeared at a similar dose (Figure 7c,d).

Isolated Aortic Tissue Preparation and Vasorelaxant Activity
Phenylephrine (1 μM) caused a contraction in endothelium intact and denuded rabbit aorta. J. samacrelaxed completely the endothelium intact aorta at a dose of 3 mg/mL, while at a similar dose, in endothelium-denuded aortic strip no vasodilation was recorded (Figure 7a). Noradrenaline (10 µ M) caused vasoconstriction in endothelium intact and denuded rabbit aorta, but J. sambac produced partial vasodilation in endothelium intact aorta as compared to PE induced contraction at a similar dose while no vasodilatory effect was observed in endothelium-denuded aorta similar to PE induced contractions (Figure 7b). In L-nitro-arginine (1 × 10 −4 M) and atropine (1 × 10 −6 M) pretreated aortic strips, the vasodilator effect of J. sambac was disappeared at a similar dose (Figure 7c,d).

Effect on Adrenaline Induced Platelet Activation and Aggregation
ADR (2 µ M) addition to the suspension of washed human platelets produced a significant decline in the optical density at 600 nm, indicating the aggregation of platelets. The aggregatory effect was recorded at 37 °C. J. sambac (100, 200 and 300 µ g/mL) dramatically inhibited the platelet aggregation ( Figure 8) in a dose dependent manner.

Effect on Adrenaline Induced Platelet Activation and Aggregation
ADR (2 µM) addition to the suspension of washed human platelets produced a significant decline in the optical density at 600 nm, indicating the aggregation of platelets. The aggregatory effect was recorded at 37 • C. J. sambac (100, 200 and 300 µg/mL) dramatically inhibited the platelet aggregation ( Figure 8) in a dose dependent manner.

Acute Oral Toxicity Dose Test
No mortality or morbidity was observed at any dose in acute oral toxicity dose test.

Discussion
As flowers and leaves generally have different phytochemical compositions, the majority of the published data is available on J. sambac flower and its essential oils biological activities [10]. ADR significantly increased the level of cardiac markers, CK-MB, LDH, troponin, CRP, ALT, AST, ALP (p < 0.001). In comparison, three groups receiving the leaf extract of J. sambac showed that the average amount of CK-MB, LDH, troponin, CRP, ALT, AST, ALP were reduced compared to those of the intoxicated group which advocates its cardioprotective potential (Figure 2). It was reported that the presence of high amounts of flavonoids in plants was responsible for their cardioprotective potentials [11,13] while, J. sambac leaf extract was found rich in flavonoids content (Table 1), [11]. Thus, it is logical to believe that the cardioprotective effect of J. sambac in rabbits might also depend on the presence of these flavonoids.
In the current study, the use of J. sambac hydroalcoholic leaf extract resulted in a significant reduction of CRP in groups receiving the extracts, which may be ascorbic acidor flavonoid-based antioxidant effect of J. sambac (Figure 2) complimenting the previous antioxidant studies [5,14]. Saponins are believed to lower the level of lipid peroxidation products in a dose-dependent manner [15,16]. The results of this study add to the body of evidence supporting previously published studies on saponins or saponin-containing plant extracts' cardioprotective effects in animal models [17,18]. Similarly J. sambac found richin saponins during phytochemcial evaluation (Table 1).
Early study [19] reported significant LPO inhibitory effect of hydroalcoholic leaf extract of J. sambac which further strengthens its claim as a cardioprotective agent as LPO is one of the major contributory factors in ADR induced MI [20].
Histopathology of heart tissues in the ADR-induced group showed remarkable damage in cardiac cell architecture for example; mononucleate cellular infiltration, interstitial oedema, disintegration and tear of muscular fibers, vacuolar disintegration, and distention of capillaries, mottled staining, haemorrhage, and obstruction of the myocardium were observed. Severe necrotic lesions were noticed in all the rabbits' cardiac tissues but in J. sambac treated groups less myocardial deterioration was observed ( Figure 6) which complements the results obtained from the biochemical investigation (Figures 3 and 4). Less inflammatory cells were observed as compared with ADR intoxicated group which is evidence to its cardioprotective effect as reported in previous studies [21].
ADR increases the Ca ++ influx by opening the calcium channels of cardiomyocytes, increases oxidative stress by increasing the workload [6]. A higher dose (2 mg/kg body weight), causes aortic and coronary vasoconstriction [6]. PE (1 µM) and noradrenaline (10 µM) causes vasoconstriction and increases the mean blood pressure primarily by stimulating the alpha-adrenergic receptor and/or opening of voltage-gated L-type calcium channels [22] which in turn increases oxidative stress (Figure 7). The vasodilator effect of J. sambac may depend on muscarinic receptor activation or release of cellular vasodilator nitric oxide because the vasodilatory effect was endothelium dependent and completely blocked when pretreated with atropine (1 × 10 −6 M) and L-NA (1 × 10 −4 M) like endothelium-denuded aorta (Figure 7). While partial vasodilator effect was observed against noradrenaline (10 µM)-induced aortic contractions, suggests the involvement of multiple pathways in its vasodilator effect [23]. Flavonoids-based vasodilator effect was reported in the plants earlier [24,25]. J. sambac found rich in flavonoid content (Table 1, Figure 1). Antithrombotic and anticoagulant drugs are well known source of cardioprotection in modern-day cardiology and ADR is a well-established procoagulant [6]. J. sambac significantly reduced the platelet aggregatory effect of ADR [ Figure 8] which adds another colour to its cardioprotective potential.

Plant Materials
J. sambac had been collected fresh from Multan. It was authenticated by an expert botanist at the Institute of Pure and Applied Biology, BZ, University, Multan. The voucher specimen (R.R. Stewart, F.W. Pak. 549) has been placed for further reference.

Extract Preparation
The fresh plant was subjected to shade dry. All the foreign adulterants and vegetative waste were eliminated through manual picking before grinding the leaf part of the plant into a coarse powder with the assistance of a special herbal grinder. The crushed plant powder is stored in airtight jars waiting for extraction. The powder attained from only one batch was used in the experiment. J. sambac (250 g) leaf powder was soaking in a hydroalcoholic solvent (70:30 v/v) for 9 days in 2.5 L amber coloured air-tight jars. Then it was filtered and the filtrate was evaporated at 37 • C under reduced pressure on a rotatory evaporator to obtain a thick paste-like consistency [26]. At the end %age yield of the crude extract was calculated using this formula; % age yield = Theoretical yield (gm)/Actual yield (gm) × 100 (1)

Animals
Male rabbits weighing 1-1.5 kg had been obtained from the animal house of the Faculty of Pharmacy, The Islamia University of Bahawalpur. They had been nourished with standard commercially available food and tap water ad libitum. The temperature was maintained at 25 • C. The experiments were performed according to the rules of the National Institute of Health [27] and approved by the Ethical Committee of the Department of Pharmacology, The Islamia University of Bahawalpur, Pakistan (BOS & BOF/10/6/20).

Chemicals
Adrenaline, methanol, phenylephrine, and potassium chloride were purchased from Sigma-Aldrich, St. Louis, MO, USA. Verapamil was purchased from Searle Lahore, PB, Pakistan (PVT.) Ltd. (Lahore, Pakistan) AST, ALP, ALT, LDH, CRP Kits obtained from GM Chemicals and Diagnostics, Multan, PB, Pakistan. All other chemicals utilized the experiment were of highest purity and of reagent analytical grade.

Preliminary Phytochemical Evaluation
Phytochemical evaluation was done for the validation of various phytochemical classes (alkaloids, anthraquinones, glycosides, tannins, flavonoids and saponins) in the hydroalcoholic extract of J. sambac by using the standard protocol [28].

HPLC Analysis
Phytochemical compounds were separated by using the Agilent HPLC (Santa Clara, CA, USA) sequence 1100 system provided with autosampler, UV/vis detector, quaternary pump, 5 um, 250 mm × 4.6 mm i.d. and C18 reversed-phase column (Thermo Electron Corporation, Waltham, MA, USA) connect to HPChemStation software [29]. An (acetic acidwater, 2:98, v/v) and B (water-acetic acid, 2:98, v/v) were the solvents used to create the mobile phase (methanol). The following elution scenarios were used: 0-2 min, 5 percent where A is the area showed in mAu and C is the concentration showed in g/mL. The correlation coefficient (R2) was 0.9999 for both standardization curvatures. All the tests were performed in triplicate to have uniform results. HPLC chromatograms of the extract attained for two variations studied are shown in Figure 1.

Acute Oral Toxicity Dose Test
The acute oral toxicity of the extract was evaluated in 12 rabbits. They were divided into 3 groups and each group contained 4 rabbits and they were kept on fasting for 24 h and dosed in the following manner 500, 1000 and 2000 mg/kg body weight. After the dosing, the rabbits had been noticed for 14 days for lethargy, jerkiness, and death [30].

Determination of DPPH Assay
DPPH assay was conducted as described by using the previously described method [31]. In short, different concentrations of J. sambac extract (4 mL) was added to DPPH solution and prepared up to 5 mL with methanol, incubated for 40 min in dark. The spectrophotometer was used to measure the absorbance of the incubated solution at 517 nm. All of the tests were carried out in triplicate, and the percent inhibition was measured in ascorbic acid equivalents. The DPPH scavenging effect is calculated as:

Acute Myocardial Infarction Study
The animals had been divided into 4 major groups each group contained 6 rabbits. Group-1 rabbit administrated ADR 2 mg/kg body weight, subcutaneously at 24 h of a gap for two successive days. Group-2 rabbits were pre-treated with 100 mg/kg of extract for the period of fourteen successive days and, adrenaline 2 mg/kg, was administrated on the 14th and 15th day at a gap of 24 h. The rabbits of group-3 were pre-treated with 200 mg/kg of extract for fourteen days consecutively and on the 14th and 15th-day adrenaline 2 mg/kg was administrated subcutaneously at a gap of 24 h. Group-4 rabbits were pre-treated with extract in an amount of 300 mg/kg for fourteen days successively and on the 14th and 15th-day, ADR 2 mg/kg was inoculated subcutaneously at an interval of 24 h while J. sambac doses were administered via oral gavages [32]. The three doses were selected based on previously reported data on the hepatoprotective potential of the J. sambac [33]. On 16th day, rabbits were anaesthetized, and the blood samples of rabbits taken from the marginal ear vein of the rabbits to assess the biochemical parameters, including CK-MB, LDH, troponin, CRP, ALT, AST, and ALP levels in serum, were measured by commercially available Kits.
Screening of Cardiac to Body Weight and Cardiac Weight to Tail Length Ratio Cardiac weight and tail length were screened by measurement of cardiac weight to body weight help to key out cardiac weight index, the tail index was assessed by segmenting heart weight by tail length [6].

Histopathology
Rabbits had been killed under intense anaesthesia and the heart was isolated for histopathological examination, then the ventricular portion of the heart swiftly transferred to a 10% formalin solution. After that tissue had been submerged in the paraffin. A 5 µm thick segment had been cut and stained with a hematoxylin-eosin dye then mounted in the xylene [34]. Microscopic observation of the ventricular portion of the heart tissues from different groups was used to evaluate the adrenaline effect on the cardiac cells structure as well as its variation by the drug test inoculation under a compound microscope and micro-images were captured.

Isolated Aortic Tissue Preparation and Vasorelaxant Activity
Thorax cavity was opened, and carefully aorta was dissected out after slaughtering the rabbits. Fat and connective tissues were carefully detached from the aorta in a Petri dish containing Krebs' solution. Aortic rings of 3-4 mm in length was cut and placed in tissue organ bath (10 mL) already contained the Krebs-Henseleit solution which is connected with thermocycler to maintain 37 • C and continuously bubbled with carbenogen gas (95% O 2 and 5% CO 2 ). One of the hooks was fixed to the bottom, and the other was connected to a force displacement transducer that connected to a Powerlab data acquisition system to record the isometric contractions. Kreb'ssolution was changed every 15 min to prevent the buildup of metabolites throughout the 40-min stabilization period at 1.0 g resting tension. After equilibration, the rings were preconstricted with 1 × 10 −6 M phenylephrine (PE) until the steady contractile curve (5-8 min), and the vasodilator effect of J. sambac was recorded on cumulative dosing manner [35,36].

Effect on Adrenaline Induced Platelet Activation and Aggregation
The blood samples were collected from the marginal ear vein of the rabbit and centrifuged (3000 rpm for 15 min) to get platelet-rich plasma. Samples were supplemented with ADR (2 µM). Impedance aggregometry was used to assess platelet aggregation and flow cytometry was used to evaluate platelet activation before and after supplementations [37]. Firstly, the effect of three different added concentrations of adrenaline on platelet aggregation was assessed (n = 5). Next, platelet aggregation was measured after the addition of ADR in combination with J. sambac at 100, 200 and 300 µg/mL concentrations (n = 5).

Conclusions
The hydroalcoholic leaf extract of J. sambac was seen to produce vasorelaxant/ hypotensive effect during ex-vivo study via stimulating the muscarinic receptors and/or releasing the local vasodilators NO. The cardioprotective effect of hydroalcoholic leaf extract J. sambac may be due to its variety of phytoconstituents. Pretreatment with J. sambac may refill cardiomyocytes with antioxidants, which are required for defense against the oxidative stress caused by adrenaline. However, the exact molecular mechanism of cardioprotection remains to be explored. Moreover, during phytochemical screening, tan-nins, flavonoids, and cardiac glycosides were discovered, which have a possible impact in cardiovascular disorders, particularly hypertension-induced ventricular hypertrophy and myocardial infarction. Conclusively, ex-vivo and in-vivo research revealed J. sambac's therapeutic potential in cardiovascular diseases. Acknowledgments: Authors are thankful to Qaisar Jabeen, Chairperson Department of Pharmacology for her administrative and technical support.