In vivo analgesic, antipyretic, and anti-inflammatory potential in Swiss albino mice and in vitro thrombolytic activity of hydroalcoholic extract from Litsea glutinosa leaves

Traditional knowledge regarding medicinal plants and their use by indigenous cultures is not only useful for maintenance of cultural traditions and biodiversity but also for community healthcare and drug development in the present and future. Therapies with synthetic tropical applications have many side effects and cannot be afforded by the people due to higher cost of the drug. For overcoming this problem plants growing around us are utilized without scientific validation. The use of higher plants and their extracts to treat infections is an age-old practice. Traditional medicinal practice has been known for centuries in many parts of the world. Herbal medicines are gaining interest because of their cost effective and eco-friendly attributes [1].

L. glutinosa belongs to the family Lauraceae and is a well-known evergreen species growing wild in the forest of Chittagong and Sylhet districts in Bangladesh. It is occasionally planted in most areas of the country [2]. Leaves are mucilaginous and considered for antispasmodic, emollient, and poultice. The leaves are also used in diarrhea and dysentery as well as for the treatment of wounds and bruises [2]. The leaves were reported for the treatment of the spontaneous and excessive flow of semen in young boys [3]. The leaf extract also shows antibacterial and cardiovascular activities [4]. The berries yield oil which is used by some tribal practitioners in the treatment of rheumatism. Tannin, β-sitosterol, and actinodaphnine are reported to be the common constituents of the species; and other constituents known are: Boldine, norboldine, laurotetanine, n-methyllaurotetanine, n-methylactinodaphnine, quercetin, sebiferine, litseferine etc. [5].

Thrombosis is the fundamental pathophysiological process that underlies the acute coronary disorders such as pulmonary emboli, deep vein thrombosis, strokes and heart attacks; which are the main causes of morbidity and mortality in developed countries [6]. This disease is characterized by the development of a blood clot (thrombus) in the circulatory system of the body due to the failure of homeostasis which leads to vascular blockade and while recovering causes fatal consequences, such as myocardial or cerebral infarction, as well as death [7]. Therefore, anticoagulation therapy is the basis of management, and the proper choice of thrombolytic drugs [8].

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage [9]. By acting in the CNS or on the peripheral pain mechanism, analgesic compounds selectively relieves pain without significant alteration of consciousness. Actually analgesics are applied when the noxious stimulus cannot be removed or as adjuvant to more etiological approach to pain [10].

Inflammation is the response of living tissues to injury. It involves a complex array of enzyme activation, mediator release, and extravasations of fluid, cell migration, tissue breakdown and repair. Non-steroidal anti-inflammatory drugs (NSAID) are among the most commonly prescribed drugs due to their consistent effectiveness in the treatment of pain, fever, inflammation and rheumatic disorders. However, their use is associated with adverse effects at the level of digestive tract, ranging from dyspeptic symptoms, gastrointestinal erosions and peptic ulcers to more serious complications, such as over bleeding or perforation [11]. Therefore to overcome the toxicity of NSAID, the development of new anti-inflammatory drugs is still necessary and the natural product such as medicinal plants could lead in discovering new anti-inflammatory drugs with less undesirable effects [12].

Pyrexia or fever is usually caused as a secondary impact of infection, tissue damage, inflammation, graft rejection and malignancy or other diseased states. The body by its natural defense mechanism creates an environment where infectious agent or damaged tissue cannot survive. Generally, infected or damaged tissue initiates the enhanced formation of pro-inflammatory mediators (cytokines like interleukin 1β, α, β and TNF-α) which increase the synthesis of prostaglandin E₂ (PGE₂) near preoptic hypothalamus area and thereby triggering the hypothalamus to elevate the body temperature [13]. Most of the antipyretic drugs normally prevent or inhibit COX-2 expression to reduce the elevated body temperature by inhibiting PGE₂ biosynthesis. Moreover, these synthetic agents irreversibly inhibit COX-2 with high selectivity which is toxic to the hepatic cells, glomeruli, cortex of brain and heart muscles, whereas natural COX-2 inhibitors usually have lower selectivity with fewer side effects [14]. L. glutinosa was selected due to its availability in Bangladesh, therefore, used in rural areas for different treatments, and not such investigations have been carried out with this plant native to Bangladesh. Our main goal was to evaluate the in vivo antipyretic, anti-inflammatory and analgesic activity, and investigate the thrombolytic property of the plant leaves to validate its traditional uses.

Thrombosis or blood clot formation is a critical event in which the damaged regions of the endothelial cell surface or blood vessel are blocked by the deposition of platelets, tissue factor and fibrin [15]. In the formation process the major role is played by platelets as the process of thrombosis is initiated when the activated platelets form platelets to platelets bonds. These activated platelets further bind to the leucocytes and bring them into a complex process of plaque formation and growth [16]. It is the thrombolytic agents that lyse clot by disrupting the fibrinogen and fibrin contained in a clot. Plasmin is one of the natural anti-thrombotic agents. The cell surface bound plasminogen is easily activated to plasmin which ultimately leads to fibrinolysis [17]. After a long process of trial and error the scientists have discovered several thrombolytic drugs from various sources. To make those drugs more site specific and effective some of them have been modified with modern recombinant technology. Streptokinase (a bacterial plasminogen activator), a widely used thrombolytic agent, is capable of converting additional plasminogen to plasmin. But this drug has several adverse effects like bleeding and embolism which lead to further complications. To overcome these complications a number of studies have been conducted by various researchers in order to discover new sources of herbs and natural foods and their supplements having antithrombotic effect with minimal adverse effect [6]. As a part of that research work we also tried to find whether the herbal preparations of L. glutinosa leaves possess clot lysis potentiality or not. When we compared the result of positive control (streptokinase) with that of negative control (water), we found that there was negligible amount of clot disruption when water was added to the clot. This prominent result encouraged us to compare four different test samples in the same manner against the negative control and observe significant thrombolytic activity. It was reported that phytochemicals like saponin, alkaloids and tannin are responsible for thrombolytic activity [18]. As the bark extract of L. glutinosa possesses saponin, alkaloids [19], therefore the possibility of the presence of these phytochemicals in the leaves extract may be the probable reason of demonstrating the thrombolytic activity.

It was observed from the study that in both analgesic activity assay models the plant extract demonstrated analgesic effects. This means that the extract may possess both peripheral and central analgesic effects. The leaves extract of L. glutinosa exhibited significant dose dependent inhibition of acetic acid‒induced writhing in mice in comparison to that of the control (saline). Acetic acid induces inflammatory pain by impelling capillary permeability [20], and releasing substances that excite pain nerve endings [21]. The peripheral analgesic effect is generally mediated by the NSAIDs through inhibition of cyclooxygenase and/or lipoxygenase (and other inflammatory mediators) or inhibition of pain responses mediated by noiceptors peripherally [22]. Therefore, it is possible that crude extract of L. glutinosa leaves may be showing analgesic effect through these mechanisms although the exact mechanism of action is yet to be discovered. Again in hot plate test the extract also showed prominent antinociceptive effect against the standard drug ketorolac. The hot plate response is a more complex supraspinally organized behavior [23]. The μ receptor is a type of receptor that is commonly known as the pain relief receptor. It has been proved to be a potent receptor in regulating thermal pain [24]. Moreover, activation of μ₂ opioid subtype receptor leads to spinal analgesia [25]. Therefore, by considering the test report, it may be assumed that the antinociceptive activity of L. glutinosa leaves extract is likely to be mediated centrally although the exact mechanism is yet to be discovered. Previous studies on different plant extracts showed analgesic effect in animal models and their effects have been attributed to the presence of alkaloids, glycosides, flavonoids and saponins [26, 27]. The phytochemical screening of L. glutinosa extract revealed the presence of most of the above‒mentioned phytochemicals [20]. Thus it is possible that the analgesic activity of L. glutinosa leaves extract can be due to the presence of these phytochemical constituents.

The present study was also conducted to evaluate the probable anti-inflammatory activity of the methanol extract of L. glutinosa leaves on mice in an acute inflammatory model. The carrageenan‒induced mice paw oedema is a suitable test for evaluating anti‒inflammatory drugs or natural products [28]. Carrageenan induced paw edema is biphasic event. In the first phase there are incidences like release of histamine, serotonin and kinins whereas the second phase of edema is imposed by release of prostaglandins, protease and lysosome. The second phase is sensitive to most clinically effective anti-inflammatory drugs [29]. The results of present study showed that the role of methanolic extract of L. glutinosa leaves against carrageenan induced acute inflammation was significant. The leaves extract at a dose of 250 and 500 mg/kg showed significant dose dependent reduction in paw size from 1–4 h and elicited anti‒inflammatory response comparable with standard drug ketorolac. This might be due to the inhibition of the biphasic response induced by the carrageenan. Therefore, it is possible that acute anti‒inflammatory effect of L. glutinosa leaves may involve multiple mechanisms like inhibition of either cyclooxygenase and/or lypooxygenase enzyme or inhibition of synthesis, release and action of above inflammatory mediators; but the exact mechanism of action needs to be discovered by further investigation.

It is known that fever is provoked by many exogenous substances in animal models, including bacterial endotoxins and microbe infection. These exogenous pyrogens are responsible for the production of different pro-inflammatory cytokines which stimulate the release of local prostaglandins (PGs) by entering into the hypothalamic circulation and therefore resetting the hypothalamic thermal set point. Thus to regulate the body temperature it is necessary to have a delicate balance between the production and loss of heat. It is the hypothalamus that regulates the set point and also controls body temperature. The non-steroidal anti-inflammatory drugs inhibit the prostaglandin synthetase within the hypothalamus and thereby demonstrate their antipyretic action [30]. The present study revealed that the methanolic extract of L. glutinosa leaves causes a significant antipyretic effect in yeast provoked elevation of body temperature. Here, the methanolic extract caused almost similar result in lowering of body temperature, in comparison to that of standard drug. It may be predicted that the extract their antipyretic action through inhibition of prostaglandin synthetase within the hypothalamus as like as the NSAIDs. Although, there is no direct evidence of L. glutinosa to interfere with prostaglandin synthesis in hypothalamus but it can be supported by a related study in which Dalbergia odorifera extract was found to inhibit prostaglandin biosynthesis [31]. Thus the present pharmacological evidence provides support for the folklore claim of L. glutinosa leaves as an antipyretic agent.

In light of the results, it can be revealed that the hydroalcoholic extracts of L. glutinosa leaves have remarkable thrombolytic, analgesic, anti-inflammatory and antipyretic activities. Therefore, it may presage further studies to better understand the mechanism of such actions scientifically.