Background
Plants are the important sources of various pharmacologically active compounds [
1]. Among World Health Organization (WHO) enlisted 252 basic and essential drugs, 11 % are solely of plant origin [
2]. It has been assessed that there are more than 250,000 flower plant species in the world [
3]. Exploration of bioactivities of medicinal plants aids to develop phytotherapeutic agents [
4]. WHO has exclusively acknowledged the significance of herbal medicine for health care system and published many strategies, guidelines and standards for these botanical medicines [
5].
Free radicals produced in living cells might result in oxidative stress, which might trigger a number of diseases. Antioxidant emerges as savior from this condition [
6]. Besides, some synthetic antioxidants such as butylated hydroxytoluene and butylated hydroxyanisole have been reported to be unsafe for human health. Thus, the pursuit for effective, nontoxic and natural antioxidants has been intensified in recent years [
7]. Mortality rate from thrombotic diseases such as myocardial or cerebral infarction is increasing recently. This situation might become more challenging in the future due to obesity, diabetes, metabolic syndromes, etc. [
8]. Currently, much effort has been concentrated on the discovery of natural products as effective antithrombotic drugs [
9]. Inflammatory processes are involved with many severe conditions, such as neurodegenerative diseases, asthma, chronic inflammatory bowel diseases, rheumatoid arthritis, type 2 diabetes, cancer, etc. [
10]. The currently available antiinflammatory drugs include nonsteroidal antiinflammatory drugs, glucocorticoids, immunosuppressant drugs and biologicals. Despite these, therapy is often hindered by side effects. Thus, the discovery of new antiinflammatory drugs is still a need of the present time [
11].
C. citrinus (Curtis.) belongs to the family Myrtaceae. It is an evergreen shrub known as Red bottle-brush or Lemon bottle-brush and available all over Bangladesh. The flower spikes of bottle-brushes form in spring and summer and are made up of a number of individual flowers. This is a woody plant and can be 3 m to 4.6 m long [
12]. This herb is popular as folk medicine to treat diarrhea, dysentery, rheumatism, cough, bronchitis, etc. [
13,
14]. It has already been reported that this plant has antimicrobial [
15], relaxant [
16] and cardioprotective [
17] properties. Previous phytochemical investigations led to the isolation of various terpenoids, flavonoids, etc. A herbicidal compound, nitisinone, was also isolated from this plant [
18].
In the present study, the leaves of C. citrinus were assessed comprehensively for its free radical scavenging, thrombolytic and membrane stabilizing activities.
Discussion
Traditional medicine has gained popularity in all regions of the developing world for meeting some primary health care needs and is also rapidly spreading in industrialized countries [
27]. WHO has estimated that 25 % of modern medicines are made from plants first used traditionally [
28]. It has been assessed that the global market for herbal medicines is currently over 60 billion United States dollar annually and growing steadily [
29]. Even in the present era of synthetic medicine, drug discovery research based on herbal medicines is successful to a significant extent [
3,
30]. With this view,
C. citrinus, a plant of Bangladesh, has been studied here utilizing crude methanol extract of leaves and its organic fractions.
Free radicals are produced in human body by various endogenous systems, exposure to different physiochemical conditions or pathological states [
7]. In excess, they can damage cellular macromolecules including carbohydrates, proteins, lipids and nucleic acids. Ultimately, they promote inflammatory damages, cataract, cancer, aging, thrombus formation and many other associated problems [
31]. To counteract these disorders, intake of antioxidants are very essential [
6]. In DPPH and hydrogen peroxide free radical scavenging assays, the methanol extract scavenged free radicals prominently among all the tested materials including the standard drug, ascorbic acid. The carbon tetrachloride fraction also had a tendency to inhibit free radical production to an extent in both assays (Figs.
2 and
3).
The analysis of crude methanol extract using UV-visible spectroscopic technique in the present study demonstrated the abundance of phenolics and flavonoids than other extractives (Figs.
4 and
5). Besides, its carbon tetrachloride fraction also displayed noticeable level of phenolic and flavonoid contents. These findings might partially explain the reason for stronger free radical scavenging ability of the crude methanol extract and its carbon tetrachloride fraction [
32,
33].
However, thrombosis occurs due to the imbalance between thrombogenic factors and protective mechanisms, which leads to many vascular complexities including stroke, myocardial infarction, deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, etc. [
34]. Tissue plasminogen activator, urokinase, streptokinase, etc. are used abundantly for treating thrombotic disorders but they are not beyond all limitations. Better thrombolytic agent is a necessity of time [
35,
36]. In this study, the petroleum ether soluble fraction displayed the highest thrombolytic activity among the tested extractives of the leaves (Fig.
6). It has been reported earlier that terpenoids might have significant potentials of displaying thrombolytic activity [
37,
38]. Leaves of
C. citrinus were reported previously to have some terpenoids [
18]. Besides, a preliminary phytochemical screening of this study (Table
1) also indicated the presence of abundant terpenoids in petroleum ether fraction. This might be one of the reasons for exhibiting noticeable thrombolytic activity of the petroleum ether fraction among the tested extractives.
On the other hand, the membrane stabilization assay using erythrocytes is used as a common tool to screen antiinflammatory materials. The erythrocyte membrane is comparable to the lysosomal membrane and its stabilization acts as a parameter to indicate the ability of an extract to stabilize the lysosomal membrane as well [
26]. Maintaining stability of the lysosomal membrane is vital to limit the inflammatory response by inhibiting the release of lysosomal constituents of activated neutrophil such as bactericidal enzymes and proteases, which cause additional tissue inflammation and damage upon extracellular release [
39]. Exposure of erythrocytes to hypotonic medium, heat, methyl salicylate, phenylhydrazine, etc., results in membrane lysis [
40]. In the present study, the chloroform soluble fraction showed the highest membrane stabilizing activity in both heat- and hypotonic solution induced hemolysis among the plant extractives (Fig.
7). It was equivalent to the membrane stabilizing action of the standard drug, acetylsalicylic acid in the hypotonic solution induced hemolysis assay and more effective than the standard drug in heat induced hemolysis assay. Numerous flavonoids and alkaloids have been reported previously to display antiinflammatory activity [
41,
42]. Chloroform fraction of the leaves of
C. citrinus was also found to contain flavonoids (Fig.
5) and alkaloids (Table
1). The persuasive membrane stabilizing antiinflammatory activity of this fraction might be related to these biosynthesized metabolites.
Acknowledgements
We are grateful to the Bangladesh National Herbarium, Mirpur Road-1, Dhaka (
http://www.bnh.gov.bd) for plant identification.