Insights in Biomedicine Open Access

Keywords

Antioxidant; Anti-inflammatory; Membrane stabilization; Cardioprotective

Introduction

The use of plant products is increasing antique, eventually increasing day by day. At present, thousands of plant metabolites are being successfully used in the treatment of a variety of diseases. According to an estimate, 80% of the world’s population is found to rely upon plants for medication [1]. For example, more than 47% of all drugs, used in Russia are obtained from plant sources [2]. In Bangladesh about 250 plant species are used in herbal medicines.

Clerodendrum viscosum Vent. is a slightly woody shrub, 2-4 feet in height under the family Verbenaceae. It is a terrestrial shrub having square, blackish stem and simple, opposite, decussate, petiolate, exstipulate, coriacious, hairy leaves with a disagreeable odour [3]. Flowers are bluish-purple often white in pyramid shaped terminal panicles. It has blunt quadrangular sterns and branches, leaves are usually three at a node, sometimes opposite oblong or elliptic, serrate, flowers are blue, many in long cylindrical thyrsus and the fruits are four lobed purple durpe, somewhat succulent with one pyrene in each lobe [4]. The plant tropical habitat and commonly distributed throughout the plains of India, mainly in West Bengal [5]. It is known as Hill glory bower and Clerodendrum in English; Bhandira and Sthauneya in Sanskrit; Bhates and Titabhamt in Hindi; Periyalam, Peruku and perigalam in Malayalam, and Ghentu or Vatia in Bengali.

Different species of Clerodendrum genus have been traditionally used over centuries for their promising biological activities in – oxidative stress, inflammation, furunculosis, echymosis, gastritis, burns, wounds, headaches, dizziness, furuncles, hysteroptosis, dyspepsia, dropsy, fever, jaundice, typhoid, syphilis, gonorrhea, ophthalmia, antidiabetic, antibacterial, anti-malaria, organ damage and so on [5-7].

In Indian folk medicine, parts of C. viscosum are used in the treatment of bronchitis, asthma, fever, wound, blood disorders, inflammation, burning sensation and epilepsy. Various parts of the plant are used by tribes in colic, scorpion sting and snake bite, tumors and certain skin diseases. The leaves are slightly bitter, cure inflammation, skin diseases and small pox [7]. The bitter substance, clerodin is widely used as a tonic and an anthelmintic agent in the North India. The leaf extract is also reported for its haemsynthesis, anti-piles, anti-diabetic, and painkiller activities Leaves and roots contain clerodolone, clerodone, clerodol, and a sterol now designated as clerosterol and are used for external applications on tumours [6,7]. In a phytochemical study, the species C. viscosum revealed the presence of saponin, clerodin (a bitter diterpene) and some enzymes. Leaves also contain fixed oils, while roots by luperol, β-sitosterol, clerosterol (5,25-sigmastadien_3β-ol, clerodolone as lup_20(30)-en-3β-diol- 12-one), clerodone (3β-hydroxylupan-12-one) and a steroidal glycoside. The plant is also evident to contain alkyl sterols, 2,-(3, 4-dehydroxyphenyl) methanol 1-O-α-2rhamnopyranosyl-(1→3)- β-D-(4-O-caffeoyl) glycopyranoside (acteoside), triterpenes, steroids and flavonoids [7].

A clot inside the blood vessel is called atherombosis, which is one of the major consequences of cardiovascular diseases. Unfortunately, recently used anti-atherothromboic drugs have a number of serious adverse effects [8], which is a stimulation to search of new and safe agents. According to [9], oxidative stress due to the overproduction of reactive oxygen and nitrogen species (ROS/RNS) triggers inflammatory and chronic human diseases, including aging and atherosclerosis.

In previous studies, C. viscosum has been found to show organo-protective along with antioxidant and anti-inflammatory capacities. Thus, this study was shot out to screen a possible anti-atherothrombosis effect of the crude methanol leaf extract of C. viscosum in human clotted blood. In this regard, after a preliminary phytochemical investigation, antioxidant and antiinflammatory potentials were re-checked along with a newly perfumed membrane stabilization test.

Materials and Methods

Research ethics

This study was conducted under the Department of Pharmacy, Southern University Bangladesh (SUB) with the ethical number #PH-9991010. For human blood, ten students of the same/ different departments of the SUB were willingly participated in this study. A specialist from a reputed diagnostic centre was invited to collect 4-5 mL blood through venous puncture.

Plant collection, identification, drying and grinding

The whole plant was collected from the Batali Hill-Tracts (Chittagong), Bangladesh and was identified at Forest Research Institute (Chittagong), Bangladesh. A voucher specimen was deposited with the number: BFRIH-2034.

The leaves were washed with running tap water and subjected for shade-drying (temperature not exceeding 50°C). Dried leaves were then ground into coarse powder with the help of a grinder and stored in an amber color airtight container until the extraction commenced.

Preparation of crude extract

Approximately 110 g of powdered materials was subjected to hot extraction with 700 mL of absolute methanol for 10 h using a Soxhlet extractor (Quickfit, England). The extract was then concentrated under an air cooler. The yield value was 15.89%.

To carry out the antioxidant assay, the crude extract was dissolved in methanol (used for extraction), while in distilled water (DW, with a 10 min hand shake) following to an overnight standing for the other tests. For antioxidant test, we used a concentration range from 3.125-100 μg/mL, while for other assays it was 125- 500 μg/mL. The standards were diluted in DW.

Source of reagents and chemicals

The standards and the other necessary reagents and chemicals were purchased from the Sigma Chem Ex. Co. St. Louis, Missouri, USA.

Preliminary phytochemical analysis of the methanol extract of Clerodenrum viscosum

Phytochemical study was conducted according to the methods described [10].

Antioxidant capacity assay (DPPH radical scavenging test)

The test for DPPH• scavenging activity was done using a modified method described by Islam et al. [11]. Briefly, 0.3 mL of sample was added to a 2.7 mL ethanolic solution of DPPH (0.5 mM). After 30 min, the absorbance was measured using a spectrophotometer at 517 nm. A similar concentration of TRO served as the positive control, while only 0.3 mL methanol was considered as a negative control group. The blank contained no sample. The DPPH radical scavenging potential was calculated using the following equation:

% inhibition of DPPH^• scavenging = [(A_br – A_ar)/A_br] × 100

Where, A_br and A_ar represent the before and after absorbance of DPPH free radicals in reaction mixtures.

Evaluation of anti-inflammatory activity (egg albumin denaturation test)

The anti-inflammatory (in vitro) of methanol extract of C. viscosum (MECVL) was carried out according to [12]. Briefly, 1% egg albumin was constituted in phosphate buffer saline solution (PBS, pH 7.4). The assay mixture contains 1 mL phosphate buffer (pH 7.4, 0.15 M), 2 mL hypo-saline (0.36%) and 0.5 mL of the test sample. Acetyl salicylic acid (ASA) and DW were taken as positive and negative (NC) controls. After the incubation at 37°C for 30 min, reaction mixtures were centrifuged and the supernatant was collected for spectrophotometric analysis at 560 nm. Activity was measured by the following equation:

% inhibition of protein naturation = 100 - [{(absorbance of test solution) ÷ (absorbance of control)} × 100]

Evaluation of membrane stabilization capacity (HRBC assay)

This test was conducted by the hypo-saline induced hemolysis (HL) method described by a modified method [13]. In this occasion, fresh human RBC was reconstituted as 10% suspension human red blood cell reconstitution (HRBC) in isosaline (0.9% NaCl, pH 7.4). The assay mixture contains 1 mL phosphate buffer (pH 7.4, 0.15 M), 2 mL hypo-saline (0.36%), 0.5 mL HRBC suspension (10% v/v) with 0.5 mL of the test sample. The ASA and DW were taken as positive and negative controls. After incubation at 37°C for 30 min, the reaction mixtures were centrifuged and the supernatant was collected for spectrophotometric analysis at 560 nm. Activity was measured by the following equation.

% inhibition HL = 100 - [{(absorbance of test solution) ÷ (absorbance of control)} × 100]

Anti-atherothrombosis assay (Clotlysis test in human blood)

The thrombolytic activity of the MECVL was evaluated [12] using streptokinase (SK) 100 IU and previously said NC, respectively. Briefly, blood was collected from 10 healthy volunteers and distributed into pre-weighed (W1) micro-centrifuge tubes (0.5 mL/tube) and incubated at 37°C for 45 min and then weight (W2) was taken. The weight of clotted blood (ΔW) was taken by subtracting the pre-weight and the weight of clotted blood containing tube. Then 100 μL of the treatments were added to the clot containing tubes marked. The MECVL was tested at concentrations of 125-500 μg/mL. On the other hand, 100 μL of streptokinase (100 IU/tube) and 100 μL of NC were added to the control marked tubes. All the tubes were then incubated at 37°C for 90 min. After incubation, fluid released was removed carefully without disrupting the clot, and tubes were again weighed for getting the weight variation among the pre-weight and final weight (W3) that was achieved for clotlyses.

Statistical analysis

Values are mean ± SD (standard deviation). The data were analyzed by means of analysis of variance (ANOVA) followed by t-Student–Newman–Keuls's as post-hoc test using the GraphPad Prism software (version 6.0) with 95% confidence interval at p<0.05.

Results

Preliminary phytochemical analysis revealed the presence of alkaloids, glycosides, flavonoids, reducing sugars and gums (Table 1).

Chemical groups	Consequences
Alkaloids	++++--
Glycosides	++
Steroids	--
Tannins	--
Flavonoids	+
Saponins	-
Reducing sugars	++
Gums	+

(+): Presence; (-): Absence; Numbers represent the number of tests performed.

Table 1: Chemical groups identified in methanol extract of Clerodenrumviscosum.

According to the Table 2, the MECVL showed significant (p<0.05) DPPH radical scavenging capacity in comparison to the NC group. However, the activity was lower than the ascorbic acid (AA, standard). The extract and ascorbic acid at 100 μg/mL exhibited highest scavenging of DPPH radical by 47.1 ± 0.8% and 94.8 ± 1.1%, respectively. A concentration-dependent radical scavenging capacity was observed by the MECVL and AA. The NC produced a negligible radical scavenging capacity.

Parameters		% scavenge of DPPH radicals
		MECVL	AA	NC
Concentration (µg/mL)	3.125	10.2 ± 0.6a	13.3 ± 1.3a	-
	6.25	24.5 ± 1.1a	32.3 ± 0.8a,b	-
	12.5	29.3 ± 2.1a	50.5 ± 1.5a,b	-
	25	33.0 ± 1.0a	90.8 ± 0.8a,b	1.8 ± 0.4
	50	34.6 ± 1.3a	93.6 ± 0.6a,b	-
	100	47.1 ± 0.8a	94.8 ± 1.1a,b	-
IC50 [CI; R2]		8.3 ± 0.2 [4.5 – 15.3; 0.8]	9.5 ± 0.4 [7.1 – 12.8; 0.9]	-

Values are mean ± standard deviation (SD); ap<0.05 compared to the NC; bp<0.05 compared to the MECVL at same concentration; ANOVA followed by t-Student–Newman–Keuls's as post-hoc test; AA: ascorbic acid; MECVL: methanol extract of C. viscosumleaf; NC: negative control (methanol); IC₅₀: Half minimal inhibitory concentration; CI: Confidence interval; R²: Coefficient of determination.

Table 2: DPPH radical scavenging capacity of crude extract and controls.

Both MECVL and the standard, acetyl salicylic acid (ASA) concentration-dependently inhibited protein denaturation capacity in egg albumin assay. At 500 μg/mL, MECVL and the ASA produced % inhibition of protein naturation by 81.5 ± 0.1% and 89.1 ± 0.1%, respectively. In comparison to the NC group, the MECVL and ASA significantly (p<0.05) inhibited egg albumin denaturation. The IC50s calculated for the MECVL and ASA were 127.7 ± 0.5 μg/mL and 91.9 ± 0.6 μg/mL, respectively (Table 3).

Test groups
NC
ASA (μg/mL)	125
	250
	500
MECVL (μg/mL)	125
	250
	500

Values are mean ± standard deviation (SD) (n = 5); ANOVA followed by t-Student–Newman–Keuls's as post-hoc test; *p<0.001 when compared to the NC; ASA: acetyl salicylic acid; MECVL: methanol extract of C. viscosumleaf; NC: negative control (DW); IC₅₀: Half minimal inhibitory concentration; CI: Confidence interval; R2: Coefficient of determination.

Table 3: Anti-inflammatory assay in egg albumin.

Table 4 says that, not only the ASA, but also MECVL concentrationdependently and strongly inhibited HL of HRBC when compared to the NC group. At 500 μg/mL , ASA and MECVL exhibited HL inhibition by 92.2 ± 0.1% and 89.4 ± 0.1%, respectively. IC₅₀s calculated for the MECVL and ASA were 123.8 ± 0.6 μg/mL and 82.2 ± 0.6 μg/mL, respectively.

Test groups		% hemolysis inhibition	IC50 [CI; R2]
NC		0.0 ± 0.0	-
ASA (μg/mL)	125	66.7 ± 0.1*	82.2 ± 0.6 [27.5 – 245.7; 0.9]
	250	84.8 ± 0.1*
	500	92.2 ± 0.1*
MECVL (μg/mL)	125	46.1 ± 0.1*	123.8 ± 0.6 [27.9 – 549.3; 0.9]
	250	69.8 ± 0.1*
	500	89.4 ± 0.1*

Values are mean ± standard deviation (SD) (n = 5); ANOVA followed by t-Student–Newman–Keuls's as post-hoc test; *p<0.001 when compared to the NC; ASA: acetyl salicylic acid; MECVL: methanol extract of C. viscosumleaf; NC: negative control (DW); IC₅₀: Half minimal inhibitory concentration; CI: Confidence interval; R2: Coefficient of determination.

Table 4: Membrane stabilization test in re-constituted human erythrocytes.

Table 5 suggests that, the MECVL has clotlysis capacity as compared to the NC group. The highest clotlysis capacity of the MECVL was observed with 500 μg/mL concentration of 81.9 ± 4.8%, while the standard drug, streptokinase (SK) by 84.5 ± 3.9% with 100 μL. The NC produced negligible clostlysis capacity.

Treatments	Per tube (100 μL)	% clotlysis	IC50 [CI; R2]
NC	-	2.7 ± 1.5	-
SK	100 IU	84.5 ± 3.9*	-
MECVL	125 μg/mL	36.8 ± 4.1*	138.7 ± 0.5 [44.4 – 433.7; 0.9]
	250 μg/mL	62.8 ± 4.7*
	500 μg/mL	81.9 ± 4.8*

Values are mean ± standard deviation (SD) (n = 5); ANOVA followed by t-Student–Newman–Keuls's as post-hoc test; *p<0.001 when compared to the NC; SK: streptokinase; MECVL: methanol extract of C. viscosumleaf; NC: negative control (DW); IC50: Half minimal inhibitory concentration; CI: Confidence interval; R2: Coefficient of determination.

Table 5: Thrombolytic activity of crude extract and controls.

Discussion

Nevertheless, till date considerable attention has been given on plant-derived products. There is no doubt that, these kinds of medicaments are increasingly popular throughout the world. In fact, plant foods are one of the common daily consumption to us [14]. Previous phytochemical studies suggest that C. viscosum contains glycosides, flavonoids, steroids and some sugars. To date, seven sugars have been demonstrated in C. viscosum such as raffinose, lactose, maltose, sucrose, glactose, glucose and fructose [6,7]. Our study demonstrates the presence of alkaloids, reducing sugars and gums along with the previously reported phytochemical groups.

Flavonoids are well-known for their promising biological activities, including immunomodulatory, anticancer, cyto- and organoprotective, anti-diabetic and so on [14]. Phytochemicals containing active hydroxyl group (-OH), especially the essential oils and flavonoids may act as multi-edged sword in various diseases, has been considered as perfect weapons in the treatment of diseases [14]. In our study, MECVL concentration-dependently exerted antioxidant and anti-inflammatory activities. Furthermore, it also exerted a strong membrane stabilization capacity in HRBCs. The determined IC₅₀s, confidence interval (CI) and coefficient of determination (R2) values suggest that MECVL has significant cytoprotective capacity. The AA and ASA are established antioxidant and anti-inflammatory agents, respectively. The AA can scavenge ROS and stimulate physiological defense systems, while ASA has a shunting effect on pro-inflammatory as well as inflammatory mediators. However, substance having radical scavenging and antagonistic capacity of inflammatory mediators may impart cellular protection. For an example, the diterpene phytol is evident to scavenge OH radical from the cell surface of Saccharomyces cerevisiae, thus protects them from OH-induced damaging effect. This diterpene is also evident to protect rat erythrocytes from hydrogen peroxide-induced oxidative stress, which is a powerful oxidizer that liberates OH radical [14]. Moreover, examples of phytochemicals with antioxidant and antiinflammatory capacities are vast [10,14].

Thrombus formation inside the blood vessel obstacles the passages of required amount of oxygen and nutrients to the cells has been detected as one of the major consequences of cardiological complications, including stroke [8]. The SK is a widely used fibrinolytic drug, which binds and hydrolytically actives human plasminogen. The ultimate result is the clotlysis. On the other hand, oxidative stress due to the overproduction of reactive oxygen and nitrogen species (ROS/RNS) triggers inflammation and chronic human disease, including aging. The ROS/NOS-mediated oxidative stress can be identified in most of the key steps in the pathophysiology of atherosclerosis and the consequential clinical manifestations of cardiovascular disease due to it involves lipid metabolism, plaque rupture, thrombosis, myocardial injury, apoptosis, fibrosis and failure [9]. In this study, we found MECVL has a clotlysis capacity of human clotted blood. In comparison to the NC group, MECVL exhibited a significant (p<0.05) clotlysis capacity. The MECVL may act through either way.

Conclusion

The findings in phytochemical screening of MECVL are the agreement of previous studies. MECVL concentrationdependently exhibited antioxidant, anti-inflammatory, membrane stabilization and clotlysis activities. Antioxidant and anti-inflammatory-mediated cytoprotective effect may be linked to its membrane stabilization and anti-atherothrombosis effects. Further researches are welcomed to isolate and investigate responsible phytochemicals and their molecular mechanisms.

Acknowledgement

We are duly thankful to the Department of Pharmacy, Southern University Bangladesh (SUB) for hosting and providing laboratory facilities to conduct this study.

Conflict of Interest

None declared.

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