The local name of C. bengalense Roxb. (Family: Burseraceae) is Dhuna Rata or East Indian Copal. The native origin of the plant is not known but the plant is abundant in Vietnam, China, Laos, Myanmar, Thailand and India-Assam (Wu et al., 2008). Extracts from leaf and root is used in bronchitis, leprosy, jaundice, cough and asthma. Anti-inflammatory, antiseptic and anti-asthmatic activities were demonstrated by leaf and bark extracts (Le et al., 2012). The species has been reported to be useful in skin rashes and snake bite (Sarkar et al., 2017). A new flavone glycoside and six known compounds with cytoprotective properties have been isolated from the stem bark of C. bengalense. This might be the reason of the traditional use of this plant in tumor and liver damage (Le et al., 2012).

It is important to investigate the medicinal plants of Bangladesh for prominent biological activities (Sharmin et al., 2017; 2018). C. bengalense is abundant in Bangladesh and very few experiments were carried out using the bark of the plant. Therefore, antioxidant, cytotoxic, thrombolytic, membrane stabilizing and antimicrobial activity assays were conducted using the crude methanol extract of C. bengalense bark extract as well as its organic and aqueous soluble fractions.

Plant materials

C. bengalense bark was collected from Mirpur botanical garden, Dhaka, Bangladesh. A voucher specimen (DACB 47558) for this collection has been maintained in Bangladesh National Herbarium for future reference.

Small pieces of the bark were sun dried for several days. Then the bark pieces were oven dried for 24 h for better grinding. High capacity grinding machine was used to powder the dried bark. 500 g powdered material was soaked in 3.0 l of methanol in a clean, ambered color reagent bottle (5.0 l) for 10 days. The bottle was occasionally shaken and stirred. Then the plant material was filtered, and the filtrate was evaporated to dryness using a rotary evaporator at 40°C and 50 r.p.m to give the crude methanol extract. The concentrated methanol extract (5 g) was fractionated by modified Kupchan partition protocol (Van Wagenen et al., 1993) and the petroleum ether (PESF, 0.8 g), carbon tetrachloride (CTCSF, 1.7 g), dichloromethane (DCMSF, 1.3 g) and aqueous (AQSF, 0.7 g) soluble materials were obtained and refrigerated until further use.

Drugs and chemicals

Beacon Pharmaceutical Ltd provided Streptokinase. All other drugs, reagents and solvents were obtained from Sigma-Aldrich, Munich, Germany.

Total phenolic content

The method developed by Harbertson and Spayd (2006) was used to determine the total phenolic content of the test samples.

Antioxidant activity

The antioxidant activity of the test samples was determined using BHT and ascorbic acid as reference standards (Brand-Williams et al., 1995).

Brine shrimp lethality bioassay

The cytotoxic potential of the plant samples was determined using single day in vivo assay. The test samples were assayed using Artemia salina. Vincristine sulphate was used as the reference standard (Meyer et al., 1982). The lethality of brine shrimp nauplii was used in this assay to determine cytotoxic activity.

Thrombolytic activity

The thrombolytic activity was determined following the method developed by Prasad et al. (2007). Streptokinase was used as positive control.

Membrane stabilizing activity

The ability of the extract and fractionates to inhibit heat and hypotonic solution induced haemolysis of human erythrocytes was assessed following the method developed by Omale et al. (2008).

Antimicrobial screening

Disc diffusion method was used to investigate the antimicrobial potential of the crude extract and its aqueous and organic soluble fractionates by observing their ability to generate zone of inhibition (Bayer et al., 1966).

Statistical analysis

Three replicates of each sample were used for statistical analysis and all of the values are expressed as the mean ± standard deviation (SD). The results were evaluated by a two-tailed non-parametric pair t-test. P < 0.05 was considered statistically significant.

Different test samples of C. bengalense demonstrated presence of phenolic components within the range of 2.75 to 14.31 mg of GAE/g of sample. Among the test samples, the crude methanol extract showed the highest value of phenolic content (14.31 ± 0.23 mg of GAE/g of sample). In free radical scavenging activity assay, the highest free radical scavenging activity was given by the crude methanol extract (IC₅₀ = 50.62 ± 0.26 μg/ml) followed by pet-ether soluble fraction (IC₅₀ = 59.42 ± 0.63 μg/ml), as compared to ascorbic acid and BHT exhibiting IC₅₀ values of 3.63 μg/ml and 20.93 μg/ml, respectively (Table 1).

In case of brine shrimp lethality bioassay, among all the test samples of the bark of C. Bengalense, the highest cytotoxic activity was given by the pet-ether soluble fraction (LC₅₀ = 8.96 ± 0.64 μg/ml) followed by the crude methanol extract (LC₅₀ = 18.37 ± 0.38 μg/ml) as shown in Table 1 .

In thromboltic activity assay, the pet-ether soluble and the carbon tetrachloride soluble fractions showed 31.08 ± 0.12% and 27.58  ±  0.55%  of  clot lysis,  respectively  as compared to 65.01% clot lysis by the standard streptokinase (Table 2).

At concentration 1.0 mg/ml, the test samples of C. bengalense protected the haemolysis of RBCs induced by heat and hypotonic solution as compared to the standard acetyl salicylic acid (0.10 mg/ml). The dichloromethane soluble fraction showed 60.94 ± 0.31% inhibition of heat induced haemolysis which is found to be more significant than acetyl salicylic acid (42.12 %). On the other hand, the aqueous soluble fraction inhibited 49.17 ± 0.31 % hypotonic solution-induced haemolysis of RBCs as compared to 72.00% by acetyl salicylic acid (Table 2).

The antimicrobial activity of C. bengalense test samples was evaluated against gram positive and gram negative bacteria and the results were compared with standard antibiotic, ciprofloxacin. Among the test samples of C. bengalense, only the carbon tetrachloride soluble fraction and dichloromethane soluble fraction revealed antimicrobial activity with zone of inhibition ranging from 7.0 to 9.0 mm.  The  highest  zone  of  inhibition (9.0 mm) was showed against Salmonella typhi by the carbon tetrachloride soluble fraction (Table 3).

A few phyto-components like sabinene, caryophyllene and α-humulene have been previously isolated from C. bengalense (Thang et al., 2004). Among them sabinene has been reported to possess antioxidant activity (Quiroga et al., 2015).  Again, β- caryophyllene from the essential oil of Aquilaria crassna, demonstrated significant antioxidant potential (Dahham et al., 2015). Therefore, these phytocomponents might have contributed to the observed antioxidant activity of the species under investigation (Table 1).

In Vietnam, C. bengalense has been used as an ingredient for making remedies of cancer and liver damage for a long period of time (Le et al., 2012). β-caryophyllene has been found to show anti-cancer activity against colorectal cancer cells (Dahham et al., 2015). Therefore, this phyto-component might be held responsible for the observed cytotoxic potential of the test samples (Table 1). In case of thrombolytic activity assay, the findings may help in the development of new cardiovascular drugs using the bark of C. bengalense (Table 2).

There are lots of similarities between human red blood cell membranes with that of lysosome. Hence, the membrane stabilizing activity can be correlated to anti-inflammatory effect (Mounnissamy et al., 2008). Sabinene has been reported to exhibit strong anti-inflammatory activity by inhibiting nitric oxide production in lipopolysaccharide and interferon gamma-triggered macrophages (Valente et al., 2013). 1% sabinene was observed to inhibit lens protein-induced inflammation in rabbit’s eye (Quan-Sheng et al., 1993). Therefore, this phytocomponent might be considered responsible for  the observed membrane stabilizing activity.

In support of the observed antimicrobial activity, it can be stated that sabinene, β-caryophyllene and α - humulene have been found to exhibit significant antimicrobial activity (Arunkumar et al., 2014; Dahham et al., 2015; Rahman et al., 2016). Therefore, the presence of these compounds in the species under investigation might be the reason of observed antimicrobial activity (Table 3).

Mankind has been fighting against many diseases such as cancer, heart diseases and neurodegenerative diseases for many years. Some medicines like antibiotics that are found to be useful now may not be found to be effective in future due to development of resistance. Therefore, mankind is continuously searching for better and more potent medicines with fewer side effects. In this investigation, the test samples of C. bengalense showed significant cytotoxic and membrane stabilizing potentials. The plant should be further analyzed for the identification of the compounds responsible for the observed activities.

The authors have not declared any conflict of interests.