Phytochemical, antioxidant and antimicrobial properties of Litsea angulata extracts

Preparation of plant extracts

The plant material was obtained from Education Forest Laboratory of Forestry Faculty, Mulawarman University, East Kalimantan, Indonesia. Three different plant parts of L. angulata (bark, branch, and leaves) were separated, ground and extracted. The successive maceration extraction method was adopted from Sruthi and Indira³, with the solvents modified. About 50 g of the air-dried powder of each plant material was extracted individually with one of the following solvents: n-hexane, ethyl acetate, and 96% ethanol. The extracts were filtered and concentrated under vacuum using a rotary evaporator until the solvent was completely evaporated. In total, nine different extracts were produced, with each solvent used to produce extract from each plant part (branch, n-hexane; branch, ethyl acetate; branch, ethanol; bark, n-hexane; bark, ethyl acetate; bark, ethanol; leaves, n-hexane; leaves, ethyl acetate, and leaves, ethanol extracts).

Phytochemical screening

A total of 60 mg of each extract were dissolved individually in 1 ml solvent that used for extraction and the solutions were used to test for qualitative phytochemical tests. The tests were done according to the standard procedures described into literature by Kokate⁴, Senthilmurugan⁵, Harborne⁶ to detect the following bioactive compounds: alkaloids, flavonoids, saponins, tannins, terpenoids, steroids, carotenoids, and coumarin.

DPPH free radical scavenging assay

The DPPH assay was performed as described by Kuspradini et al.⁷. Various concentrations of samples of each extract (12.5, 25, 50 and 100 ppm) in 96% ethanol were added with DPPH. After 20 minutes, the absorbance of the resulting solution and the blank were recorded. Ascorbic acid was used as a positive control. The absorbance was recorded spectrophotometrically at a wavelength of 517 nm. DPPH free radical scavenging activity was stated as % inhibition = (1 – Absorbance of sample/Absorbance of control) × 100. To inhibitory activity, half-maximal inhibitory concentration (IC50) values were calculated.

Determination of antibacterial activity

The minimum inhibitory concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the samples were assessed against Staphylococcus aureus and Streptococcus mutans using the 96-well microdilution and solid medium, respectively. The bacterial concentration in the inoculum was standardized at 0.5 McFarland turbidity scale, equivalent to 10⁸ CFU ml^–1. The method of MIC was adopted from the method outlined by Mohsenipour and Hassanshahian, with modifications⁸. A stock solution was prepared by dissolving 5 mg extracts in 1 ml of 40% ethanol. A total of 50 µl stock solution were serially diluted twofold in 40% ethanol to achieve the range of test concentrations (1250, 625, 312.5 and 156.25 ppm), which were added to wells of a 96-well microplate. Next, 100 µl sterile nutrient broth culture medium (NB) and 50 µl of the culture of the respective organism were added into each well. The inoculated microplates were incubated at 37°C for 24 h.

At 1 hour before the end of incubation, the bacterial growth was confirmed by adding 0.01% solution of 2,3,5-triphenyl tetrazolium chloride (TTC, Merck, Germany) (50 µl) and the plate was incubated for another hour. The viable bacterial cells reduced the yellow TTC to pink. The inhibition of growth was visually detected when the solution in the well remained clear after incubation with TTC. Positive controls (bacteria + NB + chloramphenicol), negative controls (bacteria and NB), vehicle controls (bacteria + NB + solvent), and media controls (NB) were included in each test. MBC was determined by inoculating the assay from the wells showing no microbial growth onto the surface of nutrient agar medium on the petri dish. The petri dishes were incubated for 24 h at 37°C and subjected to visual inspection. MBC was considered as the lowest concentration where there was no resumption of bacterial growth.

Statistical analysis

All experiments were conducted three times. Regression analysis was used to calculate IC50 values of antioxidant. All statistical analyses used Microsoft Excel 2010 software.

Phytochemical screening

The result of phytochemical screening showed that L. angulata contain alkaloids, flavonoids, tannins, terpenoids, carotenoids and coumarin (Table 1). It can be shown that the ethanolic extract of the L. angulata showed more number of secondary metabolites when compared with other extracts.

Antioxidant activity

All extracts could inhibit DPPH radical scavenging activity (Table 2). The IC50 values with regards to different used solvents and plant parts were, in increasing order, as follows: leaves, ethanol; bark, ethanol; branch, ethanol; branch, ethyl acetate; bark, ethyl acetate; branch, n hexane; leaves and bark, n hexane. Raw absorbance data from which IC50 values were calculated are shown in Dataset 1⁹.

Antimicrobial activity

All extracts could inhibit the growth of S. mutans and S. aureus and showed the MIC value at 156.25 ppm concentration (Table 3). The MBC value could not detect in the range of 156.25–1250 ppm concentration. It is indicated that the MBC value in this study was higher than 1250 ppm.