Phytochemical, antioxidant and antimicrobial properties of Litsea angulata extracts

Background: Litsea angulata is a plant species belonging to Lauraceae family that is distributed throughout Indonesia, Malaysia, and New Guinea. The seeds have been traditionally used by local people in Kalimantan, Indonesia for the treatment of boils; however, there is no information about the potency of its branch, bark and leaves yet. This study aimed to determine the antioxidant, antimicrobial activity as well as the phytochemical constituent of Litsea angulata branch, bark, and leaves. Methods: Extraction was performed by successive maceration method using n-hexane, ethyl acetate, and ethanol solvent. Antioxidant activity was evaluated by DPPH radical scavenging assay. The antimicrobial activity using the 96 well-plate microdilution broth method against Staphylococcus aureus and Streptococcus mutans. Results: Based on the phytochemical analysis, it showed that extract of L. angulata contains alkaloids, flavonoids, tannins, terpenoids, and coumarin. The results showed that all extracts of plant samples displayed the ability to inhibit DPPH free radical formation and all tested microorganisms. Conclusions: L. angulata contains secondary metabolites such as alkaloids, flavonoids, tannins, terpenoids, carotenoids, and coumarin. The antioxidant activity on different plant extracts was a range as very strong to weak capacity. All extracts in this study could inhibit the growth of S. aureus and S. mutans.


Introduction
Many plants species from the genus Litsea are a potential source of biologically active compounds and are used as traditional medicines such as antispasmodic, wound healing, relieving rheumatism and cold 1,2 . There is little information about the potency of Litsea angulata species. L. angulata, belonging to the Lauraceae family, which can be found in East Kalimantan, Indonesia, and to our knowledge, data are limited on its biological activities. The fruit seed of L. angulata has been reported to have antifertility properties. No previous studies report the antibacterial properties of L. angulata (bark, branch and leaves), while the related species such as L. cubeba leaf extract, Litsea petiolata Hook. f. leaves, and stem bark and leaf extracts of Litsea glutinosa (Lour.) C.B. Rob, presented antibacterial activity against S. aureus [3][4][5] . In addition essential oil of Litsea cubeba can also act as antibacterial against S. mutans 6 . Therefore the present study aimed to assess the phytochemical constituents, antioxidant, and antimicrobial of different plant part of L. angulata.

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 Indira 9 , 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 Kokate 10 , Senthilmurugan 11 , Harborne 12 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. 13 . 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 10 8 CFU ml -1 . The method of MIC was adopted from the method outlined by Mohsenipour and Hassanshahian, with modifications 14 . 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 +

Amendments from Version 1
In response to the reviewers' comments, we have updated our manuscript. A number typographical errors as pointed out by the reviewer are corrected and additions have been added on the manuscript: information on the pedoclimatic characteristic of the place where the plant are harvested, the place of determination of the Litsea angulata, statistical analysis on the discussion section, benefits and comparison of extracts, addition of some references, revision on the order of IC50 value in antioxidant activity, explanation about using different plant material and bacterias such as Staphylococcus aureus and Streptococcus mutans, confirmation about negative control, vehicle control and control machines in the results, clarification about why ethanol can extract more active components such as tannin, carotenoid and coumarin compared to n-hexane and ethyl acetate, revision on conclusion.
We have updated the affiliation for Indah Wulandari. Table 1. Secondary metabolites in L. angulata extracts.

Phytochemical screening
The result of phytochemical screening showed that L. angulata contain alkaloid, flavonoid, tannin, terpenoid, carotenoid 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. The extraction of various phytochemicals was seen to be more effectively done by ethanol (polar) than the ethyl acetate and n-hexane (semi polar and non polar) solvents.

Antioxidant activity
All extracts could inhibit DPPH radical scavenging activity (

Discussion
Plant extracts have been reported to have numerous biological activities due to their phytochemical contents, which contribute significantly towards the antioxidant and antimicrobial activities such as flavonoids, tannins, and terpenoids [16][17][18] . The secondary metabolites extraction can be performed using solvent with different polarities, depending on the type of compound structure. Thus, it can be reported that polar solvent (ethanol) could extract more secondary metabolites such as tannin, carotenoid and coumarin than ethyl actetate and n-hexane solvent (semi-polar and non polar). It may be possibly be one of the reasons for a strong antioxidant activity shown by the ethanolic extracts of L. angulata. The solubility or insolubility of the active compound(s) in the solvent used for extraction can caused the differential effects on antioxidant and antibacterial 19-21 . The IC50 value was calculated using the regression linear equation, where y is 50 (percent inhibitory value) and x is amount of inhibitory concentration value in µg. Data analysis were shown in Dataset 1. According to Blois 22 sample which had an IC50 value more than 150 ppm was a weak antioxidant, 101-150 ppm was a medium antioxidant, 50-100 ppm indicated as a strong antioxidant, while lower than 50 ppm was a very strong antioxidant. Bark-ethyl acetate extract obtaining the lowest IC50 value (2.41 ppm) in comparison to another extracts, and it indicates that this extract has a strong antioxidant. Antimicrobials are considered as bactericidal if the MBC is not more than four times higher than the MIC, and MBC value is always equal or higher than MIC 23 .

Conclusions
L. angulata can be used as a source of natural antioxidant to prevent damage associated with DPPH free radicals and antibacterial to inhibit the growth of S. mutans and S. aureus bacteria. It may be due to the presence of its active compound such as alkaloids, flavonoids, tannins, terpenoids, carotenoids and coumarin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Manuscripts entitled Phytochemicals, antioxidants and antimicrobial properties extract Litsea angulata has been written well. Revise in the keyword in italic format, add your information where Litsea angulata the species ( ) was determined. Please make sure the alkaloids contained in -hexane Litsea angulata n extract because positive false in phytochemical tested are often reported. Authors wrote that all statistical analyses used Microsoft Excel 2010 software, but in the results and discussion, we cannot find the data with statistical analysis. The authors may add the data with statistical analysis or they can explain in the discussion because in the method they used the statistical analysis for data analysis. The authors may add the data with statistical analysis or they can explain in the discussion because in the method they used the statistical analysis for data analysis.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Natural product chemistry I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.