Comparative antimicrobial studies on plant species known as

Pasak Bumi is a local name for a medicinal plant in Kalimantan, Indonesia. It is a famous medicinal plant and commonly used in traditional medicine as an aphrodisiac, as well as in the treatment of malaria. Pasak Bumi is a commercial name for (Simaroubaceae) plant species. Eurycoma longifolia Besides there are two other plant species also known Eurycoma longifolia locally as Pasak Bumi, (Rubiaceae) and Rennelia elliptica Trivalvaria (Annonaceae). This study was performed to investigate the macrophylla antimicrobial activities of the different species of Pasak Bumi and its total phenol contents. The antimicrobial activity of the ethanol extract was determined using the Agar Well Diffusion method at various concentrations while the phenol content was determined by the Folin Ciocalteu method. The results of the ethanol extract from the different root showed that the T. had the highest phenol content, and the highest activity index macrophylla (AI) was found in the (0.96 at 1000 μg concentration). The E. longifolia results of this study show that the three different Pasak Bumi have potential as antimicrobials against oral pathogen; 1 yeast: and 3 Candida albicans, bacterias: and Staphylococcus aureus, Streptococcus mutans, . Streptococcus sobrinus

Pasak Bumi is a plant used in traditional medicinal that grows in the tropical forests of Kalimantan of Indonesia. It is used by the local people as an aphrodisiac, for postpartum treatment, fever, and malaria 1,2 . In Central Kalimantan, there are three different plant species on Pasak Bumi; Yellow Pasak Bumi (Eurycoma longifolia Jack., Simaroubaceae), Red Pasak Bumi (Rennelia elliptica, Rubiaceae) and Black Pasak Bumi (Trivalvaria macrophylla, Rubiaceae) 3 . Previous research of Pasak Bumi (E. longifolia Jack) from different regions has shown activity in inhibiting the growth of microbes, however, research on the other species of Pasak Bumi such as Rennelia elliptica and Trivalvaria macrophylla are still limited. From the above information, the research aim is to compare the inhibition activities of the three different plants against one yeast: Candida albicans, and three bacterias: Staphylococcus aureus, Streptococcus mutans, and Streptococcus sobrinus. The research was also designed to extend our knowledge and help us explore the antimicrobial activities of the three different plant species.

Preparation of plant extracts
One kilogram of each plant was excavated and harvested from Katingan district, Central Kalimantan. The root was chopped and separated from its stem and leaves. The roots were sliced into small sections with a knife and allowed to dry under shade. The dried samples were crushed into powder using an electric blender. Once crushed, 50 grams of each powder of the plant root was weighed using a digital balance (Mettler Toledo, Mettler-Tokyo Group). Furthermore, the powder was extracted using successive maceration with the following solvents: n-hexane, ethyl acetate, and 96% ethanol. The ethanol filtrate was evaporated under a vacuum rotary evaporator (Eyela, N-N series) at 35°C until dry and used for the present study ( Figure 1).

Total phenol content
The total phenolic content was determined spectrophotometrically (UV Mini 1240 Shimadzu) in accordance to the Folin-Ciocalteu method 4 . The sample solution was prepared by dissolving the dry extracts (2 mg) in 100 μl DMSO and 900 μl of distilled water. The reaction mixture was made by mixing 200 μl of the extract from sample solution (200 μg/mL), 300 μl of distilled water, 250 μl of 10% Folin-Ciocalteu reagent (Merck Millipore, CAS No. 109001) and 1250 μl of 7.5% sodium carbonate. After a 90 minutes incubation at room temperature, the absorbance was determined spectrophotometrically at 760 nm. Gallic acid (Wako, CAS No. 5995-86-8) was used as a reference standard for plotting a calibration curve (concentration range: 2 to 10 μg/mL). The total phenolic content was expressed as a Gallic Acid Equivalent (GAE)/mg extract, using a standard calibration graph.
Antimicrobial activity Four pathogenic microbial strains; C. albicans (CA), S. aureus (SA), S. mutans (SM) and S. sobrinus (SS) from the Forest Product Chemistry Laboratory's culture collections, were used for the present study. The in vitro activity was screened using the agar well diffusion method in Nutrient Agar medium 5,6 . The extracts of each plant at a concentration of 10 mg/ml in 40% ethanol were prepared, and an aliquot of the test solution was put in to get a final concentration of 100, 250, 500, and 1000 μg/well. It was then placed on the inoculated nutrient agar plates and incubated for ±18-24 h at 37˚C. Ten μg/well of chloramphenicol (PT. Indofarma, Tbk., Indonesia) and 40% ethanol were employed as a positive and negative control. After incubation, the diameter of the inhibition zones was measured by a ruler. The experiment was performed in triplicate. The antimicrobial index (AI) was calculated using the formula 6,7 : Activity index (AI) = Inhibition Zone of the sample/Inhibition Zone of chloramphenicol.

Statistical analysis
All experiments were conducted three times. Regression analysis was used to make a calibration curve and calculate the total

Results
The total phenolic contents were calculated using the following linear equation based on the calibration curve of gallic acid: y = 0.0667x + 0.009; R2 = 0.9948, where y is absorbance and x is amount of gallic acid in μg (Table 1). T. macrophylla root extract obtaining higher total phenolic content in comparison to E. longifolia and R. elliptica. The extracts exhibited dose-dependent antimicrobial activities (Figure 2), and the results indicated that the in vitro antimicrobial activity of the T. macrophylla, E. longifolia, and R. elliptica extracts were ranked in the following order; SS>SM>SA>CA; SA>SM>SS>CA; and SS>SA>CA>SM, respectively. The highest activity was found in E. longifolia against S. aureus, with a maximum AI value (0.96) at 1000 μg/well concentration while the lowest activity at all concentration was found in R. elliptica extracts.

Discussion
Plant extracts with a high AI value indicates that the extracts have good antimicrobial activity against the selected pathogens 6 . The inhibitory activity of E. longifolia root extracts was in agreement with previous literature, it could inhibit S. aureus 8-10 and C. albicans 11 . R. elliptica was found to be able to inhibit the growth of C. albicans and S. aureus, which is contrary to a previous study where it was found to be inactive 12 ; however, there was no information about the extraction method for R. elliptica and the concentration used on that study. So far there have been no reports of the T. macrophylla being antimicrobial, but in this study T. macrophylla has proven to be an inhibitor for the growth of S. aureus, S. mutans, S. sobrinus and C. albicans. This is believed to be the first report to explore and compare the antimicrobial potentials of the three different Pasak Bumi plants.
The antimicrobial activity may be attributed to the high content of the phenols present. Phenolic compound such as gallic acid can causes irreversible changes (such as charge, intra and extracellular permeability, and physicochemical properties) in the properties of microbial membranes, with consequent leakage of essential intracellular constituents 13 . E. longifolia possess a higher antimicrobial activity than T. macrophylla, but its phenolic content was lower than T. macrophylla. E. longifolia extract might contain more non-phenolic compounds, or possess phenolic compounds that contain a higher number of active groups than the other extract. The interactions between chemical compounds (phenolic and non-phenolic compounds) might also be responsible for the antimicrobial effects 14 .

Conclusions
The present study performed in vitro studies of antimicrobial properties of three different Pasak Bumi (E. longifolia Jack, R. elliptica and T. macrophylla) on oral pathogens which gave positive results and different degree of activity.

Data availability
Underlying data is available form Open Science Framework The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1.

Introduction
Introduction part is very poor. The rationale of the need of comparative antimicrobial study of these plant species should have been elaborated.
No literature about the microbial infections has been cited. Authors needs to add some information about the microbial infections and about the role of herbal plants in treating microbial infections.
Authors must include the literature about the chief constituents present in these plant species.
Authors reported that Pasak Bumi is a local plant in Kalimantan, Indonesia. Authors must include other geographical regions where this plant grows.
Authors must include some information that why only yeast Candida albicans and Staphylococcus and bacterial strains were taken for the aureus, Streptococcus mutans, Streptococcus sobrinus study?

Methods
For identification of the phytoconstituents present in these plant species authors must have included the preliminary phytochemical screening, (a qualitative identification) or must have reported the literature on the phytochemical screening of these plants.
Why 40% ethanol was used to prepare different concentrations of the plant extracts?
3. In figure 1 authors have reported that ±50 gram of sample powder was taken for further maceration with n-hexane. Is there any variations in the initial weight of the powder? Why symbol ± is added? Is there any standard deviation or SEM?

Results
The activity index (Fig. 2) shows very little difference in concentration 500 and 1000 µg/well. As the concentration 1000 µg/well is double of the 500 µg/well so the difference in activity index should have been more. This suggests that something is wrong with the assays and/or with the presentation of the data. Authors need to analyze and discuss this point critically.

Grammatical errors/Corrections
Eg. In conclusion The present study performed studies of antimicrobial properties of three different Pasak Bumi ( in vitro E. Jack, and ) on oral pathogens which gave positive results and longifolia R. elliptica T. macrophylla different degree of activity.
The above statement needs to be revised "The present study performed studies…………? in vitro Is the work clearly and accurately presented and does it cite the current literature? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Partly
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: Microbial Infections, Natural Products I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. 07