Influence of Hibiscus tilliaceus Extract and Suspension on Echerichia coli and Staphylococcus aureus Growth

. This research aims to know the influence of suspension compared to its crude extract on antibacterial activity, especially for Escherichia coli and Staphylococcus aureus . An independent t-test was used as statistical analysis. The yield of the thick extract obtained was 11.153 % ± 1.032 % and it gave an inhibition zone of 7.878 mm ± 0.301 mm for Escherichia coli and 7.484 mm ± 0.636 mm for Staphylococcus aureus . While the suspensions gave an inhibitory zone of 6.293 mm ± 0.754 mm and 5.140 mm ± 0.121 mm for Escherichia coli and Staphylococcus aureus in sequence. The extract's particle size followed by suspension was 951.167 mm ± 113.662 nm and 474.000 mm ± 38.500 nm. The independent t-test results showed that the extract and suspension significantly influenced the growth of Staphylococcus aureus and Escherichia coli with sig values of 0.012 and 0.028. In conclusion, the extract had a greater effect than the suspension on the growth of Staphylococcus aureus and Escherichia coli .


Introduction
The problem with using extracts is that the active substance is difficult to penetrate the lipid membrane of body cells because it has a large molecular size and low water solubility, which causes poor absorption and bioavailability.This causes many plants that have potential active substances that cannot be used in vivo even in vitro tests to have good results.There are efforts to improve the effectiveness of extract by reducing its particle size [1,2].Some of the advantages obtained by reducing the particle size of the extract are increasing the extract yield, the amount of active substance, the solubility of the extract in water, antidiabetic activity, surface area, viscosity, antibacterial activity, herbal therapeutic effects, and reduced toxicity [2][3][4][5][6][7][8].Based on the facts, the researchers varied the particle sizes of the extracts to obtain optimal extract activity.
Several methods for generating low particle size compounds are high-pressure homogenization, complex coacervation, co-manipulation, salting-out, nanoprecipitation, diffusion-emulsification of solvents, supercritical liquids (including the fast expansion of supercritical solutions (RESS), and fast expansion of supercritical solutions into a liquid solvent (RESOLV)), self-assembly, dialysis, etc.These methods must be matched with each type of compound being made [2].
To produce low-sized particle extracts, people usually used ionic gelation methods.The advantage of this method is that the preparation is simple, without using harmful organic solvents, and also without heating so that it doesn't damage the active substances in the extract.In the process, this method produces a suspension.Some studies use suspensions directly, and other studies use extracts obtained from suspension drying for further testing.Several pharmacological activity tests for suspension, among others analgesic activity, drug delivery activity, antioxidant activity, etc.The suspension consists of crude extract, sodium tripolyphosphate, acetic acid, ethanol, and chitosan.All of these constituents have antimicrobial activity for either bacteria or fungi.So that raises the question, how is the antimicrobial activity of the suspension because its constituents also have antimicrobial activity.Therefore, this research aims to know the influence of suspension compared to its crude extract on antibacterial activity, especially for Escherichia coli (Migula 1895, Castellani and Chalmers 1919) and Staphylococcus aureus (Rosenbach 1884).E. coli represents gram-negative bacteria whereas S. aureus represents gram-positive bacteria.

Methods
The method used in this research is outlined in Figure 1.Antibacterial activity testing detail begins with inoculation of the E. coli and S. aureus bacteria with Nutrient agar media and incubation for 24 h.Amount 1 mL of the E. coli and S. aureus suspension each was put into a petri dish and added with Mueller-Hinton agar.After hardening, a well hole is made and filled with thick extract or suspension.Incubation was carried out for 1 × 24 h at 37 ºC.
To determine the significance of the influence of suspension compare to its thick extract on antibacterial activity, especially for E. coli and S. aureus was used independent t-test as statistical analysis.

Results and discussion
H. tiliaceus leaf extraction was carried out by remaceration repeatedly until the filtrate was close to colorless.The goal is for maximum withdrawal of active substances.The yield obtained from the thick extract was approximately 11.153 % ± 1.032 % close to the yield that has been reported in the study of the Acute Toxicity Test of H. tiliaceus Leaves Extract in White Male Mice", which was 11.9881 % with ethanol 70 % solvent [9].Polarity which does not differ much between methanol and ethanol 70 % causes the resulting yield to be almost the same.However, further testing of the active substance levels is needed.
From its appearance, the thick extract's color is dark green-black and very thick while the suspension's color is light green-brown and liquid-like shown in Figure 2. The color of the suspension obtained was different from the color of the filtrate from the maceration process (dark green like a leaf).Heating in the evaporation process can cause color fading due to the oxidation of some of the active substances contained.

Fig. 2. The appearance of thick extract and suspension
Preparation of the suspension with the ionic gelation method is considered could produce extracts with a low particle size.To prove it, the particle size of the thick extract and the suspension was measured.The particle sizes of thick extract and suspension are presented in Table 1.  1, it can be seen that the particle size of the thick extract is about two times larger than that of the suspension.The advantages of using ultrasonic waves in producing low particle size extracts compared to conventional methods are speeding up the process and producing higher yields [10].The presence of ultrasonic waves can vibrate the extract particles so that they do not aggregate to form a larger particle size.
All of the constituents to prepare the suspension have antimicrobial activity for either bacteria or fungi.H. tiliaceus leaves showed very good results against all gram positives and gram-negative bacteria tested [11].Sodium tripolyphosphate excellent in inhibits the growth of several gram-negative and positive bacteria [12].Acetic acid has been shown to have good antibacterial activity against micro-organisms such as Pseudomonas aeruginosa [13].Ethanol is commonly used as antimicrobial agent.Meanwhile, chitosan has a broad antimicrobial spectrum so that it can inhibit gram-negative and gram-positive bacteria, and also fungi [14].It is expected that by using all materials that have antibacterial activity, it can produce suspension which has superior antibacterial activity.The antibacterial activity of thick extract and suspension is presented in Table 2 as inhibition zone data.Inhibition zone obtained from thick extract have larger than the suspension for both E. coli and S. aureus.This is because the active substance in the extract which is filled into the well's hole is larger for the thick extract than the suspension.The addition of other compounds to make up the suspension proved unable to increase the antibacterial activity compared to the thick extract.Meanwhile, the ability of the extract and suspension to inhibit the growth of E. coli was greater than that of S. aureus.This is because gramnegative bacteria (E.coli) have thinner cell walls than gram-positive bacteria (S. aureus) so they are easily damaged by the presence of antibacterial compounds.The cell wall of grampositive bacteria consists of several layers of peptidoglycan which form a thick and rigid structure contains a cell wall substance called teichoic acid, while the cell wall of gramnegative bacteria consists of one or more thin peptidoglycan layers, so the cell wall of gram-negative bacteria is more susceptible to physical shocks, such as giving antibiotics or other antibacterial agents [15].The antibacterial strength classification according to Davis and Stout (in 16] is that extracts with an inhibition zone diameter of more than 20 mm are in the very strong category, a diameter of 10 to 20 mm are in a strong category, a diameter of 5 to 10 mm are in the moderate category and diameter of less than 5 mm are in the weak category.Based on the data in Table 2, it can be concluded that the antibacterial strength of both thick extract and suspension is in the moderate category.For further research, it is necessary to compare the antibacterial activity of thick extract with dried suspension.
The significance of the influence of suspension compare to its thick extract on antibacterial activity especially for E. coli and S. aureus was tested by independent t-test as statistical analysis.The results showed that the extract and suspension significantly influenced the growth of S. aureus and E coli with sig values of 0.012 and 0.028.

Conclusion
The extract had a greater effect than the suspension on the growth of Staphylococcus aureus and Escherichia coli.

Fig. 1 .
Fig. 1.The research methodIn detail, the process of making the suspension was carried out by mixing 1 g of the H. tiliaceus thick extract with 12.5 mL of 96 % ethanol then sonicated at 40 kHz for 10 min (called Mixture A).Then 0.013 g of sodium tripolyphosphate were mixed with 12.5 mL of aquadest and sonicated at 40 kHz for 5 min (called Mixture B).After that 0.225 g of chitosan were mixed with 75 mL of 1 % acetic acid solution and then sonicated at 40 kHz for 25 min (called Mixture C).Mixture A was then poured into Mixture B while sonicated at 40 kHz for 20 min (called Mixture D).The process is ended by pouring Mixture D into mixture C then sonicated at 40 kHz for 50 min.Antibacterial activity testing detail begins with inoculation of the E. coli and S. aureus bacteria with Nutrient agar media and incubation for 24 h.Amount 1 mL of the E. coli and S. aureus suspension each was put into a petri dish and added with Mueller-Hinton agar.After hardening, a well hole is made and filled with thick extract or suspension.Incubation was carried out for 1 × 24 h at 37 ºC.To determine the significance of the influence of suspension compare to its thick extract on antibacterial activity, especially for E. coli and S. aureus was used independent t-test as statistical analysis.

Table 1 .
The particle sizes of thick extract and suspension

Table 2 .
The inhibition zone of thick extract and suspension