MATERIALS AND METHODS

Study area: The study was carried out during a period of 5 months, from February, 2019 until the end of June, 2019. The crude extracts of T. crispa stem was prepared in the laboratory of Environmental Chemistry, Department of Environmental Health and Industrial Safety, Universiti Kebangsaan Malaysia, Malaysia.

Preparation of plant: Tinospora crispa stem powder was obtained from Yuda Company Sdn Bhd at Rawang Selangor. The whole sample (1 kg) per packet was stored in a dry place at room temperature. Sixty gram of T. crispa stem powder was weighed using electronic weighting balance and used in this study.

Extraction: Three different types of solvents were used in two different extraction methods namely maceration and ultrasonication. The solvents used in this study consist of solvents from low polarity to high polarity (chloroform, methanol and aqueous).

Maceration: Tinospora crispa stems powder was extracted with 600 mL of chloroform, methanol and aqueous solution at a ratio of 1:10 and left to macerate for 3 days at room temperature with occasional shaking using orbital shaker. Then, extracts were filtered through Whatman paper No.1. The filtrates were concentrated under reduced pressure at 40-45°C using rotary evaporator and the dried crude extracts were weighed to calculate the yield and stored at (-20°C) until used for analysis.

Sonication: Extraction was performed using chloroform, methanol and aqueous solution by immersing the extracts into ultrasonic bath. Sonication was run for 30 min with ultrasound frequency >20 kHz. The filtrates were then concentrated using rotary evaporator to obtain the crude extracts. All collected extracts were stored at (-20°C) until use.

Preparation of sterile discs: Whatman filter paper No.1 was punched into 6mm discs and sterilized. Sterile discs were loaded with 10 μL of extracts (100 mg mL^–1) and left for hours in a sterile condition. The final concentration used for the tests was 1 mg/disc.

Test Microorganisms: Six species of bacteria (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonela typhimurium) and 3 species of fungi (Aspergillus niger, Candida albicans and Saccharomyces cerevisiae) were tested in this study. Bacteria and fungi were obtained from Department of Biomedical Sciences , Faculty of Health Sciences Universiti Kebangsaan Malaysia (UKM) whilst S. cerevisiae was purchased from Faculty of Sciences and Technology, Universiti Kebangsaan Malaysia Bangi.

Microorganism culture: Each of the bacteria strains was cultured onto nutrient agar plate and incubated for 18-24 h at 37°C while fungi were cultured onto Sabouraud Dextrose agar plate for 2-3 days at 32°C. Two or three colonies were then cultured in 5 mL of Mueller Hinton Broth for 4 h. The optical density of bacteria culture was adjusted to 0.5 McFarland standard (1.0×10⁸ CFU mL^–1) while for fungus was 1.0×10⁶ CFU mL^–1 using spectrophotometer measurements.

Determination of antimicrobial activity: Antimicrobial activity was determined by disc diffusion method. Bacterial and fungal culture adjusted to 0.5 McFarland standard was used to lawn Mueller- Hinton and Sabouraud Dextrose agar plates using sterile swabs respectively. These plates were then left to dry for 2-3 min before sensitivity test. The discs which were impregnated with extracts were placed on the Mueller-Hinton and Sabouraud Dextrose agar. Each of the test plate consisted of 5 discs, including 1 positive control (Gentamicin and Vancomycin) for bacteria while Nystatin for fungus, 1 negative control (DMSO 5%) and 3 extract-treated discs. The plate was incubated for 24 h at 37°C for bacteria and 2-3 days at 32°C for fungus. Then, the diameter of inhibition zone was measured by using ruler. The test was done in triplicates.

Minimum inhibitory concentration (MIC): Minimum inhibitory concentration (MIC) was determined using 96-well microtiter plates. Briefly, 50 μL of the methanolic extracts was mixed with 50 μL of Mueller-Hinton Broth before adding the standard inoculated bacterial culture into the mixture. The lowest extract concentration that did not show any bacterial growth for B. cereus and S. aureus was considered as MIC value.

Minimum bactericidal concentration (MBC): The test mixture concentration which did not show any growth of bacteria in S. aureus and B. cereus obtained from determination of MIC were subcultured onto Mueller Hinton Agar and incubated for 24 h at 37°C. The least concentration with no growth of these bacteria was considered as the MBC value.

RESULTS

The percentage yield of T. crispa stem extract from maceration using aqueous solution was the highest (11.0%), followed by methanol (3.0%) and chloroform (2.02%) respectively as shown in Table 1. Meanwhile, sonication method produced the highest percentage of extracts from aqueous extraction (11.78%), followed by methanol extraction with 3.15% and chloroform extraction with 2.68%. Sonication yielded a slightly higher percentage of extract as compared to maceration for all the solvents used as shown in Table 2.

From the antimicrobial tests, it was observed that methanol extracts obtained from maceration alone showed antibacterial activity against 2 g positive bacteria (B. cereus and S. aureus) but did not show any activity against B. subtilis. Diameter of inhibition zone for the methanol extract were 4.0±0.5 mm for B. cereus and 8.3±0.6 mm for S. aureus at the concentration of 100 mg mL^–1 (1.0 mg/disc). However, chloroform and aqueous extracts did not show any antibacterial activities against Gram positive bacteria. Furthermore, all T. crispa extracts showed no inhibition against Gram negative bacteria (E. coli, P. aeruginosa and S. typhimurium). Extracts from sonication using all extraction solvents exhibited no inhibition against all tested microorganisms. Moreover, T. crispa stem extracts from both maceration and sonication also failed to exhibit any antifungal activity against the tested fungal species as shown in Table 3.

Minimum inhibition concentration (MIC) test was performed for methanol extract against selected bacteria i.e., B. cereus and S. aureus. The MIC value for B. cereus and S. aureus using standard antibiotics was 1.0 mg mL^–1 and using methanol extract was 100 mg mL^–1 . Methanol extract showed MBC value at 5.0 mg mL^–1 against S. aureus alone.

Table 1:	Percentage of T. crispa crude extract using maceration method

Table 2:	Percentage of T. crispa crude extract using sonication method

Table 3:	Diameter of inhibition zone by T. crispa stem extract prepared using maceration method

DISCUSSION

The basis for choices of solvents used in this study is based on their polarity. The polarity index of the each selected solvent is shown in Table 2. Extraction of T. crispa stem resulted in highest percentage yield of crude extracts using aqueous solvent compared to methanol and chloroform for both maceration and sonication methods. Aqueous solvent is effective in extracting dissolved chemical compounds because water has a high polarity and shorter chain which helps in increasing the ability to extract polar compounds¹⁴. The percentage of aqueous extracts for the sonication method (11.78%) was slightly higher compared to the maceration method (11.0%). The sound waves generated during the sonication process can help cavitation that will increase the mass transfer of the cell contents¹⁵.

From this study, methanol extract from maceration method showed weak inhibitory activity against S. aureus and B. cereus with average diameter of the inhibition zone <9 mm. The results from this study were similar to the findings by Islam et al.¹⁶. The study showed a little inhibition zone of methanol extract at the concentration of 0.4 mg/disc against S. aureus (7.9 mm), B. cereus (4.6 mm), E. coli (2.8 mm) and P. aeruginosa (3.5 mm). Another study showed no inhibition zone against selected microorganisms (B. cereus, E. coli, P. aeruginosa and Candida albicans) at the concentration¹⁷ 0.5, 1.0, 2.5, and 5.0 mg disc^–1.

There are several factors that could have contributed to the negative results seen for disc diffusion study here. Bioactive compounds in the crude extracts can only show antibacterial activity at sufficient amount of concentration¹⁸. In addition, Sulaiman et al.¹⁷stated that there is a probability that the bioactive compounds extracted through maceration are insufficient to inhibit the growth of tested microorganisms.

Sonication method showed no antibacterial activity against all the bacterial cultures. The bioactive compounds in crude extract of T. crispa are inactivated by sound waves generated during sonication process¹⁹. This is due to the formation of free radicals that can damage the molecular structure of bioactive compounds²⁰.

Meanwhile, chloroform and aqueous extracts did not show any antibacterial activity against the bacteria tested for both extraction methods. The percentage yield of aqueous extract was higher than methanol and chloroform extraction but only methanol extracts showed antibacterial activity in this study. This shows that antibacterial activity is unaffected by the higher yield of the crude extracts but depends on the polarity of solvent which can influence the bioactive compound extracted from the T. crispa stem²¹. Methanol extract showed antibacterial activity against B. cereus and S. aureus alone suggesting that bioactive compounds in T. crispa stems can be extracted through methanol alone²². Methanol may dissolve polar compounds such as flavonoids, polyphenols, tannins and saponins²³. Tannins extracted using methanol had shown antimicrobial activity. Tannin can damage the polypeptide compound on the cell wall of bacteria and interfere with the transmission of protein sources to inhibit bacterial growth²⁴.

Inhibition showed in this study was more sensitive to Gram positive bacteria compared to Gram negative bacteria. The results of this study were similar with study conducted by Cowan²³, who reported that Gram negative bacteria are more resistant. According to Von Staszewski et al.²⁵, bacterial cell wall of Gram negative is more complex comprising of phospholipid membranes and peptidoglycan layers that prevent antimicrobial substances from penetrating them. Nevertheless, no antibacterial activity was demonstrated by all the crude extracts against B. subtilis (Gram positive). The results of this study were supported by Gami and Parabia²⁶ who reported that methanol extract of Tinosporacordifolia stem is inactive against B. subtilis. These bacteria are less sensitive due to their ability to form endospores that make them resistant to the environment²⁷.

No antifungal activity was shown for both extraction method using all extracts against selected fungi. The resistance of the fungus to the extract was likely due to the differences in mechanisms between fungi and bacteria.

According to Ghannoum and Rice²⁸, fungus has a distinct diploid structure consisting of ergo sterol, the most important component of the cell membrane that prevents antifungal materials from penetrating the fungal cells.

CONCLUSION

The percentage of yield for aqueous extract of T. crispa using maceration and sonication methods was higher compared to that from chloroform and methanol extraction. Sonication method was a better extraction method whereby the percentage of yield obtained for all the solvents was slightly higher than for the maceration method. The methanol extract obtained using maceration alone showed low antibacterial activity against S. aureus and B. cereus with inhibition zones of 4.0±3.5 and 8.3±0.6 mm respectively, at the concentration of 100 mg mL^–1 (1.0 mg/disc). The methanol extract had an MIC value of 100 mg mL^–1 against S. aureus and B. cereus and an MBC value of 5.0 mg mL^–1 against S. aureus. The methanol extract acted as a bacteriostatic agent against B. cereus and as bactericidal agent for S. aureus.

SIGNIFICANCE STATEMENT

This study discovered the active compounds in the crude extracts of T. crispa can be beneficial for human body to act as anti-therapeutic agent and antimicrobial agents. Various studies show that T. crispa extracts contain many phytochemicals that can be used to treat various kind of diseases. This study will help the researchers to uncover the critical areas of other parts of T. crispa that many researchers were not able to explore. Thus, the new and safer antimicrobial agents from medicinal plants can be developed.

ACKNOWLEDGMENT

We would like to thank the Malaysian Ministry of Higher Education for the financial support via grant (grant number: GGP-2017-082) and the laboratory staff of Environmental Health and Industrial Safety Programme as well as Biomedical Science Program, Faculty of Health Sciences, Universiti Kebangsaan Malaysia for the technical support.