The potency of ethanolic extract from corn silk as natural antibiotics for acne-related bacteria: A preliminary study

Introduction: Corn silk or known as herb name stigma maydis is an important medicinal botanical in many traditional medicines worldwide, including jamu, an Indonesia traditional medicine. The exploration of corn silk to treat acne vulgaris is still lacking, therefore the current research was conducted to analyze the activity of ethanolic extract from corn silk (EECS) against 3 acne-related bacteria, Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus. Materials and Methods: The antibacterial activity of EECS at concentration range of 10 to 100% v/v was evaluated using the disk diffusion method. As comparation, distilled water was used as a solvent control, while 1% clindamycin was used as a positive control. Results: Shinoda’s test showed that flavonoid was detected in the EECS. The higher concentration of EECS exhibited higher diameter of inhibition zone indicating higher antibacterial activity on P. acnes, while the antibacterial activity of S. epidermidis was not increased at similar concentrations of EECS. The antibacterial activity of EECS against S. aureus decreased at the higher EECS concentration (>70%). Conclusion: Taken together, EECS is a potential as a bioactive source to inhibit the growth of acne-related bacteria P. acne, S. epidermidis and S. aureus. Further investigation is needed to explore the corn silk or stigma maydis as a medicinal botanical in jamu targeted to treat acne vulgaris.


Introduction
Corn (Zea mays L.) or "jagung" is the second staple food after rice, which supports food security in Indonesia. In 2018 the corn production in Indonesia reached 30 million tons. 1 Corn grains are usually utilized as food and feed sources, whereas other parts of the corn plant like corn silk is a biological secondary product of corn cultivation. The utilization of corn silk as a botanical medicine can be an added value of corn to improve the income of farmer in Indonesia.
Corn silk or known as herb name stigma maydis is a female inflorescence of corn in form of fine soft thread 10-20 cm long. Corn silk contained several compounds including flavonoids, tannins, terpenoids, steroids, alkaloids, saponins, carotenoids, anthocyanins. [2][3][4] Nowadays, corn silk is reported as an important medicinal botanical in many traditional medicines worldwide. 5,6 Bioactive constituents such as flavonoids and terpenoids showed potential antidiuretic 7 , hypoglycemia 8 , anti-cancer 9 , hypocholesterolemia 10 , hypopigmentation 11 , hypothyroidism 12 , antifungal, antiviral and antibacterial activity 3,[13][14][15] . In Indonesian traditional medicines, stigma maydis is used as one of ingredients in jamu claimed to treat urinary related problems, to relieve pain and inflammation of arthritis, to reduce uric acid, and to reduce body fat. 16 The exploration of corn silk to treat acne vulgaris is still lacking, although antibiotic resistance in acne patients raises a concern worldwide. 17 Acne vulgaris is one the skin-related neglected tropical diseases which showed a highly prevalent inflammatory skin condition in up to 90% of teenager. 18 The disease has been associated with colonization of bacteria Propionibacterium acnes in sebaceous areas, Staphylococcus epidermidis and Staphylococcus aureus in moist human skin areas. [19][20][21][22] The aim of the current research was to analyze the antibacterial activity of ethanolic extract from corn silk and compare the activity with standard antibiotic clindamycin in the 3 acne-related bacteria. The results can be used to provide an evidence of the antibacterial activity of the corn silk extract as lowcost herbal antibiotic to treat acne vulgaris.

Extraction preparation
Fresh corn silks were collected from the corn farmer in Grobogan, Purwodadi, Central Java, Indonesia. The silk was harvested at maturity stage (30 days after silking). The colors of silk were yellow-brown (Fig. 1a). A given amount (2 kg) of fresh corn silks was cleaned with tap water and was thereafter dried in an oven at 50 °C for 5 days to reach a constant weight. The oven-dried corn silk (Fig. 1b) was milled using an electric blender. The fine powder was extracted using 70% ethanol for 1 week at room temperature. The extract solution was filtered through a filter paper (Whatman No. 1) to remove insoluble material. The filtrate obtained was then dried using rotary evaporator and N 2 gas. The ethanolic extract were stored at 0-4 °C until analysis. The antibacterial activity of ethanolic extract of corn silk (EECS) was evaluated on the 3 different strains of bacteria using the disk diffusion method. 23,24 A suspension of 200 ml of EECS was used as a 100% of EECS concentration, while EECS concentration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% was diluted from the 100% EECS concentration with distilled water. As comparation, distilled water was used as a solvent control (0% EECS), while 1% clindamycin (CDM) was used as a positive control. A volume the 20 µL of each test solution were applied into paper disk of 6.0 mm diameter, then the disk was placed on the agar surface. Upon incubation at 37 °C for 24 hours, diameter of inhibition zone was measured. The absence of a zone inhibition indicates the absence of antibacterial activity. Five replications of these tests were performed for each strain of bacteria evaluated.

Statistical analysis
Data of concentration-inhibition zone data were fitted with an interleaved bars graph as mean values ± SEM. Since the data were not normally distributed, comparisons between multiple groups were analyzed by Kruskal-Wallis test followed by a post hoc Dunn's Multiple Comparison Test. Statistically, p < 0.05 was considered significant. All analyses were done in GraphPad Prism software (version 5.00 for Windows, GraphPad software, San Diego, USA).

Flavonoid test
Shinoda's test showed that flavonoid was detected in the crude ethanolic extract from corn silk (EECS). EECS was extracted using 70% ethanol as reported by Limmatvapirat et al. (2020) that flavonoids content was higher in corn silk extracted using 40% v/v ethanol than those extracted by water. 2 Table 1 shows flavonoid content in corn silk revealed from the reported studies. Apart from flavonoid, ethanolic extract of corn silk contained flavonoids, tannins, terpenoids, steroids, and phenolic compounds like anthocyanins, p-coumaric acid, vanillic acid, quercetin, etc. 2,4,25 The phytochemical constituents of corn silk were influenced by corn hybrid, maturity stage of corn silk, cultivation region, and solvent polarity. 2,25 Therefore, a future research to quantitatively analysis the phytochemicals constituents of corn silk from Indonesia would be relevant to explore it as Indonesia's indigenous botanicals. The assessment will be of interest considering the high number of corn cultivation in Indonesia, and the fact that Indonesia government flags to develop Indonesian modern drug/ Obat Modern Asli Indonesia (OMAI). 26

Antibacterial activity of ethanolic extract (EECS) from corn silk
To assess the antibacterial activity of EECS, 3 acne-related bacteria Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus were exposed to increasing concentrations (10 to 100%) of EECS for 24 h. Figure 2 depicts the antibacterial activity of EECS against P. acnes increased in a concentration-dependent manner. The higher EECS exhibited higher diameter zone indicating higher antibacterial activity on P. acnes, while the antibacterial activity of S. epidermidis was not increased at similar concentrations of EECS. In the other hand, the antibacterial activity of EECS against S. aureus decreased at the higher EECS concentration (>70%).   31 The promising antibacterial effect of flavonoid in EECS can be attributed to apoptosis induction via the ROS-mediated mitochondrial pathway in the anticancer activity of corn silk extract against human breast cancer (MCF-7) cells. 9 Moreover, it has been recognized that flavonoids are very effective antioxidants [2][3][4]27 which may contributed to the antibacterial activity. Further research, probably involving co-cultured of the 3 bacteria assays, would be needed to analyze the antagonism effect of EECS against P. acne, S. epidermidis and S. aureus and to determine at which acne-related bacteria the EECS are effective in treatment of acne vulgaris. The mechanism of antagonism S. epidermidis to P. acne is reported by excretion of succinic acid 32 or polymorphic toxins 33 .

Conclusions
Taken together, EECS is a potential as a bioactive source to inhibit the growth of acne-related bacteria Propionibacterium acne, Staphylococcus epidermidis and Staphylococcus aureus. Further investigation is needed to explore the corn silk or stigma maydis as a medicinal botanical in jamu targeted to treat acne vulgaris.