Antibacterial activity of Magnolia alba flower extracts on Staphylococcus epidermidis and Staphylococcus aureus

The white champaca (Magnolia alba) plant has been reported possess antioxidant and antimicrobial activity. The aim of this study was to investigate the antibacterial activities of n-hexane, ethyl acetate, and methanolic Magnolia alba flower extracts on Staphylococcus epidermidis and Staphylococcus aureus. In this study, we also determined the secondary metabolites of the extracts by the phytochemical screening assay. The antibacterial activity of the Magnolia alba flower extracts was determined by the Kirby-Bauer diffusion method. The phytochemical screening assay showed that n-hexane extract contained of flavonoids, terpenoids, and steroid, while the ethyl acetate and methanolic extracts contains of alkaloids, flavonoids, terpenoids, and steroid. The antibacterial activity of the n-hexane, ethyl acetate, and methanolic Magnolia alba flower extracts was determined at four different concentrations of 5, 10, 20, and 50%. Results indicated that n-hexane extract had no activity against Staphylococcus epidermidis and Staphylococcus aureus. Meanwhile, ethyl acetate and methanolic extracts had antibacterial activities against Staphylococcus epidermidis and Staphylococcus aureus. The diameter zones of inhibition exhibited by the ethyl acetate extract against Staphylococcus epidermidis and Staphylococcus aureus ranged between 10.45 - 21.03 mm and 10.26 - 26.13 mm respectively. Meanwhile, the diameter zones of inhibition exhibited by the methanolic extract against Staphylococcus epidermidis ranged between 11.96 - 18.01 mm and against Staphylococcus aureus ranged between 7.23 - 13.9 mm. In conclusion, the ethyl acetate Magnolia alba flower extracts gave higher antibacterial activity against Staphylococcus epidermidis and Staphylococcus aureus.


Introduction
Natural antimicrobial substances, especially from plant origin, have attracted much attention in recent years [1]. A lot of efforts have been made to search for novel natural antimicrobials from various plant sources or natural products. Previous researchers revealed that Magnoliaceae family such as Magnolia champaca possesses antimicrobial activity [2,3,4]. The antimicrobial activities of Magnoliaceae family are due to containing secondary metabolites and their essential oil composition including alkaloids, sesquiterpene and terpenoids [5]. The extract of the bark of Magnolia champaca has been known contains sesquiterpene lactone, alkaloids parthenolide, and beta-sitosterol, while the flower and fruit extracts contain essential oil [6,7]. Nursa'adah reported that n-hexane extract of Magnolia champaka contains of phenols, terpenoids, and steroids. Although, the antimicrobial activity of Magnolia 2 champaka has been reported, studies pertaining to the antimicrobial activity of the flowers of white champaka (Magnolia alba) are still limited [8]. Magnolia alba also known as white champaca belonging to the Magnoliaceae family, have fragrant flowers (Fig. 1). Magnolia alba is widely used for medicine, all parts of this plant such as root, bark, leaves and flowers provide various benefits as medicine. The flowers of Magnolia alba are used for treatment of headache, sinusitis, cough, inflammation, flatulence, nausea, and vaginal discharge [5]. The flowers and leaves of Magnolia alba contain the essential oils that are used to relieve ophthalmic and gout [9]. Hsing mentioned that the extract of Magnolia alba leaves contain annonaine, a bioactive benzylisoquinoline alkaloid compound. He also showed that, this compound has strong activity in inhibiting the growth of bacteria and fungi [10]. Besides that, Sree showed that n-butanol flower extract of Magnolia alba has strong antimicrobial activity against Staphylococcus aureus by a diameter of the inhibit zone formed was 17 mm [11]. In the present study, we investigated the phytochemical screening and antibacterial activity of various flower extracts (n-hexane, ethyl acetate, and methanolic extracts) of Magnolia alba on Staphylococcus epidermidis and Staphylococcus aureus.

Plant Material and Identification
Fresh flowers of white champaca (Magnolia alba) were obtained from Desa Sukaramai, Baiturrahman District, Banda Aceh, Indonesia. The white champaca was identified in the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Darussalam, Banda Aceh, Indonesia.

Extracts Preparation
The flowers of Magnolia alba were first washed with tap water to remove the impurities and dried at room temperature for 26 days without exposure to the sunlight to produce a simplicial of flowers of Magnolia alba. The simplicial was weighed and broken down into small pieces by using a blender, than dried in the oven (Memmert thermostatic, universal oven UF55) at 50°C. The simplicial was then ground into fine powder, and macerated using multistage maceration method. The multistage maceration was performed by using three different solvents based on their polarity degree of solvents such as n-hexane, ethyl acetate and methanol. The extracts obtained were concentrated at reduced pressure to dryness using rotary evaporator (Rotaryvapor R-100, Buchi) until the solvent completed evaporation; the crude extracts obtained were dissolved and prepared in different concentrations: 5, 10, 20 and 50%. The concentrated extracts were stored at 4°C in labelled sterile screw-capped bottles till further analysis [12].

Characterization of Extracts
The crude flower extracts of Magnolia alba were characterized on the basis of water and ash contents, as well as solubility in water and ethanolic by using standard procedures [13].

Phytochemicals Screening
The crude flower extracts of Magnolia alba were used for phytochemical screening assay to detect the secondary metabolites such as alkaloids, flavonoids, saponins, tannins, and streoids/terpenoids by using standard procedures of phytochemical examination [14].

Antibacterial Assay
The antibacterial activity of n-hexane, ethyl acetate, and methanolic extract of Magnolia alba flower extracts was conducted using the agar disc diffusion (Kirby-Bauer) method. Mueller Hinton Agar (MHA) was poured in sterile Petri dishes and allowed to solidify, the test cultures of Staphylococcus epidermidis and Staphylococcus aureus were spread all over surface on the solidified MHA agar using a sterile cotton bud. The Whatmann paper disc of approximately 6 mm in diameter was placed on the surface of inoculated agar medium. Each disc was filled with 12 μl of each extract, the paper disc of clindamycin was used as positive control, while the solvents such as n-hexane, ethyl acetate, and methanol were used as negative control. The plates were incubated at 37°C for 24 h, the inhibition zones surrounding the agar disc were measured in millimeters [15].

Characterization of the Extracts
The results showed that the percentage moisture content of n-hexane, ethyl acetate, and methanolic flowers extracts of white champaca were 20.3; 39.96; and 25.42% respectively (Table 1). It is important to analyse the moisture content of the extract because higher moisture accelerates the growth of the microbes and can destroy the secondary metabolites in the extract. Voight (1994) reported that the moisture content in the plant extracts should not exceed 30% [16]. Table 1 showed that the moisture content of n-hexane and methanolic extracts were less than 30%, while ethyl acetate extract was not qualified as predetermined requirements. The average of water soluble contents of n-hexane, ethyl acetate, and methanolic flower extracts of white champaca were 0; 15; and 65.66%, respectively. Meanwhile, the ethanol soluble content of nhexane, ethyl acetate, and methanolic flower extracts of white champaca were 18.37; 56.33; and 79%, respectively. Determination of water and ethanol soluble contents was performed to determine the amount of the compounds soluble into a polar solvent. These results also indicated that ethyl acetate and methanolic extracts contain semi polar and polar compounds.
Determination of total ash was performed to determine the internal and the external of mineral contents of the extracts. The results showed that total ash of n-hexane, ethyl acetate, and methanolic flower extracts of white champaca were 2.25; 10.6; and 0.031%, respectively. These results showed that all extracts tested had low total ash contents. Total ash of the plant extracts is influenced by the physiological and environmental factors of plant [13].

Phytochemical screening of extracts
The phytochemical screening of n-hexane, ethyl acetate, and methanolic of Magnolia alba flower extracts are presented in Table 2. The preliminary results showed that phytochemicals including, alkaloids terpenoids, and steroids were present in the n-hexane of Magnolia alba flower extract, while flavonoids, saponins, tannins, terpenoids, and steroids were available in ethyl acetate of Magnolia alba flower extract. The phytochemical screening results also showed that all secondary metabolites tested were available or present in methanolic of Magnolia alba flower extract.  Non-pathogenic Staphylococcus does not ferment mannitol and medium remains red [15]. This is in accordance with our results which revealed that Shaphylococcus epidermidis on MSA medium was unable to change the medium from red to be yellow, while Staphylococcus aureus was able to change the medium to be yellow on MSA. The antibacterial activity of n-hexane, ethyl acetate, and methanolic extracts of Magnolia alba flower towards Staphylococcus epidermidis and Staphylococcus aureus are  Table 3 and Fig. 2. In this assay, the antibacterial assay of the extracts were performed at concentrations of 5, 10, 20 and 50%, the results showed that n-hexane of Magnolia alba flower (white champaca) extracts had no activity against Staphylococcus epidermidis and Staphylococcus aureus at concentrations used. This results were not in accordance with the results reported by Murniana which mentioned that n-hexane of Magnolia alba DC. flower (yellow champaca) extract has atibacterial activity against Staphylococcus aureus [17]. This is may be caused by difference in species of Magnolia alba that were used in this study.
From Table 3, it was evident that ethyl acetate extract of Magnolia alba flower had antibacterial activity against Staphylococcus epidermidis and Staphylococcus aureus at all concentrations tested. The data showed that the diameter of the inhibition zone increased by increasing the concentration of the extracts. According to Pelczar and Chan, the higher concentration has stronger activity in inhibiting the growth of microbes; this is because the secondary metabolites that penetrated into the cell of the bacterial at higher concentration will be greater [18].  Based on the antibacterial activity above, we could conclude that the greatest antibacterial activity was obtained from ethyl acetate extract, and followed by methanolic extract, while n-hexane extract did not show antibacterial activity to both of the bacteria tested. The antibacterial activity of ethyl acetate and methanolic extracts may be caused by the secondary metabolites contained in both extracts such as flavonoids, saponins, tannins, terpenoids and steroids. The antibacterial activity of ethyl acetate extract was greater than methanolic extract probably because the concentration of active compounds extracted in ethyl acetate extract was higher, and therefore more active to inhibit the growth of the bacteria. Further research should investigate the specific compounds that inhibit growth or kill the bacteria. In addition, the secondary metabolites contained in ethyl acetate extract are thought work synergistically to inhibit the growth of both bacteria tested.
Alkaloids act by interfering with peptidoglycan layer of the cell, so the cell wall layer of the bacteria is not fully formed which causes cell death. The flavonoids act as antimicrobial by forming complex compounds with extracellular proteins, so the bacterial cell membrane will be destroyed due to discharge of the intracellular contents from the cell. While, the saponins act by reducing surface tension of the cell membrane, the cell permeability will increase and leakage, followed by release of intracellular compounds. Tannins act by shrinking the cell wall or cell membrane thus disrupting the cell permeability of the bacteria [19]. Terpenoids interact with purine or transmembrane protein in the outer membrane of the bacterial cell wall, forming a strong polymer bond resulting in damage to the purine [20]. Meanwhile, the steroids act by inhibiting bacterial growth by destroying bacterial cell membranes that can cause bacteria to become lysed [21].

Conclusion
This study has demonstrated the antimicrobial activity of n-hexane, ethyl acetate, and methanolic of Magnolia alba flower extracts against Staphylococcus epidermidis and Staphylococcus aureus. It was conclusive that ethyl acetate and methanolic extracts possessed antibacterial effect towards Staphylococcus epidermidis and Staphylococcus aureus at concentrations tested, while n-hexane extract had no antibacterial activity against these bacteria. This results suggest that Magnolia alba flower can be used to develop bioactive substances that may have promising effect on the treatment of some diseases caused by Staphylococcus epidermidis and Staphylococcus aureus.