Anti-microbial Properties of Thai Traditional Flower Vegetable Extracts Wunwisa Krasaekoopt and Areeya Kongkarnchanatip Faculty of Biotechnology, Assumption University Bangkok, Thailand Abstract In this research, three types of Thai traditional flower vegetables, Sesbania grandiflora, Senna siamea and Telosma minor, were used due to their claim on helping person who has stomach disorder. Ratio of flower to water of 1:2 was used for water extraction with shaking condition for seven days. The crude extracts were then examined for anti-microbial properties using disc diffusion test on three types of bacteria, Bacillus cereus, Escherichia coli and Staphylococcus aureus. The results indicated that the seven-day extraction provided the highest anti-microbial properties of these three flower vegetables on all bacteria, especially for S. aureus that had the highest inhibition zone. In addition, the anti-microbial activity of Senna and Sesbania were higher than that of Telosma extract. Using column chromatography, the crude flavonoid was separated. The percentage of flavonoid found in Sesbania flower, Senna flower and Telosma were 8.4, 8.6 and 3.4%, respectively. The anti-microbial properties of these crude flavonoids were also investigated and the obtained results were corresponding to those of crude extracts. Keywords: Stomach disorder, crude extract, flavonoid, Senna, Sesbania Telosma 1. Introduction In Thailand, there are many kinds of flower vegetables that have medicinal properties. are flowers of traditional vegetable plants that have flowers and have been used for cooking since ancient time. It was believed that consumption of these flower vegetables can cure illness and diseases. They also help people who suffer from diarrhea, which indicates the antimicrobial activity of these vegetables (Somanapan 1990; Vachirasup 1995; Boonyaprapatsara 1996 and 2000). In addition, the countries in Central Africa, West Africa and Indonesia used Khilek flower (Senna siamea) to treat patients who had stomachache (Grieve 1981). The flower vegetables are composed of various chemical compounds that can be grouped as flavonoid, anthraquinone and glycoside. Flavonoid is expected to be the main component that plays a major role in microbial inhibition. This research was aimed to study the extraction of flavonoid from three types of flower vegetables, namely: Thai copper-pod or Khilek flower (Senna siamea), cork-wood tree or Dok Khae flower (Sesbania grandiflora) and Telosma or Khachon flower (Telosma minor). In addition, the anti-microbial activity of these flower vegetables was investigated. 2. Materials and Methods 2.1 Extraction of Flavonoid from Flower Vegetables Three kinds of flower vegetables were used in this experiment, namely: Thai copperpod or Khilek flower (Senna siamea), corkwood tree or Khae flower (Sesbania grandiflora) and Telosma or Khachon flower (Telosma minor). They were blend with distilled water with the ratio of flower to water as 1:2. The mixtures were left to stand for seven days with occasional shaking. The samples were collected everyday and then filtered using Whatman No. 4. The supernatants were kept in brown bottles in the refrigerator. These samples were then tested 71
for anti-microbial activity as described in Section 2.3. 2.2 Column Chromatography Separation Flavonoids in the extracted solutions were separated using column chromatography. Cellulose powder (Himedia RM 126) was used as absorbent, while butanol (Fluka) was used as diluents (Cassidy and Gomes 1991). The obtained solutions were then put in the oven at 50 C to evaporated butanol from the samples. The flavonoids were then kept in the brown glass bottle in the refrigerator. 2.3 Anti-microbial Activity Testing Flavonoid extracted from flower vegetables was examined for anti-microbial activity on Escherichia coli, Staphylococcus aureus and Bacillus cereus, which were the representatives. for rod, cocci, gram-positive and gram-negative bacteria. The test was done using disc diffusion test. A plate of nutrient agar (NA) was inoculated with 0.1 ml of bacterial suspension. Before incubation, a small absorbent paper disc, impregnated with extracted flavonoid from flower vegetables, was placed on the plate. The plate was then incubated at 37 C for 24 hr. After that the inhibition zone was measured in mm. 2.4 Statistical analysis A completely randomized design and analysis of variance (ANOVA) were applied in this experiment. Comparisons of means were carried out using least significant difference (LSD) test. Table 1. Anti-microbial activity of crude solutions extracted from Sesbania, Senna and Telosma flowers on bacteria Flower vegetable Time(day) E.coli B.cereus S.aureus Sesbania grandiflora 1 6.5 i * 7.2 h 7.3 h 2 7.3h 7.2 h 9.3 f 3 8.5 g 7.2 h 10.3 bc 4 8.6 g 9.1 f 10.3 bc 5 10.0 c 9.7 de 10.5 b 6 10.3 bc 9.8 d 10.5 b Senna siamea Telosma minor 7 10.5 b 9.8 d 11.3 a 1 7.5 j 7.3 j 7.3 j 2 7.6 ij 7.6 ij 7.5 ij 3 8.0 ghi 8.2 gh 7.7 ghij 4 8.8 ef 8.3 fg 9.7 bc 5 9.0 de 10.0 bc 9.7 bc 6 9.5 cd 10.0 bc 9.8 bc 7 10.2 b 10.0 bc 11.7 a 1 6.8 h 7.5 ef 7.3 fg 2 7.0 gh 7.5 ef 7.3 fg 3 7.2 fg 8.5 cd 7.5 ef 4 8.2 d 8.8 c 7.8 e 5 8.7 c 9.5 ab 8.2 d 6 8.8 c 9.5 ab 9.7 a 7 9.3 b 9.7 a 9.7 a 72
* The same letter presents non-significant different at 95% confidential level. The statisticalanalysis was done separately for each type of flower vegetable components that have anti-microbial activity. 3. Result and Discussion These occurred in all types of flower vegetables. The crude solution of Sesbania exhibited the 3.1 Extraction of Flavonoid from Flower highest inhibition activity on S. aureus (11.3 Vegetables mm), followed by E. coli and B. cereus as 10.5 and 9.8 mm, respectively. For Senna flower From the preliminary experiment, the extracted solution, the highest inhibition was crude solutions were extracted using both found in S. aureus, while the others were nonalcohol and water extraction. It was noticed significantly (P>0.05) different, with the value that the extracted solution using alcohol of 10.0-10.2 mm. Moreover, the crude presented low anti-microbial activity on three extracted solution from Telosma flower types of microorganisms used, namely: E. coli, showed the highest inhibition on both B. cereus B. cereus and S. aureus, compared with those and S. aureus. of water extraction. These occurred in all types In addition, it was recognized that all of of flower vegetables used in this experiment. the crude solutions extracted from all types of Therefore, the water extraction was used. flower vegetables had the ability to inhibit the The anti-microbial activity of each flower vegetable was investigated for seven days of extraction. The results are presented in Table 1 growth of S. aureus, which is gram-positive cocci pathogenic bacteria. Although there were no significant for all types of flower vegetables. differences (p>0.05) of each bacterial It was recognized that as the time of extraction increased, the anti-microbial activity significantly (p<0.05) increased. This was indicated that the extraction time directly inhibition among all types of flower vegetables after seven days of extraction (Table 2), the bacterial inhibition of crude solution extracted from Sesbania and Senna flowers was slightly influenced the concentration of extracted higher than those of Telosma flower. Table 2. Anti-microbial activity of crude solution extract for seven days of Sesbania, Senna and Telosma flowers on bacteria. E. coli B. cereus S. aureus Sesbania flower 10.5 c * 9.8 b 11.3 a Senna flower 10.2 c 10.0 b 11.7 a Telosma flower 9.3 c 9.7 b 9.7 a * The same letters present the non-significant difference at 95% confidential level. The statistical analysis was done separately for each bacterium. 3.2 Column Chromatography of Flavonoid Extracted From Flower Vegetables The crude solutions extracted from flower Table 3. Percentage of yield of flavonoids extracted from flower vegetables vegetables were then passed through the column chromatography in order to separate flavonoid from the crude solution. The percentages of flavonoid in each type of flower vegetables are presented in Table 3. 73
% yield of flavonoids Sesbania flower 8.4 a * Senna flower 8.6 a Telosma flower 3.4 b * The same letter presents no significant difference at 95% confidential level The column chromatography method was used to separate and determine the mount of flavonoid in flower vegetables. The adsorbent was cellulose powder, which was used as stationary phase and to separate all types of flavonoid. The water-saturated bunatol was the eluent, which was used as a mobile phase of column chromatography. The flavonoid was eluted and butanol was then evaporated at 50 C.Sesbania and Senna flowers contained flavonoid about 8.4-8.6%, which were significantly higher (p<0.05) than those of Telosma flower(3.4%).therefore, this may be the reason to support that the crude solution extracted from Sesbania and Senna flowers had higher anti-microbial activity than that of Telosma flower. The anti-microbial activity of crude flavonoid of flower vegetables was also investigated (Table 4). Crude flavonoid from all flowers has the highest anti-microbial properties on S. aureus. Moreover, crude flavonoid from Senna flower illustrated the better microbial inhibition than the others. This result was also parallel to those of crude extracted solution. Table 4. Anti-microbial activity of crude flavonoid extracted from Sesbania, Senna and Telosma flowers on bacteria E. coli B. cereus S. aureus Sesbania flower 14.3 b 13.7 c 15.3 a Senna flower 14.3 b 14.0 bc 16.7 a Telosma flower 13.2 c 14.0 bc 14.2 bc * The same letters present the non-significant difference at 95% confidential level 4. Conclusion The appropriate extraction process of flavonoid from flower vegetables was water extraction for seven days with shaking condition. The crude extracted solution of Sesbania grandiflora and Senna siamea flowers had higher microbial inhibition than that of Telosma flower due to higher flavonoid contents. Moreover, the crude extracted solution of these flower had the highest anti-microbial activity against Staphylococcus aureus, which is one type of food poisoning bacteria. In addition, the crude flavonoid extracted from these flower also had the similar anti-microbial activity to the extracted solution. 74
5. References Boonyaprapatsara, N. 1996 and 2000. Thai Traditional Herbal Medicine Plant. Vols. 1 and 4. Prachachon Publ., Bangkok, Thailand. Cassidy, G.; and Gomes, H. 1991. Adsorption and Chromatography, 1 st ed. Interscience, New York, NY, USA. Grieve, H. 1981. The Herb: New Plants. http://www.botanical.com/botanical/mgmh/c omindx.html. Somanapan, A. 1990. Medicine from Herbal Plant, 1 st ed. Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. Vachirasup, T. 1995. Senna plant in Thailand, 1 st ed. Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. 75