Activity of Kalanchoe integra against Selected Pathogenic Bacteria

Chemicals, Plant and Microorganisms

All chemicals and reagents utilized were analytical grade. They were purchased from Sigma-Aldrich (USA) and Hi Media Laboratories (Mumbai, India). K. integra leaves and stem were collected from the INES- Ruhengeri botanical garden. The microbes used were S. typhi, S. sonnei, H. influenza, E. coli, S. pneumonia and S. aureus. They were from Rwanda biomedical center.

Preparation of Kalanchoe Integra Extract

Leaves and stem were washed and dried at room temperature (24ºC) for one week. They were then put in hot air oven for 30 min at 100ºC for full dryness. 4 kg of K. integra leaves and 2 kg of stem were blended differently to form powder using sample mill machine. The three solvent used were 95% methanol, 96% petroleum ether, and distilled water. 20 g of leaves powder was dissolved in 200 ml of each solvent. The same procedure was repeated with stem powder. They were allowed to stand on macerated machine for 5 days until soluble matter was dissolved. Each mixture was sieved by using triple layer of gauze to remove pigments. Haploid machine was used to evaporate solvent [14].

Screening of Phytochemicals

The qualitative phytochemical analysis of K. integra crude extracts was performed to check the presence of alkaloids, saponins, glycosides, resins, phenols, tannins and flavonoids. Phenols were detected by adding 0.5 ml of ferric chloride into stem crude extract and another 0.5 ml of ferric chloride into 1 ml of leaves extract. The presence of phenolic compounds was indicated by the occurrence of a bluish green color. Tannins were detected by pouring 2.5 ml of distilled water into 0.5 g of leaves extract and another into 0.5 ml of the stem extract. After that, the solution was filtered and divided into two parts. 1% lead acetate was added to the first part and the presence of white sediment was a positive test. 1% of ferric chloride solution was added to the second part and the appearance of bluish green color was positive test [15].

Saponins were detected by adding 5 drops of extract into water. After shaking, the mixture was left to stand for 15 min. The presence of saponins was indicated by the foam formation. Flavonoid concentrations were determined by combining 50 mg of powder, 5 mL of distilled water, and 2 mL of NaOH. The presence of flavonoid was indicated by a color change, from yellow to colorless, after adding few drops of HCl. For glycosides, 10 ml of H2SO4 was mixed into 1 ml of extract. Fehling’s solution (10 mL) was added. The absence of brick red precipitate indicated a positive test. Alkaloids were detected by mixing 50 mg of powder with 10 ml HCl. The negative test was shown by the absence of turbidity after adding 2 drops of Mayer’s reagent [15].

Sub-culturing of Microorganisms

H. influenza, S. aureus and S. sonnei, E. coli were sub- cultured on nutrient agar (NA). S. pneumoniae was sub- cultured on blood agar and S. typhi was sub-cultured on agar containing xylose, lysine, and deoxycholate. Incubation was performed on the inoculated Petri dishes at 37ºC for 24 h.

They were stored at 4ºC.

Antimicrobial Activity of Kalanchoe integra Leaves and Stem Extract Against Pathogenic Bacteria

Microbial suspension was prepared by mixing 2 loops full of colony with 4 ml of normal saline in sterile tube. It was then compared with 0.5 McFarland standards for checking the turbidity. Mueller hinton agar (MHA) media were prepared and sterilized. Agar well diffusion method was performed by puncturing the holes of 6 mm in the MHA plates. After that, microbial suspension was inoculated in to punctured media. Prepared extracts were put in all holes according to their concentrations. The positive control used was levoflaxin (5µg/ml) for all the pathogenic bacteria, while solvent without extract was used as negative control. The Petri dishes were incubated for 24 h at 37℃. Inhibition zones were measured in mm.

Screening of Phytochemicals from Kalanchoe integra.

In the present study, the activity of K. integra against selected pathogenic bacteria was assessed. The plant parts selected were leaves and stem (Table 1). Similarly, stem [12, 13] and leaves [9, 10, 14, 16, 17, 18, 19] were also used. However, roots were also utilized by some authors [9, 16, 17]. Therefore, to extract phytocompounds of K. integra that may have antimicrobial activities, any plant part can be utilized. Methanol, petroleum ether, and water were used as extraction solvents (Table 1). Methanol as polar solvent was the solvent of choice in most cases [8, 9, 10, 11] followed by non-polar solvent like petroleum ether [9, 16, 17]. Abdellaoui, et al. [8] and Kolodziejczyk-Czepas, et al. [20] and Stochmal preferred to use ethanol, whereas water was used by Biswas, et al. [9] and Richwagen, et al. [13] Thus, various solvents are tried during screening, and the ones with high yield of phytocompounds are chosen. Many researchers prefer to use polar and non-polar or a mixture of them.

Medicinal plants are the most useful for curing human disease because of phytochemical constituents’ presence [21]. After screening, using methanol and water as solvents, the leaves of K. integra revealed the presence of flavonoids, tannins, phenols, and saponins. Flavonoids, tannins, and phenols were found in the stem with water and methanol solvents. However, by using petroleum ether, there was no presence of any phytochemical (Table 1). Similarly, flavonoids were reported as chief phytosubstance extracted in Kalanchoe sp. [8, 9, 10, 16, 17, 19]. In leaves and stem, saponins [17] and phenols were also seen [8, 12, 22]. Only Quazi, et al. and Rashmi, et al. [16, 17], reported the presence of tannins. Although K. integra lacked alkaloids and glycosides (Table 1), alkaloids and glycosides [8, 16, 17] were present in other Kalanchoe sp. Like in the present study, the presence of resins was not reported by any researcher. Thus, K. integra can be screened with polar solvents like water and methanol in order to get phytochemicals that may have antimicrobial activities.

Antibacterial Activity of Kalanchoe Integra against Selected Pathogenic Bacteria

Phytochemicals of Kalanchoe sp. were reported as antimicrobial, anti-asmathic, anti-inflammatory, antihelmenthic, antioxidant, and anti-tumor agents [9, 10, 12, 13, 14, 16, 18, 19]. The purpose of this study was to show that K. integra is effective as an antibacterial agent against S. aureus, E. coli, S. pneumoniae, S. typhi, H. influenza, and S. sonnei. Leaf extracts showed a high inhibition zone compared to the stem extracts. Indeed, leaves methanol extract inhibited all selected bacteria except S. sonnei. Tables 2 & 3 show the antimicrobial activities of K. integra. The inhibition zones of 12.5 mm on E. coli and 18.5 mm on S. aureus were recorded with methanol leaf extracts at 100%. Similar inhibition zones of 18, 11, and 12 mm were noted for S. typhi, H. influenza and S. pneumonia, respectively [16].

The extracts of K. integra inhibited also S. aureus [11, 13, 18], E. coli [10, 11], Klebsiella pneumonia [13], S. typhi and S. pneumoniae [23]. In contrast, a largest inhibition zone of 52 mm was observed against E. coli, but S. aureus showed resistance [18]. The biggest inhibition zone seen could be attributed to high concentration used of leaves powder (300 mg/ml). The Table 4 shows antibacterial activity within three solvents of leaf extracts. Significance was seen within solvents, but not for bacteria. Antibacterial sensitivity results of stem and leaf extracts by agar well diffusion methods showed that leaf extracts were more effectives compared to the stem extract. Leaves and stem by using water solvent showed only antimicrobial activity against E. coli. (Tables 2-5). Contrastingly, methanol extracts of K. integra had no effect E. coli [13]. Sarkar, et al. [11] extracted phytochemicals from K. blossfeldiana that were active against E. coli, S. aureus, and S. pneumoniae at low concentration of 0.25 mg/ml. In a study conducted by Biswas, et al. [9], root extracts were found to be the most effective antibacterial agents. Therefore, from the present results, methanol leaf extract can be used as antimicrobial agent against the tested bacteria. Table 6 shows comparison of methanol extract of leaves and stem effect on bacteria.