Structure elucidation of β-sitosterol with antibacterial activity from the root bark of Malva parviflora
© The Author(s) 2016
Received: 15 April 2016
Accepted: 21 July 2016
Published: 29 July 2016
The powder of root bark of Malva parviflora (Malvaceae) was successively extracted with petroleum ether (b.p. 60–80 °C), chloroform and ethanol. The chloroform extract showed antibacterial activity against Staphylococcus aureus and Escherichia coli, whereas the ethanolic extract showed antibacterial activity against only S. aureus. The chloroform extract, after column chromatographic separation on silica gel using petroleum ether:chloroform (3:1) as eluent, furnished 98 mg of white crystalline compound. The yield of the compound is 0.316 % (w/w). The compound has a melting point of 134–136 °C and the Rf value 0.56 in benzene:chloroform:acetone (1:15:1) on silica gel TLC. The compound was characterized as β-sitosterol by physical properties, chemical test, spectral analysis (FTIR, NMR and MS) and comparing the data obtained from the literature.
Populations throughout Africa, Asia and Latin America use traditional medicine to meet their primary healthcare needs (WHO 2002). Although animal parts and minerals have been used, the primary source of traditional medication is herbal medicines (WHO 2000). The majority of Ethiopians depend on herbal medicines as their only source of healthcare, especially in rural areas. Medicinal plants and knowledge of their use provide a vital contribution to human and livestock healthcare needs throughout the country areas (World Bank 2004). Malva parviflora is one of the most widely used herbs in Ethiopia.
Malva parviflora has been widely used in many parts of the world for curing various diseases. In South Africa, Xhosa people use a poultice made from the whole plant parts of the plant to treat boils, inflammed purulent wounds and swellings (Afolayan et al. 2008). A hot poultice of leaf is used to treat wounds and swellings in La Reunion, and tea of the leaf is taken as a nervine tonic and used as a taenicide and for profuse menstruation (Sharma and Ali 1999). A dried powder or an infusion of the leaves and roots is used to clean wounds and sores in Lesotho (Afolayan et al. 2008). Tea of the leaf is used for treating dry, irritative cough and bronchitis (Ishtiaq et al. 2012). A decoction of the leaves and roots is also used as a hair rinse to remove dandruff and to soften the hair, and tea of the leaf is also used to clean out the mother’s system after childbirth (Mukul 2012). Seeds are demulcent, used to treat cough and ulcers in the bladder (Sharma and Ali 1999). In Ethiopia, the fresh root bark of M. parviflora is chopped into small pieces and applied on the damaged skin surfaces to treat furuncles, carbuncles, wound infections and other related ailments.
The skin infections such as furuncles and carbuncles are universally caused by Staphylococcus aureus (McCaig et al. 2006). The most common bacteria causing wound infection is S. aureus followed by Escherichia coli (Shittu et al. 2002; Ahmed et al. 2007). Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that can live as a commensal organism on the skin and in the nose and throat (Ryu et al. 2014). Human beings are a natural reservoir for S. aureus; and asymptomatic colonization is far more common than infection (Chambers 2001). Approximately 30 % of healthy people are asymptomatically colonized by S. aureus (Ryu et al. 2014). A transmission of S. aureus may occur by direct contact to a colonized carrier (Chambers 2001). Escherichia coli is a Gram-negative bacillus that belongs to the Escherichia genus which is made up of species present in the human and other animal intestine. When eliminated in the environment together with feces, E. coli contaminates water, soil and food. E. coli strains may cause various infections including infections of the skin wounds (Moş et al. 2010).
Although M. parviflora plays a significant role in traditional medicine in many countries including Ethiopia, only a limited study has been done to scientifically explore antibacterial activity, and there have been no previous reports on the isolation and structural characterization of the bioactive compounds present in this plant. Thus, the present study aimed to evaluate antibacterial activity of root bark of M. parviflora for further structural characterization of bioactive compound(s).
Collection of the plant material and description the study area
Fresh plant material was collected at the agricultural farmlands area, around Shanto Town (latitude 07°01′28.2″N and longitude 037°55′09.9″E), Damot Pulassa Woreda, Wolaitta Zone, SNNPR, Ethiopia, in October 2012 towards the end of the rainy season. A voucher specimen (083488) has been deposited at the National Herbarium, Addis Ababa University, Addis Ababa, Ethiopia.
Preparation of the plant material
The root parts were washed with tap water followed by distilled water. The bark part was then separated from the root and dried under shade. The dried samples were powdered using a local coffee grinder.
The powdered root bark of M. parviflora was successively extracted by Soxhlet extraction with petroleum ether (b.p. 60–80 °C), chloroform and ethanol using standard procedure as described by Jones and Kinghorn (2006). Fifteen grams of the powder was extracted in 300 mL of the solvents. The extractions were carried out for 6 h by petroleum ether, 10 h by chloroform and 15 h by ethanol. The solvents in the filtrates were then evaporated completely using water bath at their boiling points. The dried crude extracts were weighed, and the percentage (%) yields of the extractions process were calculated. The processes were repeated a total of three extractions.
Preparation of extracts solution for antibacterial activity assay
The solutions (each 30 mg/mL) of the ethanolic and chloroform extracts were prepared by dissolving their crude extracts in sterile distilled water and dimethylsulfoxide (DMSO) respectively. The solvents were hence used as a negative control. Serial twofold dilution of each extract was prepared in a concentration ranging from 25 to 2.5 mg/mL for the determination of minimum inhibitory concentration (MIC) value. The antibacterial activity of the root bark of M. parviflora was compared to gentamicin (Ivee Aqua Epz Ltd, Kenya) which is a commercial synthetic antibiotic to S. aureus and E. coli pathogens (Ghalem 2014).
Two bacterial species namely S. aureus (ATCC 25923) and E. coli (ATCC 20922) were used for antibacterial activity assay. The bacteria pathogens were obtained from Ethiopian Health and Nutrition Research Institute (EHNRI), Addis Ababa, Ethiopia.
Antibacterial activity assay
The antibacterial activity of the extracts was evaluated by Agar Well Diffusion method as described by Mattana et al. (2010) with little modification. Both bacteria strains were inoculated into 10 mL of sterile nutrient broth [Bulux Laboratories (P) Ltd, India] in respective conical flasks and incubated at 37 °C for 24 h. Nutrient agar (Merck KGaA, Germany) medium was prepared, poured into sterilized Petri dishes and allowed to solidify inside the biological safety cabinet. The cultures were then swabbed on the surface of sterile nutrient agar plates using a sterile cotton swab. Three wells (6 mm in diameter) were drilled into each plate, and the solutions of the extracts were added, until wells were filled. After in upright position incubation at 37 °C for 24 h, the diameter of inhibition zones were measured in millimeter (mm). The antibacterial activity assay was determined as the MIC value, which is the minimum concentration of the extract that could inhibit the growth of tested bacteria.
Isolation and purification
Column chromatographic separation of chloroform extract
Volume collected (mL)
Rf value for major spot
Number of spots
PE (100 %)
CHCl3 (100 %)
CHCl3 (100 %)
PE:CHCl 3 (3:1)
CHCl 3 :Act (15:1)
Fractions (80–89), showed a major spot with two faint spots, were kept in a fridge at 4 °C for 4 h after addition of a little methanol. The crystals were formed at the bottom of the flask and separated from the mother liquor. The light yellow crystals were further purified by crystallization from methanol (white crystal, 98 mg). The yield of the compound is 0.316 % (w/w). The purity of isolated compound was determined by TLC and melting point before submitting to the spectral analysis.
Thin layer chromatography (TLC)
A small amount of crystals was dissolved in chloroform and spotted on the TLC plate using pre-coated aluminium with 0.20 mm thick silica gel 60 F254 (20 × 20 cm, Merck KGaA, Germany). Then the TLC plate was run by the solvent system of benzene:chloroform:acetone (1:15:1), respectively. After drying at room temperature, the spot was visualized by placing the plate in iodine vapor. The retardation factor (Rf) value was then measured.
Melting point measurement
A melting point (m.p.) measurement was performed by procedure as described by Brittain (2009) on a SA-300H digital melting point apparatus. A few crystals of the compound were placed in a thin walled glass capillary tube and then inserted into the side of the heating block via the hole provided in the apparatus. The compound was then heated until it was melted. The temperature at which the solid began to melt, and that at which it was completely liquid, was recorded as the melting point range of the compound.
FTIR spectrum was recorded on a Perkin Elmer 1330 spectrometer with KBr pellets. 1H and 13C NMR spectra were measured on a Bruker DPX 400 MHz and 100.06 MHz spectrometer respectively. High-resolution electrospray ionization mass spectrometry (HR–ESI–MS) measurement was carried out on a Q-Tof Micro YA263 spectrometer in the positive ion mode.
Test for steroid
The test for steroid was performed by Liebermann–Burchard reaction as described by Rajput and Rajput (2012). A few crystals of isolated solid compound were dissolved in chloroform, and a few drops of concentrated sulfuric acid were added to the solution followed by the addition of 3 drops of acetic anhydride. The solution turned violet blue and finally green.
The extraction efficiency of the solvents and the zones of inhibition induced by the plant extracts against tested bacteria were given as mean ± SD (where SD is standard deviation) of three replicates. Difference between means zones of inhibition and the standard was determined using students’ test (t-test). The level of statistical significance was set at P ≤ 0.05.
Results and discussion
Petroleum ether gave light yellow coloured solid (1.18 ± 0.07 %), chloroform yielded light brown coloured solid (3.22 ± 0.42 %) and ethanol resulted yellow coloured amorphous solid (9.62 ± 0.84 %) crude extracts. The results revealed that different solvents have been found to extract different active principles depending on their polarity, and ethanol could extract the highest amount of materials present in the plant which indicates that the root bark of M. parviflora contains the largest proportion of polar components. The percent yield of petroleum ether is very low and could not allow further chemical study. In the present study, we conducted only the antibacterial activity evaluation on chloroform and ethanolic crude extracts.
Antibacterial activity assay
There have been a few previous reports on the antibacterial activity of M. parviflora. The root of M. parviflora inhibited the growth of S. aureus and E. coli with the zones of inhibition ranged between 0.20 and 0.43 mm (Shale et al. 2005). According to Tadeg et al. (2005) report, the root of M. parviflora showed zone of inhibition (20 ± 0.0 mm) against S. aureus, but no zone of inhibition was noted against E. coli. Furthermore, Kalayou et al. (2012) performed antibacterial activity on the leaves of M. parviflora, and the zones of inhibition were 9.70 ± 1.10 mm for S. aureus and 10.25 ± 2.20 mm for E. coli. In general, the zones of inhibition obtained in the present study are different from results in the previous reports. This is due to several variables which influence the bioactive plant constituents against tested bacteria such as the environmental and climatic conditions under which the plant grow, choice of plant extracts, choice of extraction methods and antimicrobial test method as well (Ncube et al. 2008).
TLC, melting point and steroid test
The isolated compound showed a single spot with Rf value 0.56 and has m.p. of 134–136 °C. A sharp m.p. (just a narrow range of 1–2 °C) indicates that the high purity of the isolated compound (Brittain 2009). The m.p. result of the compound is very close to the literature value (134–135 °C) for β-sitosterol (Hang and Dussault 2010). In steroid test, the compound showed a violet-blue colour, which finally turned into green in Liebermann–Burchard reaction indicating the presence of steroid (Rajput and Rajput 2012).
The observed 1H and 13C NMR spectra data in CDCl3 with a drop of methanol-d 4 at 400 and 100.06 MHz, respectively
Chemical Shift, δ (ppm) value
1H NMR (multiplicity)
5.37 (overlapping, t)
0.94 (overlapping, d)
0.84 (overlapping, d)
0.82 (overlapping, t)
β-Sitosterol is a natural micronutrient found in the cells and membranes of all oil producing plants, fruit, vegetables, grains, seeds and trees (Sen et al. 2012). It is commercially available in preparative amounts only as mixtures with other phytosterols typically stigmasterol and campesterol (Hang and Dussault 2010). β-Sitosterol has been proven to be a safe, nontoxic, effective nutritional supplement and has amazing potential health benefits in many diverse applications including antibacterial activity (Sen et al. 2012). Earlier experimental studies have shown that β-sitosterol has antibacterial activity against different bacteria species including S. aureus and E. coli. According to Sen et al. (2012) and Joy et al. (2012) reports, β-sitosterol inhibited the growth of S. aureus (17.83 ± 0.58 mm) and E. coli (14.5 ± 1.84 mm) and S. aureus (13 mm) and E. coli (14 mm) respectively. So, the study suggested that the presence of β-sitosterol in chloroform extract of the root bark M. parviflora might contribute to its potency of growth inhibition against tested bacteria.
The antibacterial activity assay showed that the ethanolic and chloroform extracts of the root bark M. parviflora possess active compound to inhibit the growth of bacteria species: S. aureus and E. coli. This is in agreement with the use of this plant in traditional medicine for the treatment of furuncles, carbuncles, wound infections and other related ailments. In chromatographic separation, β-sitosterol was isolated from chloroform extract of the root bark of M. parviflora. Although β-sitosterol is a known natural product, this is the first report of the isolation from this plant and its structural characterization.
MMO collected and prepared the sample, designed experiments, interpreted the data and prepared the manuscript. MKC and AHD are supervisors of this research and guided the progress of the work. All authors read and approved the final manuscript.
This work was financially supported by Ministry of Education of the Federal Democratic Republic of Ethiopia.
The authors declare that they have no competing interests.
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