In this study, methanolic extracts from the stems, leaves and flowers of Ipomoea pes-caprae that were collected in Xuan Thuy National Park were evaluated for their antibacterial and antioxidative activities, and their ability to inhibit plant growth. The DPPH free radical scavenging activity of crude methanol extracts was evaluated.
JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol 58, No 9, pp 139-145 This paper is available online at http://stdb.hnue.edu.vn BIOLOGICAL ACTIVITY OF METHANOLIC EXTRACT DERIVED FROM Ipomoea pes-caprae (L.) COLLECTED IN XUAN THUY NATIONAL PARK Dao Thi Sen, Dao Van Tan and Hoang Thi La Faculty of Biology, Hanoi National University of Education Abstract Ipomoea pes-caprae (L.) Sweet (Convolvulaceae), a common species in Xuan Thuy National Park, Nam Dinh Province, is thought to be a potential source of bioactive compounds In this study, methanolic extracts from the stems, leaves and flowers of Ipomoea pes-caprae that were collected in Xuan Thuy National Park were evaluated for their antibacterial and antioxidative activities, and their ability to inhibit plant growth The DPPH free radical scavenging activity of crude methanol extracts was evaluated The extract of flower had the highest radical scavenging activity (IC50 = 0.33 mg/mL) while stem extract showed the lowest activity (IC50 = 3.39 mg/mL) Antibacterial activity was tested using the agar diffusion method and it was found that the extracts showed antibacterial activity against Bacillus subtilis and Vibrio parahaemolyticus The stem extract showed the highest activity against V parahaemolyticus (D, 14.7 mm) and was the only extract that inhibited E coli growth However, no extract had an inhibitory effect on Vibrio spp or Staphyloccus aureus Extracts of all parts of the plant inhibited the growth of the roots of Raphanus salivus seedlings 44 - 73% Keywords: Ipomoea pes-caprae (L.) Sweet, DPPH, Antibacterial activity, bioactivity Introduction Plants have been used as an important source of medicine for thousands of years Medicinal plants are the most important source of life saving drugs for the majority of the world’s population Recently, several distinct chemicals derived from plants have been used as a component in important drugs The World Health Organization (WHO) has estimated that up to 80% of the human population has used traditional herbal remedies as medicine [12] The genus Ipomoea (Convolvulaceae) consists of over 200 species which are widely distributed in tropical and subtropical countries Some of them are used in folk medicine to Received May 24, 2013 Accepted November 26, 2013 Contact Dao Thi Sen, e-mail address: daosenk53sinh@yahoo.com 139 Dao Thi Sen, Dao Van Tan and Hoang Thi La treat diseases [6,13] Ipomoea pes-caprae grows profusely in low-lying broad coastal sand dunes It is known to inhibit sand erosion and it thrives in a marine environment of high salinity, high temperature, continuous exposure to the sun, incessantly tidal fluctuation, intense rain and wind, etc In Vietnam, I pes-caprae is a commonly used traditional medicine Dried leaves and stems are used to treat stomach pain, fever and rheumatoid arthritis This plant has also been used to treat inflammation, skin disease, burn, wounds and ulcers [7] I pes-caprae has been used as a herbal remedy in other countries to treat inflammations, colic, diuretic disorders and gonorrhoea [8] Preclinical and clinical investigations have identified pharmacological properties of this plant [11] It has been reported that I pes-caprae inhibited the contraction of guinea pig ileum relative to its concentration The same extract also showed considerable anti-inflammatory action when evaluating in different experimental models [11] In Vietnam, recent studies on I pes-caprae focused primarily on identification and ecological characterization Therefore, the determination of antioxidant and antibacterial activity, and inhibition of growth is necessary to characterize pharmacological properties and possibly lead to future applications In this study, the methanolic extract from various parts including roots, stems, leaves and flowers of Ipomoea pes-caprae that were collected in Xuan Thuy National Park, Nam Dinh Province, were evaluated for their antibacterial and antioxidative activities and possible ability to inhibit plant growth 2.1 Content Material and methods * Plant materials: Fresh stems, leaves, flowers of I pes-caprae, free from disease, were collected in Xuan Thuy National Park, Nam Dinh Province, in September, 2012, and they were washed thoroughly - times under running tap water, shade-dried and powdered before being used for extraction * Preparation of methanolic extract: Shade-dried powder of I pes-caprae was soaked in methanol for 24 h at room temperature and transferred to a Soxhlet apparatus with methanol until a colorless extract was obtained on the top of the extractor (extracted - 10 times) After complete methanol evaporation, each of the methanol extracts was weighed and subjected to bioactivity assays * DPPH (1,1-diphenyl-2-picrylhydrazy) radical scavenging activity: The hydrogen atom or electron donating ability of the extracts was measured from bleaching with a purple colored ethanol solution of DPPH using the method of Hwang et al (2001) [5] The degree of decoloration is directly proportional to the antioxidant activity of the extract A 10 µL aliquot of the extract was added to 190 µL of DPPH in ethanol (100 µM) in a 96-well microtitre plate After incubation at 30 ◦ C for 30 min, the absorbance was measured at 517 nm using an Epoch microplate (Biotek, USA) Capability to scavenge the DPPH radical was calculated using the following equation: 140 Biological activity of methanolic extract derived from Ipomoea pes-caprae (L.) collected SC% (Scavenging capacity) = 100 - (Ac - At )/Ac × 100 where Ac is the absorbance of control and At is the absorbance of the sample Antioxidant activity of the extracts was expressed as IC50 The IC50 value was calculated based on the logarithmic regression equation of scavenging capacity against sample concentration The quality standard used is mM ascorbic acid ascorbic * Antibacterial activity: The bacterial strains Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Vibrio sp including Vibrio parahaemolyticus were obtained from the National Institute of Hygiene and Epidemiology Antibacterial activity was determined by cup diffusion method on an MPA medium The sterile medium (20 mL) was poured into a cm petri plate The medium was allowed to cool in sterile conditions and plates were then incubated with × 10 CFU cultures of test bacteria The concentration of bacterial cells in the suspension was adjusted to a minimum of 1×108 CFU/mL in the MPA solution Agar cups 10 mm in diameter were made in the plates The extracts were prepared by first being reconstituted in methanol (30 g shade-dried powder), then diluted in 10 mL of sterile distilled water An 100 µL aliquot of each dilution was introduced in three wells into MPA plates All test plates were incubated at 37 ◦ C for 24 h [10] Negative controls were prepared using sterile distilled water Chloramphenicol 0.4% was used as the positive reference to determine the sensitivity of each bacterial species tested * Inhibition of plant growth [3]: Seeds of Raphanus salivus L were grown to produce seedlings at 30 ◦ C for day The roots of seedlings were selected to be of the same size The roots were embedded in crude extracts for hour (The extracts were prepared by first being reconstituted in methanol (30 g shade-dried powder), then diluted in 10 mL of sterile distilled water) In negative controls, distilled water was prepared instead of the crude extracts The root length was measured after 24 hours The inhibition of root growth was evaluated through percentage of inhibition (I%) and calculated using the following equation: I% = (Lc – Ls ) × 100/Lc where I%: Percent inhibition; Lc : Average length (cm) of roots in the control; Ls : Average length (cm) of roots in the test * Data process: Each experiment was conducted in triplicate Data were processed using statistics generated by Microsoft Excel and SPSS software 2.2 Results and discussion 2.2.1 Antioxidant activity The results of the antioxidant activity assay are given in Table A DPPH radical was used as a substrate to evaluate the free radical scavenging activity of the I pes-caprae extracts A serial dilution of 4, 10, 20, 50 and 100 folds was used for each extract 141 Dao Thi Sen, Dao Van Tan and Hoang Thi La Antioxidant activity of the extracts was expressed as IC50 Antioxidant activity of the extracts was compared with the standard quality (5 mM ascorbic acid) Table Antioxidant activity of Ipomoea pes-caprae extracts Plant parts IC50 (mg/mL) % acid ascorbic Stems 3.39 0.48 Leaves 2.99 0.49 Flowers 0.33 2.26 The IC50 value of all extracts ranged between 0.33 and 3.39 mg/mL The highest activity was observed for the extract from flowers (IC50 0.33 mg/mL) and lowest activity was shown by the extract from stems (IC50 3.39 mg/mL) There is a tight relationship between DPPH free radical scavenging capacity and extract concentration Figure The relationship between DPPH free radical scavenging capacity and extract concentration The generation of oxygen radicals such as superoxide radicals (O− ), hydroxyl radicals and non-free radical species such as hydrogen peroxide and singlet oxygen (1 O2 ) are associated with cellular and metabolic damage, aging and a number of illnesses including cancer, cardiovascular disease, neurogenic disease and inflammation [9] On average, every cell in the body comes under the attack of free radicals once every ten seconds Although the body possesses defense mechanisms such as enzymes and antioxidant nutrients, continuous exposure to chemicals and contaminants may lead to an increase in the amount of free radicals in the body beyond its capacity to control them and cause irreversible oxidative damage [4] The free radical damage can be counteracted by the consumption of antioxidants Antioxidants act by donating an unpaired electron to free radicals and thereby inhibiting the oxidation of macromolecules and cellular structures [2] Vitamin C, vitamin E, carotenoids and glutathione are some of the basic antioxidants in the body Plants produce lot of antioxidants to protect themselves against oxidative stress caused by direct exposure to the sun and oxygen Mangrove trees and some halophytic herbs grow under such stressful conditions characteristic to estuarine ecosystems It has been found that phenolics are the major compounds present in mangrove environments that are responsible for antioxidant activity and it might be that the antioxidant activity of Ipomoea pes-caprae is due to a group of phenolic compounds 142 Biological activity of methanolic extract derived from Ipomoea pes-caprae (L.) collected 2.2.2 Antibacterial activity The methanolic extracts of plant stems, leaves, flowers was assayed in vitro using the agar disc diffusion method against five bacterial species All of the extracts had antibacterial activity against Bacillus subtilis and Vibrio parahaemolyticus The stem extract showed the highest activity against V parahaemolyticus (D, 14.7 mm) and was the only extract to inhibit E coli growth However, none of the extracts had any effect on Vibrio spp and Staphylococcus aureus Table Antibacterial activity of Ipomoea pes-caprae extracts (Zone of inhibition, D mm) Plant parts Stems Leaves Flowers E coli 7.0 ± 1.4 - B subtilis 9.0 ± 1.4 7.0 ± 1.4 5.0 ± 1.4 V parahaemolyticus 14.7 ± 1.2 10.0 ± 0.0 6.0 ± 0.0 Vibrio spp - S aureus - Note (-) not having a zone of inhibition Figure Antibacterial activity of Ipomoea pes-caprae extracts against Vibrio parahaemolyticus Stems: extract from stems; leave: extract from leaves; (+): Chloramphenicol 0,4%; (-): water I pes-caprae exhibits anti-inflammatory activities and is used as a traditional medicinal plant in Vietnam for the treatment of various types of inflammation including jellyfish sting and dermatitis The results here confirm that the use of this species in folklore has a scientific basis [7] Pathogenic bacteria are considered to be a major cause of morbidity and mortality in humans Even though pharmaceutical companies have produced a number of new antibiotics, resistance to these drugs has increased and this has become a global concern [1] Due to the increase of resistance to antibiotics, there is a pressing need to develop new and innovative antimicrobial agents Among the potential sources of new agents, plants are an excellent candidate for investigation because they contain many bioactive compounds that can be of interest in therapeutics In this study, we evaluated the preliminary antibacterial activity of the crude methanol extract of Ipomoea pes-caprae with strains of bacteria and found that it had an inhibitory effect on E coli, B subtilis and V parahaemolyticus Therefore, a detail evaluation of antibacterial activity 143 Dao Thi Sen, Dao Van Tan and Hoang Thi La as well as the extraction of pure compounds and further investigation of other pathogenic bacteria should be carried out 2.2.3 Inhibition of plant growth Seeds of Raphanus salivus were grown to produce seedlings at 30 ◦ C for day The roots of seedlings were embedded in crude extracts for one hour then were returned to the medium to germinate for 24 hours The length of roots was measured The inhibitory effect of extracts on root growth of seedlings was assessed through the value I (%) The results are shown in Table Table Growth inhibition (%) of Ipomoea pes-caprae extracts Plant parts Stems Leaves Flowers I% 73.60 ± 3.47 73.44 ± 2.54 44.56 ± 4.30 Figure The inhibitory effect of methanol stem extract on the growth of Raphanus salivus L roots All parts of the plant had an inhibitory effect on the growth of seedling roots, ranging from 44.56 - 73.60% The highest degree of inhibition was observed with the extract from stems (I% 73.60) while the lowest value was found with the extract from flowers (I% 44.56) The inhibition is generally an inhibition of mitotic activity Further studies are needed to prove anticancer properties of this plant Conclusion Methanolic extracts from stems, leaves and flowers of Ipomoea pes-caprae collected in Xuan Thuy National Park, Nam Dinh Province, exhibited antibacterial and antioxidative activities and the ability to inhibit plant growth The results of the present study support the use of Ipomoea pes-caprae in traditional medicine Acknowledgement This work was supported by the project: The Study of Antibacterial and Antioxidative Activities and the Inhibitory Effect on Plant Growth of some Plants in Xuan Thuy National Park, Nam Dinh Province Implementing institution: Hanoi National University of Education (Code number: SPHN-12 - 129) 144 Biological activity of methanolic extract derived from Ipomoea pes-caprae (L.) collected REFERENCES [1] G Adwan, M Mhanna, 2008 Synergistic effects of plant extracts and antibiotics on staphylococcus aureus strains isolated from clinical specimens Journal of Scientific Research,Vol 3, pp.134-139 [2] J C Dekkers, L J Van Doornen and H C Kemper, 1996 The role of antioxidant vitamins and enzymes in the prevention of exercise-induced muscle damage Sports Med., Vol 21, pp 213-238 [3] T T Dat, B T Lien, T V Thanh, 1999 Preliminary studies on anti-mitotic effects of some medicinal plants used as anti-cancer drugs by experienced folk Journal of Medicine, Vol 11, pp 6-7 [4] B Halliwell, 1994 Free radicals and antioxidants: A personal view Nutr Res., 52: 253-265 [5] B Y Hwang, H S Kim, J H Lee, Y S Hong, J S Ro, K S Lee and J J Lee, 2001 Antioxidant benzoylated flavan-3-ol glycoside from Celastrus orbiculatus J Nat Prod., Vol 64, pp 82-84 [6] C P Locher, M Burch, H F.Mower, J Berestecky, H Davis, B Van-Poel, A Lasure, D.A Vanden-Berghe, D.A and Vlietinck, A.J 1995 Anti-microbial activity of extracts obtained from selected Hawaiian medicinal plants J Ethnopharmacol, Vol 49, pp 23-32 [7] D.T Loi, 1977 The medicinal plants and remedies in Viet Nam Publisher of scientific and technical, Ha Noi [8] M M Khan, F Ahmad, A K Rastogi and J R Kidwai, 1994 Insulinogenicand hypoglycaemic activities of Ipomoea pes-caprae Fitoterapia, Vol 65, pp 231-234 [9] J Mimi-Oka, D V Simz and T P Simi, 1999 Free radicals in cardiovascular diseases Sci J Facta Univ Ser Med Biol., Vol 6, pp 11-22 [10] P R Murray, E J Baron, M A Pfaller, F C Tenover and H R Yolken, 1995 Manual of clinical microbiology ASM Press [11] U Pongprayoon, P Baeckstrom, U Jacobsson, M Lindstrom and L Bohlin 1991a Compounds inhibiting prostaglandin synthesis isolated from Ipomoea pes-caprae Planta Med, Vol 57, pp 516-518 [12] R Shanmugapriya, T Ramanathan, G Renugadevi, 2012 Invitro studies on the production of secondary metabolites in Ipomoea pes-caprae (L.) R Br International Journal of Advances in Pharmaceutical Research, Vol 1, No 1, pp 9-11 [13] M M Souza, A M Oliveira, F V Cechinel, C Berti, R A Yunes and R Krogh, 1998 Estudos preliminaries dapropriedate antinociceptiva de Ipomoea pes-caprae emmodelos farmacologicos especificos Alcance (UNIVALI) Vol 5, pp 53-57 145 ... this study, the methanolic extract from various parts including roots, stems, leaves and flowers of Ipomoea pes-caprae that were collected in Xuan Thuy National Park, Nam Dinh Province, were evaluated... activity of Ipomoea pes-caprae is due to a group of phenolic compounds 142 Biological activity of methanolic extract derived from Ipomoea pes-caprae (L.) collected 2.2.2 Antibacterial activity The methanolic. .. Antioxidative Activities and the Inhibitory Effect on Plant Growth of some Plants in Xuan Thuy National Park, Nam Dinh Province Implementing institution: Hanoi National University of Education (Code number: