Antioxidant and antibacterial activity of Ipomoea mauritiana Jacq.: a traditionally used medicinal plant in Bangladesh

In an attempt to explore the scientific basis for the pharmacological benefits the antioxidant and antimicrobial activities of Ipomoea mauritiana whole plant methanol extract were assessed. The total phenolic and flavonoid content were determined using standard method while antioxidant activity was determined by DPPH free radical scavenging activity method. The antimicrobial activity was evaluated by disk diffusion method and compared with standard kanamycin (30 μg/disc). The results revealed that I. mauritiana extract contains tannin, saponin, terpenoids, alkaloid and flavonoids. In DPPH, ascorbic acid and extract showed highest scavenging activity and it was 90.96% at concentration 800 μg/mL and I. mauritiana methanol extract showed 72.28% at a concentration of 800 μg/mL. The extract was able to reduce the stable free radical DPPH with an IC50 of 275.084 μg/mL while that of ascorbic acid was 230.09 μg/mL. Total phenolic constituent of the extract was 59.302 ± 3.289 mg/g as gallic acid equivalent. The flavonoid content of methanolic extract of I. mauritiana was 27.212 mg of QE/g. In case of antimicrobial screening, crude extracts of I. mauritiana showed notable antibacterial activity against tested microorganisms. The extract and standard showed the highest mean zone of inhibition ranging from 13 to 19 mm and 37 to 42 mm, respectively at a concentration of 400 μg /disc and against the gram positive bacteria (Bacillus cereus-19.25 mm) showed highest zone of inhibition. The results indicate that I. mauritiana possesses considerable antioxidant and antimicrobial activity.


Introduction
Ipomoea mauritiana (I. mauritiana) Jacq. (Synonyms: Ipomoea digitata; Ipomoea eriosperma; Ipomoea paniculata; Convolvulus paniculatus) is a vine of the Convolvulaceae family. The plant has ethnomedicinal importance and found in many parts of the world. In Ayurvedic industries, I. mauritiana is popularly used as "Vidari" instead of the permitted raw drug Pueraria tuberose (Roxb. ex Willd). The Ayurvedic Pharmacopoeia of India correlates I. mauritiana as Kshiravidari. Tubers are used in almost 45 Ayurvedic formulations, and in many instances also used as single drug. This is also an important component of the popular ayurvedic formulation Chyavanaprasha [1]. Traditionally the tubers are also extensively used by local communities in different parts of Bangladesh to treat various ailments including pain in spinal cord, to increase breast milk, body strength and sperm count, to control tuberculosis, as blood purifier in jaundice and to treat biliary disorders [2][3][4][5]. The leaves are also used to treat leucorrhea and diabetes with obesity and in infrequent urination by the folk medicine practitioners of Kurigram and Jhalokati districts of Bangladesh [6].
The pharmacological investigations reported analgesic, hypoglycemic, hypocholesterolemic, and hepatoprotective activities of I. mauritiana tuber root [7,8]. Additionally, the antibacterial activity against Escherichia coli which caused catheter associated urinary tract infections has also been reported by Pavan [9]. A number of phytochemical have been isolated from I. mauritiana tuber root methanol extracts which includes taraxerol, taraxerol acetate, βsitosterol, scopoletin and 7-O-β-D-glycopyranosylscopoletin (scopolin) [10]. Literature showed that, scopoletin isolated from Lasianthus lucidus possessed antibacterial activity against Pseudomonus aerigenosa [11]. Plants are major source of natural antioxidants that can serve as possible drug candidates for different chronic ailments such as neurological diseases, swelling and diabetes [12,13]. The wide range of potential benefits of natural antioxidants has increased the curiosity largely in medicine to ascertain new molecules to combat oxidative stress caused by free radicals [13].
Infective microbes are one of the major public health threats causing morbidity and mortality worldwide [14]. Several life-threatening microorganisms have now become resistant to many commercial antibiotics [15]. Novel, safe, potent and wide-spectrum antimicrobial molecules are therefore urgently needed [16]. Even though the plant has been widely used as traditional medicine, however there is inadequate scientific evidence on the potential benefits of the whole plant. Therefore, the current study was designed to determine the antioxidant and antimicrobial activities of I. mauritiana whole plant methanol extract.

Plant collection
I. mauritiana was collected from Tangail district of Bangladesh in July 2018 and was identified by Bangladesh National Herbarium. The collected plants (as a whole) were cleaned using tap water to remove the soil and washed with distilled water followed by shade drying. The dried plants were grounded into a fine powder.

Plant extract preparation
Powdered whole plant material having a weight of about 50 g were taken in one amber glass container and soaked in 500 mL methanol, for 3 days with occasional shaking and stirring. The extractive was then filtered using Whatman filter paper number 1 to attain a clear filtrate. The filtrate extract was concentrated and dried in vacuum at 40°C and stored at 4°C until further use.

Preliminary phytochemical screening
The presence of different phytochemicals such as alkaloid, steroids, glycosides, flavonoid, tannins, saponin, and terpenoids in crude methanol extract was determined following standard methods [17,18]. Any color change or precipitation formation indicated positive reactions to these tests.

Test for steroids and Triterpenoids
In the extract solution, few drops of acetic anhydride added and boiled. After cooling, concentrated sulphuric acid was slowly poured into the test tube. Formation of violet to blue or green color (Liebermann-Burchard reaction for steroid) indicated the positive reaction. Formation of green color of the upper layer and reddish-brown color in the bottom layer confirmed a positive test for steroids and terpenoids respectively [17,18].

Test for glycosides
The plant extract solution was dissolved in a mixture of glacial acetic acid (5%) and Ferric chloride solution and one or two drop of concentrated sulphuric acid was added. A brown ring or violet ring or greenish ring formation coloration indicated a positive test for glycosides [19].
One mL of distilled water was added to 10 drops of the extract dissolved in isopropyl alcohol (20 mg/mL) in a test-tube. The tube was shaken vigorously and observed for persistent froth formation and allowed to stand for 10 min. A positive result indicate a forth formation which is stable as long as 15 min [19].

Test for alkaloids
Few drops of Mayer's reagent (potassium mercuric iodide solution) was mixed with I. mauritiana extract solution. A yellowish-white like precipitation indicates a positive reaction for the presence of such metabolites [19].

Ferric chloride test for tannins
The plant extract solution of 10 mg was dissolved in ethanol (1 mL). Then distilled water (2 mL) was added followed by 4 drops of ferric chloride aqueous solution (10% w/v). The occurrence of phenols was confirmed by the development of dark blue or green color [20].

Total phenolic content
Total phenolic content of I. mauritiana extract was measured using a Folin-Ciocalteu colorimetric method spectrophotometrically as described by Idowu [21] with slight modification. Gallic acid was used as standard (concentration: 6.25-200 μg/mL) while I. mauritiana whole plant extract was 200 μg/mL. Firstly, 1 mL of the extract or standard gallic acid solution was used in screw cap tube and 5 mL of Folin-Ciocalteu reagent was added. Then, 4 mL (7.5%) of anhydrous sodium carbonate was added followed by 30 in incubation at 40°C. The vehicle solvent was used as blank solution. UV absorbance was taken with a UV-VIS spectrophotometer (Shimadzu, Japan) at 765 nm. Total phenolic content was calculated as gallic acid equivalent (GAE) using the equation as follows: Where C = Total phenolics (mg/g plant extract in GAE), c = concentration of sample obtained from calibration curve (mg/mL), V = volume of the sample, and m = sample weight (g).

Total flavonoid content
To determine the total flavonoid content, the method of Rahman [22] was adopted, quercetin was the standard and different concentrations were used (6.25-200 μg/mL) while I.mauritiana methanol extract was 200 μg/mL. Initially, in a volume of 1 mL of extract or 1 mL quercetin solution of different concentrations, 3 mL of methanol was added. After that, 200 μL of 10% aluminum chloride and 200 μL of 1 M potassium acetate solution were added followed by the addition of 5.6 mL distilled water. Then the mixture incubated at room temperature for 30 min. Absorbance at 415 nm was recorded against the blank (water). The total flavonoid content was calculated as per the formulae bellow: Where C = Total flavonoid content (mg/g plant extract in Quercetin), c = concentration of sample obtained from calibration curve (mg/mL), V = volume of sample, and, m = sample weight (g). Measurement was taken triplicate.

DPPH radical scavenging activity
To determine the antioxidant activity, the method of described by Braca [23] was adopted with slight modification. At first, 100 μL of plant extract and different concentrations of standard (ascorbic acid) were taken in test tubes. Then, 3 mL of 0.004% DPPH (2,2-diphenyl-1-picrylhydrazyl) solution in ethanol was added to each test tube followed by 30 min incubation. Later absorbance was taken at 517 nm spectrophotometically against methanol (blank).
The absorbance values were used to calculate the percentage of antioxidant activity (% inhibition) using the formula below: Where A 0 = Absorbance of DPPH solution only; A 1 = Absorbance in the presence of test sample and standard ascorbic acid in DPPH solution. Measurement was taken triplicate.

Antimicrobial activity of I. mauritiana Microorganisms
Four gram-positive and six gram-negative bacteria were selected as test organisms. Gram positive; Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus and Gram negative; Escherichia coli, Salmonella typhi, Salmonella paratyphi, Pseudomonas aeruginosa, Vibrio parahemolyticus and Shigella dysenteriae were used for antimicrobial activity test. Each of the stock cultures was collected as pure culture from the Institute of Nutrition and Food Science (INFS), University of Dhaka, Bangladesh.

Antibacterial activity
Disk diffusion method was used to perform the antibacterial test [24]. The sample solutions (400 and 200 μg/ mL) were made by dissolving in methanol. Sterilized and dried filter paper discs (6 mm diameter) were then soaked in the crude extracts (400 and 200 μg/mL/disc) using micropipette and the solvent residue evaporated to dryness. Kanamycin (30 μg/disc) and blank discs (soaked with methanol followed by evaporation) were positive and negative control, respectively. The plates were inverted and refrigerated at 4°C for 24 h for maximum diffusion. The following day plates were placed in the incubator at 37°C for another 24 h for optimum bacterial growth. The antimicrobial activity was observed with inhibited growth of the microorganisms giving a clear, distinct zone of inhibition around the discs. Finally, the diameter of zone of inhibition was measured. The data was obtained from three individual experiments (n = 3).

Statistical analysis
A statistical analysis was used to interpret the antimicrobial and antioxidant results. The experiment was conducted in completely randomized design with 3 replicates. The results are presented as means ±standard error of means using MS excel [25].

Result and discussion
The results of preliminary phytochemical screening of I. mauritiana are presented in Table 1. Results revealed that the methanolic extract of I. mauritiana contains terpenoids, saponins, flavonoids, steroids, and alkaloids. The presence of these classes of compounds of this plant could be responsible for its various medicinal uses [26,27].
On the other hand, the total phenolic content of I. mauritiana was evaluated as expressed by gallic acid equivalents per gram of extract which was found as 59.302 ± 3.289 mg of GAE/g. The value was obtained from regression equation of the calibration curve (y = 0.004x + 0.105; r 2 = 0.992) as shown in Fig. 1. Subsequently, the total flavonoid content of methanol extract of I. mauritiana was 27.212 ± 0.51 mg of QE/g which was obtained from the regression equation (y = 0.003x + 0.023; r 2 = 0.995) as shown in Fig. 2.
Similarly, DPPH free radical scavenging activity of I. mauritiana and ascorbic acid is shown in the  Table 2. The DPPH scavenging activity of I. mauritiana methanol extract was lesser than that of standard ascorbic acid. The IC 50 value denotes the minimum sample concentration required to scavenge 50% free radical. The antiradical activity of the extract may be due to their high content of phenols, as polyphones which contribute to the antioxidant activity in living systems by chelation and electron transfer or hydrogen donating ability of hydroxyl groups in ortho-and para-positions thus neutralizing the free radicals [28,29].
The results of antibacterial activity of the extract and standard kanamycin with respect to each of the test organisms are presented in Table 3. The mean zone of inhibition of kanamycin was between 37 to 42 mm and the extract which was between 13 to 19 mm, respectively at a concentration of 400 μg /disc. Zone of inhibition of standard was larger than extract. The extract showed the highest zone of inhibition against the gram positive; Bacillus cereus (19.25 ± 1.020 mm) and Sarcina lutea (18.5 ± 0.0813 mm) and gram negative Shigella boydii (18.25 ± 3.06 mm) and had no activity against Escherichia coli. Gram positive strains were more sensitive to the extract than the gram negative ones. However, the extract had the lowest antibacterial effect to Pseudomonas aureus. These results indicate that I. mauritiana extract is   Plant has been a very significant source of medicine for decades and many plants have been tested for their potential bioactive compounds [30].
Free radicals are significant contributor in many pathological manifestations. Antioxidants can maintain good health and protect, either by counteracting these free radicals or defending the body's antioxidant defense mechanisms. Plants and herbs, being the potential sources for a number of antioxidants in addition to their therapeutic properties, have appealed the concern of scientific community to meet up the growing interest for raw materials with potential natural antioxidant compounds. Phenolic or polyphenolic compounds are one of the major classes of compounds having strong antioxidant properties. The antioxidative action of phenolic compounds is primarily due to their redox properties, [31,32] which can hydrolyze and neutralize free radicals, purify singlet and triplet oxygen, or decompose peroxides.
On the other hand, ongoing demand for new antibiotic is present due to the sustained emergence of infectionresistant strains. About 80% of the drugs available in many developing countries originated from medicinal plants. In the industries of many countries plants comprise the raw material for processes that synthesizes pure chemical derivatives [33].
In the present study, prominent antioxidant activity was observed for the whole plant extract of I. mauritiana with potential antibacterial activity against a number of Gram (+) ve and Gram (−) ve bacteria. Different parts of the plant may vary with their antioxidant activity. For instance, a study revealed that, the root and stem bark, leaf and flower parts of Tabebiua pallida methanol extract were tested for antioxidant activity where the leaf extract was found to show highest antioxidative and free radical scavenging property which was also rich in total phenols and flavonoid contents [34]. The current study thus was conducted with the whole plant of I. mauritiana. It is clear from the results that, total phenolic and flavonoid contents and DPPH scavenging activity signifies the potential bioactivity of I. mauritiana. This result is consistent with the report of Sulaiman [35]. Moreover, phenolic compounds demonstrates potential antibacterial activity. Rahman and collegues stated that, 3,4-dihydroxybenzoic acid obtained from Cananga odorata was active against a number of Gram (+) and Gram (−) bacteria [36].
In the same way, the antibacterial activity of I. mauritiana against gram positive bacteria such as Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus and gram negative bacteria such as Escherichia coli, Salmonella typhi, Salmonella paratyphi, Pseudomonas aeruginosa, Vibrio parahemolyticus and Shigella dysenteriae was evaluated. These are important pathogens and can rapidly develop antibiotic resistance as  antibiotic use increases. However, a previous report stated that, the leaf freeze dried extract of I. mauritiana did not show any activity against Streptococcus mutans, S. mitis, Staphylococcus aureus, and a fungi Candida albicans in both agar disk and agar well diffusion tests [37]. This study reveals the promise of antibacterial activity of I. mauritiana against gram positive bacteria in particular.

Conclusion
The results demonstrated that the methanolic extract of I. mauritiana Jacq shown antioxidant and antimicrobial activity. These findings could be a scientific evidence to use this plant as a potential source of antioxidant and antibacterial agents. However, future studies are necessary to determine the mechanisms of these pharmacologic properties. Moreover, phytochemicals characterization of possible bioactive compounds in I. mauritiana Jacq is also required.