INTRODUCTION:

Ipomoea batatashas played an important role as a phytochemical source in human nutrition and animal feeding^1,2. Ipomoea batatas continues to be of remarkable economic value as the sixth most plentiful food crop in the world^3,4. This tuberous root is a high-quality source of carbohydrates, dietary fiber, vitamin A (as β-carotene), vitamin B6, vitamin C, manganese, copper, potassium, and iron². Recently, studies on sweet potato leaves have focused on its antioxidant capacities due to the high content of phenols, flavonoids, β-carotene, anthocyanins, and caffeoylquinic acid derivatives. Other reports have focused on its medicinal use, specifically its antidiabetic, anticancer and antiviral properties^5,6.

In present scenario, cancer is one of the dreadful diseases and its occurrence is increasing. In medical science, the methods available to treat a cancer patient mainly includes surgery, chemotherapy and radiotherapy etc. As these known methods are very costly and have side effects with limitations of their use, there is need for effective and acceptable cancer therapeutics agents that would be non-toxic, highly efficacious against multiple cancers, palatable and cost effective. Medicinal plants have created great interest among researchers as they have been proved to possess anticancer activity⁷and have no side effects.

In recent days, the greatest challenge for human mankind is drug resistant microbes specially bacteria. Pathogenic bacteria developed resistance against latest drugs. So there is an increasing interest about medicinal plants, which are known to be useful in treating infection from the very early stage of human history. Till today these plants are being used widely across India. These medicinal plants are gaining important interest not only for their antimicrobial activity but also for their antioxidant, antidiabetic and cytotoxic activity. Besides these activities most of the medicinal plants are rich in several nutrients which make them suitable for human consumption.

Despite of the remarkable use of its tuberous roots, there is a lack of detailed data related to the phytochemical composition and biological properties for sweet potato leaves. The aim of this research was to perform phytochemical, antioxidant and antimicrobial activities on the crude extract of sweet potato leaves.

MATERIALS AND METHODS:

Sample collection and extraction:

Sweet potato leaves were collected from VIT University nursery and were extracted by soxhlet method with hexane and methanol for 3-4 hours. The extracted material was later collected and solvent was evaporated under vacuum. The dried sample was stored at 4°Cfor further experiments.

Phytochemical Analysis:

Detection of alkaloids:

Five mg of solvent free extract was stirred with two ml of diluted hydrochloric acid and filtered. The filtrate was tested carefully with various alkaloidal reagents⁸.

A. Hager’s Test: Filtrates were treated with Hager’s reagent (saturated picric acid solution). Presence of alkaloids was confirmed by the formation of yellow coloured precipitate.

B. Wagner’s Test: To a few ml of filtrate, few drops of Wagner’s reagent were added along the side of test tube. Formation of reddish brown precipitate indicates positive test⁹.

Wagner’s reagent: [iodine (1.27g) and potassium iodide (92 g) was dissolved in 5 ml of water and made up to 100 ml with distilled water]

Detection of carbohydrates and glycosides:

Five mg of leaf extract was dissolved in 5 ml of water and filtered. The filtrate was subjected to the following tests¹⁰.

A. Fehling’s Test: 1 ml of filtrate was boiled on water bath with 1 ml of each of Fehling’s solutions A and B. Appearance of red precipitate indicates the presence of sugar.

Fehling’s solution A: Copper sulphate (34.66 g) was dissolved in distilled water and made up to 500 ml using distilled water.

Fehling’s solution B: Potassium sodium tartrate (173 g) and sodium hydroxide (50g) was dissolved in water and made up to 500 ml.

B. Molish Test: 2 ml of filtrate and two drops of alcoholic solution of α napthol were mixed, the mixture was shaken well and 1 ml of conc. sulphuric acid wasadded slowly along the sides of test tube and allowed to stand. A violet ring confirmed the presence of carbohydrates.

Detection of phytosterols:

LibermannBurchard’s Test: The extract (5 mg) was dissolved in 2 ml acetic anhydride. To this, one or two drops of conc. sulphuric acid were added slowly along the sides of the test tube. An array of colour changes indicates the presence of phytosterols¹¹.

Detection of phenolic compounds:

Ferric chloride test: The extract (2 mg) was dissolved in 5 ml of distilled water. To this, few drops of neutral 5% ferric chloride solution were added. Appearance of green colour indicates the presence of phenolic compounds¹².

Detection of flavonoids:

Alkaline Reagent Test: Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, affirms the presence of flavonoids.

Lead acetate Test: 1ml of the plant extract was added in a test tube and to this1ml of 5% lead acetate was introduced and the mixture was allowed to stand for few minutes. The formation of precipitates in the samples confirmed the presence of flavonoids.

Detection of Tannins:

About 0.5 g of extract was stirred with about 10 ml of distilled water and then filtered the extract. Few drops of 1% ferric chloride solution were added to 2 ml of the filtrate.Formation of a bluish-black, green or bluish-green precipitate indicated the presence of tannins.

Thin Layer Chromatography:

TLC was done to separate the compounds of sweet potato leaves methanol and hexane extracts. Combination of solvents (hexane:acetone; 60:40) was used as mobile phase to separate the compounds according to their mobility. Separated spots were visualized under UV light and Rf factor was calculated by the following equation.

Rf factor = Distance travelled by solute front/Distance travelled by solvent front.

Gas Chromatography – Mass Spectrometry:

Methanol and hexane extract of sweet potatoleaves were analyzed by GC-MS. Perkin Elmer Clarus 680 gas chromatographic instrument equipped with a mass spectrometer detector (Clarus 600 model) and an Elite-5MS (30.0 m, 0.25 mmID, 250 μmdf)column was used. The carrier gas used was helium at a flow rate of 1 ml min^-1. The following temperature program was used: initially the oven temperature was held at 60°C for 2 min and then ramped from 10°C/min to 300°C withhold time for 4 min, total run time 30 min. The temperature of the injector was maintained at 300°C. The ion trap was operated at 70 eV with a scan range of m/z from 50 to 600. A sample of 1 µl was injected in split mode (10:1). The intermediate and end product was identified based on the Wiley registry of mass spectral data¹³.

Antimicrobial studies:

Antimicrobial activity of Ipomoea batatas leavesmethanol and hexane extract was checked against nine clinical pathogens by agar well diffusion method. Muller-Hinton agar plates were prepared and inoculated with respective pathogens. 8 mm well was cut on the agar plate surface. Four different concentrations (25 mg/ml,50 mg/ml, 75 mg/ml, and 100 mg/ml) of the extract were added to the well and respective solvent was used as negative control. The plates were incubated for 24 hrs at 37°C. After incubation the plates were observed for clear zone¹⁴.

Antioxidant activity:

The antioxidant activity of the extracts was evaluated by DPPH radical scavenging assay which was originally described by Blois¹⁵. DPPH (2, 2-diphenyl-1-picrylhydrazyl) is a synthetic free radical with deep violet colour when is in form of solution with λ_max at 517nm. It can accept an electron or a hydrogen radical to become stable diamagnetic molecule and appear as light purple in colour which indicates the scavenging of DPPH and the substance has antioxidant activity of the substance.

Methanol extracts were prepared for all the three extracts. Methanol solution of DPPH was used as negative control. 500µl of each sample and 500 µl of DPPH solution was allowed to react and incubated at room temperature for 30mins under dark conditions. Absorbance was taken at λ_max i.e. 517nm against a blank which was 500µl of methanol. Percentage inhibition was calculated by the following equation to conclude the presence of antioxidant activity of the extracts.

Percentage of inhibition = (OD control – OD sample / OD control) x 100

Anticancer study:

Cell line

The human osteosarcoma cell line (MG 63) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% foetal bovine serum (FBS). The cells were maintained at 37°C, 5% CO₂, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.

Cell treatment procedure:

The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells were counted using a haemocytometer and diluted with medium containing 5% FBS to give final density of 1x10⁵ cells/ml. One hundred microliters per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 37^°C, 5% CO₂, 95% air and 100% relative humidity. After 24 h the cells were treated with serial concentrations of the test samples. They were initially dissolved in dimethylsulfoxide (DMSO) along with aliquot of the sample solution which was diluted to twice the desired final maximum test concentration with serum free medium. Additional four serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 µl of these different sample dilutions were added to the appropriate wells containing 100 µl of medium, resulting in the required final sample concentrations. Following sample addition, the plates were incubated for an additional 48 h at 37^°C, 5% CO₂, 95% air and 100% relative humidity. The medium containing without samples were served as control and triplicate was maintained for all concentrations.

MTT assay:

After 48 h of incubation, 15µl of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37^°C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and then measured the absorbance at 570 nm using micro plate reader.

The percentage of cell viability was then calculated with respect to control as follows

Percentage of cell viability = [A] Test / [A]control x 100

The percentageof cell inhibition was determined using the following formula.

Percentage of cell inhibition = 100- Abs (sample)/Abs (control) x100.

Nonlinear regression graph was plotted between percentage of cell inhibition and Log concentration and IC50 was determined using GraphPad Prism software^16,17.

RESULT AND DISCUSSION:

Phytochemical study:

Sweet potato leaves are source of various bioactive compounds. Result ofphytochemical studies of methanol extract is presented in table 1. Different test confirmed the presence of different compound like flavonoids, tannins, sugar, phenols, alkaloids etc. Alkaloids have been reported to show potent cytotoxic activity against human leukaemia cells, prostate cancer cell lines, viral and clinical pathogens¹⁸. From therapeuticpoint of view the most important role of flavonoids are their antioxidant properties, which is result of direct scavenging of reactive oxygen species. Flavonoids are also reported to be anti-inflammatory agents as the result of diminished formation of pro-inflammatory mediators (prostaglandins, leukotrienes, reactive oxygen species, and nitric oxide)¹⁹. Phenols and tannins are reported to possess and antioxidant activity by David et al.²⁰. Yokozawa et al.²¹ showed that the scavenging activity of tannins increases with an increase in the number of galloyl groups and molecular weight and in the presence of an ortho-dihydroxy structure: the hydroxyl groups are responsible for the chelating and radical scavenging properties of these compounds.

Table 1: Phytochemical analysis of Ipomoea batatas leaves methanol extract.

Sl no	Phytochemical test	Result
1	Alkaloids
	Hager’s test	+
	Wagner’s test	+
2	Carbohydrates
	Fehling’s test	-
	Molish Test	-
3	Phytosterols
	LibermannBurchard’s Test	+
4	Phenols
	Ferric chloride test	-
5	Flavonoids
	Alkaline Reagent Test	+
	Lead acetate Test:	+
6	Tannin	-

Note: presence: + and absence: -

Table 2: Rf value of separated spots of Ipomoea batatas leaves on TLC plate.

Sl no.	Extracts	Mobile phase	Solute front	Solvent front	Rf value
1	Methanol	Solvent system 1	2.8	4.2	0.66
2	Hexane	Solvent system 2	1.5	3.4	0.44
		Solvent system 3	2.3	3.4	0.67

Thin Layer Chromatography:

TLC was performed to identify each components of the extract by its characteristic Rf values. One unknown compounds was present in solvent system 1, 2 compounds in solvent system 2, three in solvent system 3,which was further analysed by GC-MS. The Rf values of different spots are presented in table 2.

Gas Chromatography – Mass Spectrometry:

GC-MS chromatogram of the hexane extract showed 12 peaks indicating the presence of the phytochemical constituents, out of which six were characterized and identified. In case of Methanol extract 6 peaks were obtained and only four components were identified with specific activity. The chromatogram of hexane and methanol extract with different peak area is shown in fig 1 and fig 2 respectively. The components from hexane and methanol extract showed potent antimicrobial, anti-viral, anti-oxidant activities. N-hexadecanoic acid (palmitic acid), found in methanol extract of sweet potatoleaves which possess antibacterial and cholesterolaemic effects, selective toxicity to human leukaemia cells. It also has shown in vivo antitumor activity in mice by making a target to DNA topoisomerase I^{22, 23}. Whereas activity of Z,Z-6,28-hepatatriactontadien-2-one present in both methanol and hexane extract remained unknown. Ascorbic acid present in methanol extract are reported to possess strong anti-oxidant properties. Differing biological effects of ascorbic acid enantiomers and diastereomers have also been shown on the growth of human leukaemia cells.

Antimicrobial Activity:

Antimicrobial activity of sweet potato Ipomoea batatas leaves was checked against both Gram positive and Gram negative bacteria where Gram negative strains was found to be susceptible showing larger inhibition zone²⁴. The zone of inhibition for both hexane and methanol extract of sweet potato leaves is provided in table 3 and table 4 respectively. Hexane and methanol extract of sweet potato leaves has shown highest activity against Klebsiella sp. In comparison to hexane extract, methanol extract has shown better antimicrobial activity.

Fig 2: GC-MS chromatogram of methanol extract of Ipomoea batatas.

Table - 3: Antimicrobial activity of hexane extract ofIpomoea batatas.

Sl.no	Strains	Zone of inhibition
Sl.no	Strains	25mg/ml	50mg/ml	75mg/ml	100mg/ml
1	Pseudomonas aeruginosa	0.5	0.6	0.8	0.9
2	Serretia sp.	0.6	0.9	1.0	1.2
3	Proteus mirabilas	0.5	1.2	1.5	1.8
4	Shigelladysentaria	-	1.0	1.6	1.8
5	Staphylococcus aureus	0.8	0.8	1.7	1.9
6	Klebsiella sp.	0.6	1.0	1.9	2.0
7	Enterococcussp	0.4	1.2	1.9	1.9
8	Salmonella sp.	0.5	0.8	1.2	1.6
9	Escherichiacoli	0.3	0.5	0.9	1.1

Table - 4: Antimicrobial activity of methanol extract of Ipomoea batatas.

Sl.no	Strains	Zone of inhibition
Sl.no	Strains	25mg/ml	50 mg/ml	75 mg/ml	100 mg/ml
1	Escherichia coli	-	2.0	2.5	2.5
2	Klebsiella sp.	1.0	1.0	2.5	3.5
3	Shiegelladysentaria	0.8	1.5	1.9	1.9
4	Proteus mirabilas	0.5	0.7	1.0	1.2
5	Serratia sp.	1.0	1.0	2.0	2.5
6	Pseudomonas aureginosa	1.0	2.0	2.8	3.0
7	Salmonella sp.	0.9	1.5	1.9	2
8	Enterococcus sp.	1.0	2.0	2.0	2.5
9	Staphylococcus aureus	1.3	1.5	1.7	1.8

Antioxidant activity:

Antioxidant activity was evaluated by DPPH scavenging method. The total antioxidant activity was higher for hexane extract than methanol extract. Previous study has reported that presence of anthocyanin compound in sweet potato leaves imparts the antioxidant activity, which is able to scavenge free radicals ²⁵. The percentage of methanol and hexane extract of sweet potato leaves is presented in table 5.

Table 5: Antioxidant activity of Ipomoea batatasleaves extract.

Sample	Absorbance	Average	Percentage of inhibition
Standards	0.80
Methanol 1	0.637	0.519	35.12%
Methanol 2	0.402
Hexane 1	0.296	0.314	60.75%
Hexane 2	0.332

Anticancer study:

Anticancer study of Ipomoea batataswas done against human osteosarcoma cell line (MG 63). Anticancer activity of hexane and methanol extract was checked. The activities of hexane and methanol extract are shown in fig 3 and fig 4 respectively. For hexane extract cell viability is decreased with increased concentration of extract, which indicates the moderate activity of the extract. On the other hand, percentage of inhibition is found to increase with increasing concentration of methanol extract. It indicates the presence of anticancer activity in both methanol and hexane extract of sweet potato leaves. Sweet potato induced apoptosis has a mitochondrial mediated component, which was attenuated by pre-treatment with cyclosporine A. Alterations of apoptosis regulatory molecules such as inactivation of Bcl2, up regulation of BAX, cytochrome c release and activation of downstream apoptotic signalling was also reported²⁶.

Fig 3: Anticancer activity of hexane extract of Ipomoea batatas against MG 63 cancer cell line.

Fig 4: Anticancer activity of methanol extract of Ipomoea batatas against MG 63 cancer cell line.

CONCLUSION:

The extract of Ipomoea batatas showed considerably high activity against most of the test clinical pathogens, and antioxidant activity as the DPPH was scavenged by the sample. From thin layer chromatography, different unknown components of the extract was separated which was further confirmed by GC-MS analysis.Sweet potato leaves has also shown moderate anticancer activity against human osteosarcoma cell line (MG 63). So sweet potato leaves can be considered as useful replacement of usual drugs, though purification of the compounds is needed before final clinical uses.

CONFLICT OF INTEREST:

There is no conflict of interest .

REFERENCES:

1. Shih M, Kuo C, Chiang W. Effects of drying and extrusion on colour, chemical composition, antioxidant activities and mitogenic response of spleen lymphocytes of sweet potatoes. Food Chemistry.117; 2009:114–121.

2. Dini I, Tenore GC, Dini A. Saponins in Ipomoea batatas tubers: Isolation, characterization, quantification and antioxidant properties. Food Chemistry.113;2009:411–419.

3. Mors WB, Rizzini CT, Pereira NA. Medicinal plants of Brazil. Algonac: Reference Publishing Inc; 2000.

4. Jha A, Bhattacharya A. Preparation and evaluation of sweet potato starch-blended sodium alginate microbeads. Asian Journal of Pharmaceutics. 3; 2009: 299–303.

5. Miyazaki Y, Kusano S, Doi H, et al. Effects on immune response of antidiabetic ingredients from white-skinned sweet potato (Ipomoea batatas L.). Nutrition. 21; 2005:358–362.

6. Kwon HC, Jung CM, Shin CG, et al. A new caffeoylquinic acid from aster scaber and its inhibitory activity against human immunodeficiency virus-1 (HIV-1) integrase. Chemicaland Pharmaceutical Bulletin. 48; 2000:1796–1798.

7. Gaidhani SN, Singh A, Kumari S, et al. Evaluation of some plant extracts for standardization and anticancer activity. Indian Journal of Traditional Knowledge. 12 (4); 2013:682-687.

8. Evans WC. 1997. Pharmacology. Harcourt Brace and Company. Asia, Singapore. 226.

9. Wagner H. 1993. Pharmazeutische Biology (5th Ed.) AUFI. 15 BN 3- 437-20 498-X. Gustav fisher Vwelag. Stuttgart.Germany. 184.

10. Burma DP, Chakravorty M, editors. From Physiology and Chemistry to Biochemistry. Pearson Education India; 2011.

11. Finar IL. 1986. Stereo chemistry and chemistry of natural products. 2: Longman, Singapur. 682

12. Mace ME. Histochemicallocolization of phenols in healthy and diseased banar roots. Physiologiaplantarum. 16;1963: 915-925.

13. Abraham J, Chakraborty P, Chacko AM,Et al. Cytotoxicity and Antimicrobial effects of Pistiastratiotes Leaves.International Journal of Drug Development and Research. 6(4); 2015: 208-217.

14. Hammer KA, Carson CF, and Riley TV. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology. 6(6); 1999: 985-990.

15. Blois MS, Zhao XY. Antioxdant determinations by the use of a stable free radical. Nature. 181; 1958: 1199- 1200.

16. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 65; 1983: 55-63.

17. Monks A, Scudiero D, Skehan P, et al. Feasibility of high flux anticancer drug screen using a diverse panel of cultured human tumour cell lines. Journal of National Cancer Institute. 83; 1991: 757-766.

18. Nagappan T, Ramasamy P, Wahid ME, et al. Biological Activity of Carbazole Alkaloids and Essential Oil of Murrayakoenigii Against Antibiotic Resistant Microbes and Cancer Cell Lines. Molecules. 16(11); 2011: 9651-9664.

19. Robak J, Gryglewski RJ.Bioactivity of flavonoids. Polish Journal of Pharmacology. 48(6); 1996:555-559.

20. Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian medicinal plants. African Journal of Biotechnology. 4; 2005: 685-688.

21. Yokozawa T, Chen CP, Dong E, et al. Study on the inhibitory effect of tannins and flavonoids against the 1, 1-diphenyl-2 picrylhydrazyl radical. Biochemical Pharmacology. 56(2); 1998:213-222.

22. Harada H, Yamashito U, Kurilara H, et al. Antitumor activity of palmitic acid found as a selective cytotoxic substance in a marine red algae. Anticancer Research. 22;2002: 2587-2590.

23. Saxena M, Faridi U, Srivastava SK, et al. A cytotoxic and hepatoprotective agent from Withaniasomnifera and biological evaluation of its ester derivatives. Natural Product Communications. 2;2007: 1-4.

24. Islam S. Antimicrobial activities of Ipomoea batatas (L.) leaf. Journal of Food Agriculture and Environment. 6; 2008:14–17.

25. Philpott M, Gould KS, Lim C, et al. In situ and in vitro antioxidant activity of sweetpotatoanthocyanins. Journal of Agriculture Food and Chemistry. 52(6); 2004:1511-13.

26. Karna P, Gundala SR, Gupta MV, et al. Polyphenol-rich sweet potato greens extract inhibits proliferation and induces apoptosis in prostate cancer cells in vitro and in vivo. Carcinogenesis. 32(12); 2011:1872-1880.

Received on 27.06.2017 Modified on 15.07.2017

Research J. Pharm. and Tech 2018; 11(7): 2741-2746.

DOI: 10.5958/0974-360X.2018.00506.1