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Qualitative Phytochemical Analysis and Antibacterial Activity of Dioscorea alata L.: A Nutraceutical Tuber Crops of Rural Odisha

Published Date: December 06, 2017

Qualitative Phytochemical Analysis and Antibacterial Activity of Dioscorea alata L.: A Nutraceutical Tuber Crops of Rural Odisha

Kumar S1, Mahanti P2, Rath SK 3 and Patra JK4*

1Ambika Prasad Research Foundation, Cuttack, Odisha, India

2Directorate of Environment and Climate Change, Thiruvananthapuram-24, Kerala, India

3Department of Life Science, Ravenshaw University, Cuttack, Odisha, India

4Research Institute of Biotechnology and Medical Converged Science, Dongguk University-Seoul, Gyeonggi-do, Republic of Korea

 

*Corresponding author: Jayanta Kumar Patra, Research Institute of Biotechnology and Medical Converged Science, Dongguk University-Seoul, Gyeonggi-do, Republic of Korea, E-mail: jkpatra.cet@gmail.com

Citation: Kumar S, Mahanti P, Rath SK, Patra JK (2017) Qualitative Phytochemical Analysis and Antibacterial Activity of Dioscorea alata L.: A Nutraceutical Tuber Crops of Rural Odisha. J Alt Med Res 3(1): 122.

 

Abstract

Odisha is the state of rural and tribal therapeutic systems. People living in Odisha use most of the plant in dual form as food and medicine. The rural and local communities use different types of tuberous plants in their daily meals. Among them, Dioscorea alata, locally known as Desia Aalu is used as the staple food in their regular recipes called Dalma. Dioscorea species are rich in source of phenolic compounds. By considering the above important nutritious values, an attempt has been made to evaluate the secondary metabolites present in the plant parts of Dioscorea alata. Also to check the antibacterial activity of tuber extracts against Vibrio cholerae (MTCC 3909), Salmonella typhimurium (MTCC 1252), Shiegella flexneri (MTCC 1457), Streptococcus pyogenes (MTCC 1926) and Streptococcus mutans (MTCC1497). The results showed that the plant parts are rich in diverse secondary metabolites. It was observed that the tuber extract has highest activity against S. pyogenes. With the presence of bioactive compounds and antibacterial potentials along with more edibility made this tuber crop a strong nutraceutical food for rural and tribal community in Odisha, India.

 

Keywords: Antimicrobial activity; Bioactive compounds; Dioscorea alata; Tuber 

 

Introduction

 

Tubers are the modified plant structures that are enlarged parts to store nutrients. Plants take up nutrients from the tubers for self sustenance during unfavorable conditions and also to germinate through asexual reproduction with the help of stored nutrients. There are two types of tubers i.e. stem and root tubers. Major tuber crops are cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), yam (Dioscorea spp.), edible aroids (Colocasia esculenta and Xanthosoma spp.), and andean root [1–3]. Root tuber crops provide a substantial part of the world's food supply, and are also important sources of animal feed and industrial products. The consumption of root and tuber crops as food in developed countries is considerably less when compared to developing countries, but their use as animal feeds is relatively higher. Apart from food values these tuber crops are great source of medicinal compounds [3]. They find extensive use in the ethnomedicinal uses of tribal communities all over the world [2,3].

Genus Dioscorea belongs to the family Dioscoreaceae having approximately 600 species throughout the world [2,3]. It is also known as Yam. Plants are botanically monocot vines, stout or climbers. Genus is popular due to its twining characteristic of stem. Stem twining is a key for the taxonomic characterization of the Dioscorea species. Tubers and bulbils are edible in all developing countries including India. In Odisha there are about 12 species found in forest areas. Among 12, one species Dioscorea alata L. is cultivated and use as a rich garden crop of rural and tribal mass of the state. Rests of the 11 species are growing wild, they are D. bulbifera, D. wallichii, D. pubera, D. spinosa, D. hispida, D. glabra, D. bellophylla, D. hamiltonii, D. tomentosa and D. oppositifilia. These wild species are bitter in taste but by using traditional methods people will remove bitter portion and consume their tubers. D. alata L. is not bitter which is a suitable vegetable and raw snack. The tribal and rural communities of Odisha have been using its parts as food plus medicine. It is locally known as “Ma?ti A?lu” or “Desia A?lu”. D alata L. has very good agronomic characteristics. It is commonly known as water yam. Botanically it is perennial and climbing stout. Stems are sometimes four angled and twisted as metallic screw; compressed or strongly 3–4 angled at base; sometimes with scattered soft brown prickles. Leaves are mostly opposite but sometimes alternate and possess glabrous characters. Lower leaves are broadly ovate-cordate with a very broad sinus; upper leaves are smaller and narrow [1–3].

D. alata L. is a plant of the hot humid Tropics. It is distributed in South Pacific Islands, South America, West Africa, East Africa and Asiatic regions [4]. The Asiatic Dioscorea alata L., introduced to West Africa some hundred years ago, is fairly widely grown. In India, it is mainly distributed in Eastern Ghats, Northeastern Himalayas and Western Ghats. In southern part of India, it is cultivated as secondary food. In Odisha, it is distributed throughout the state. It is rich in Similipal Biosphere Reserve, Tumudibandha area of Phulbani district of state, Koraput, Malkangiri and other parts of the state [5].

Tuber of D. alata L. is macerated with water and the juice used as cooling agent during summer among the tribal communities of the Southern and Eastern part of Odisha. It is also the main ingredient of “Dalma”, a popular traditional food of urban, rural and tribal Odisha. In this traditional food, tuber of D. alata L. is most important constituent food material. There are some scientific reports available on the food values of this tuberous plant, but not more reports on pharmacological properties of the plant parts of Dioscorea alata L. Keeping this in view, an attempt has been made to identify the key characters for proper identification, qualitative phytochemical screening of leaves, bulbils, stem and tuber and antibacterial activity of selected tuber extracts using agar cup plate method along with Minimum inhibition concentration (MIC) against two Gram-positive bacteria, Streptococcus pyogens and Streptococcus mutans; and three Gram-negative bacteria, Salmonella typhimurium, Shigella flexneri and Vibrio cholerae.

 

Material and Methods

 

Collection of Plant Materials

Tuber of D. alata are collected from Kalika Parsad, an adjoining area of Similipal Biosphere Reserve, Odisha. The key characters for identification were observed from wild. Collected tubers are planted in experimental garden, Department of Botany, Ravenshaw University, Cuttak. For experiments, plant parts of planted vine were used.

Preparation of Plant Extracts

Soxhlet method was adopted to obtain the plant extract [6]. Fifty grams of powdered tuber was fed into the apparatus for each extraction. Extracts were collected with acetone, hexane, methanol and water after 5–6 siphons. The extracts were then dried over a period of 24 hours at room temperature. The dried samples were then weighed and stored in Eppendorf tubes under refrigeration.

Microbial Strains Used

The microbial strains employed in the study included Vibrio cholerae (MTCC 3909) Salmonella typhimurium (MTCC 1252), Shiegella flexneri (MTCC 1457), Streptococcus pyogenes (MTCC 1926) and Streptococcus mutans (MTCC1497), all of them procured from IMTECH, Chandigarh. All strains were maintained on nutrient agar slants. The first three are Gram negative strains and the last two are Gram positive.

Media Used

Nutrient broth was used to maintain broth cultures. The constituents of the nutrient broth included 0.5 g Peptone (Qualigens) and 0.3 g Beef (Qualigens) per 100 ml. An additional 1.8 g of Agar (Qualigens) made up the nutrient agar medium.

Agar Cup Plate Method

Agar Cup Plate method [7] was followed to test the antibacterial activity of various extracts against the five bacterial strains. Nutrient agar plates were prepared as per manufacturer’s instructions. One day before 100 µl of nutrient broth cultures of the test microbes prepared were poured in the plates uniformly and a lawn culture was prepared using a sterile spreader in a laminar hood. Wells of 0.8 mm diameter were punched over the agar plate using a sterile cork borer. Hundred micro liters of each of the four plant extracts were poured into the wells. The plates were incubated at 37°C for 24 hours. Only the solvent (DMSO) was poured into the wells in another set of plates as part of negative control. The positive control set consisted of standard antibiotics like Kanamycin, Neomycin, Gentamycin and Gatifloxacin in place of plant extracts. Zones of inhibition free of microbial growth appeared around each well in the form of clear rings which confirmed the antimicrobial activity of the respective extract. Those extracts which did not have any inhibitory effect on the microbe did not form any clear ring. In this way, the antibacterial activity of the extracts was confirmed. The cleared zone around each well was measured and average diameter was taken.

Phytochemical Analysis

Test for tannin: In 50 ml of distilled water 0.7 ml of the extracts were dissolved and heated for 10 minutes. After cooling few drops of 1% ferric chloride was added. Colour change was observed [8].

Test for saponin: Five milli liter of extract was dried and added 1 ml of Ethyl acetate. Then the ethyl acetate was removed and added distilled water, mixture was shaken vigorously to observe for persistent foam which lasted for at least 15 minutes.

Test for flavonoids: Some amount of tuber extract was taken in a flask and dissolved in 10% NaOH. Few drops of HCl was added and the colour change was observed.

Test for terpenoid: One ml of extract was mixed with 400 μl chloroform. Then the mixture was added by drop of sulphuric acid. A reddish brown interface indicates terpenoid present.

Test for alkaloids: Some quantity of each portion of extract was stirred with 5 ml of 1% aqueous HCl on water bath and then filtered. Then 1 ml filtrate was taken individually into 2 test tubes. To the first portion, few drops of Dragendorff’s reagent were added; occurrence of orange-red precipitate was taken as positive.

Test for phenolic compounds: Extracts were treated with 3–4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenolic compounds.

Test for steroids: To the 0.2 g of extract, 2 ml of chloroform was added; the solution was cooled well in ice followed by the addition of conc. H2SO4 carefully. Two layers developed of red and green indicated the presence of a steroidal ring.

 

Results and Discussion

 

The exploration work, literature survey and experimental work revealed that D. alata is very common and popular medicinal food crop amongst the masses. Tuber and bulbils are edible and very palatable. It showed morphological variations with respect to diverse ecology. Present study added some important taxonomic key for earlier work such as scattered prickles on stem which are brown in colour or sometimes greenish brown young leaves are light green with brownish apical end (). The leaves are too large in wild condition. Vine sometimes become large climber and bulbils grown as potatoes with several vegetative eyes. The wualitative analysis of phytochemicals revealed that Dioscorea alata possesses diverse pharmacological compounds such as tannins, flavonoids, phenolic compounds, alkaloids and saponins in tubers. The analysis further revealed presence of saponin, tannin, flavonoids, reducing sugar, glycosides in leaves, presence of tannin, terpenoids, steroids, flavonoids and reducing sugar in the stem and saponin, tannin, terpenoids, reducing sugar was detected in bulbils of Dioscorea alata ().

The curative properties of medicinal plants are perhaps due to the presence of various secondary metabolites like alkaloids, flavonoids, glycosides, phenols, saponins, sterols etc. [9]. Phenolic compounds like tannins found in plant cells are potent inhibitors of hydrolytic enzymes used by plant pathogens. Active constituents of plants usually interfere with growth and metabolism of microorganisms in a negative manner [10]. Plants synthesize these compounds to protect themselves from pathogens or disease or environment [11]. Other compounds like saponins also have antifungal properties. Many plants release phenolic compounds that are toxic to microbial pathogens [12]. Steroids were found to be present in all plants. It must be noted that steroidal compounds are of importance and interest in pharmacy due to their relationship with such compounds as sex hormones [13].

The antimicrobial activity of tuber extracts showed prominent zones of inhibition against Salmonella typhimurium (MTCC 1252), Shigella flexneri (MTCC 1457), Vibrio cholera (MTCC 3909), Streptococcus pyogenes (MTCC 1926) and Streptococcus mutans (MTCC 1497) at different concentrations and compared with standard antibiotics (Table 2–5, Figure 2). Streptococcus pyogenes (MTCC 1926) possess excellent activity of aqueous extract at concentration 1 mg/ml (Figure 3).

Antimicrobial activity of Dioscorea alata solvent extracts (n-hexane, acetone, methanol, aqueous) was studied against five human pathogenic bacteria (Salmonella typhimurium, Shigella flexneri, Vibrio cholera, Streptococcus pyogenes and Streptococcus mutans) by Agar cup method. Activity was observed at three different concentrations (0.25, 0.5 and 1 mg/ml). Least activity was observed at 0.25 mg/ml concentration of tuber extract. Except n-hexane extract against S. pyogenes and S. mutans (0.63 cm and 0.62 cm respectively). None of the extract exhibited activity against the five pathogenic bacteria. At 0.5 mg/ml all the solvent extracts exhibited activity against five pathogenic bacteria. Acetone and methanol extracts showed higher activity against the pathogenic strains in comparison to n-hexane and aqueous extracts. Highest inhibition zone (1 cm) was observed for acetone extract against S. pyogenes whereas least inhibition zone (0.62 cm) was observed against Vibrio cholera for all the four solvent extracts. At 1 mg/ml, inhibition zone of solvent extracts were more than 0.25 and 0.5 mg/ml. Aqueous extract exhibited highest inhibition zone (1.3 cm) against S. pyogenes whereas least inhibition zone (0.8 cm) was observed against S. mutans for acetone and methanol extracts as well as against Vibrio cholera for aqueous extract. Overall the antimicrobial activity of different solvent extracts increased with increasing concentration. This reveals the dose dependency of bioactive compounds present in solvent extracts of Dioscorea alata.

The effectiveness of active components present in the plant extracts is responsible for the production of inhibition zones around the wells. Lack of activity can only be proven by taking large doses. Some plant extracts were unable to show any antibacterial activity. Negative results do not indicate the absence of bioactive components, not even that the plant is inactive. It may be possible that these bacterial strains may have some kind of resistance mechanism like enzyme inactivation, target site modification or decreased intracellular drug accumulation [14]. Even if the active components are present in high quantities, there could be other constituents exerting anatagonistic effects on the bioactive compounds. It may also be due to the masking of antibacterial activity by presence of some inhibitory compounds in the extracts [15].

The antibacterial activity exhibited by the selected plant extracts may be due to the presence of phytochemicals like alkaloids, tannins, flavonoids, glycosides, saponins, terpenoids and sugars present in plant extracts [16]. The tannins form irreversible complexes with proline rich proteins, resulting in the inhibition of cell protein sysnthesis of bacteria [17]. Flavonoids containing one carbonyl group form complexes with extracellular and soluble proteins and with bacterial cell wall [18,19]. Since the selected plant in the present investigation is rich in bioactive phytochemical constituents, hence it showed prominent properties against the human pathogenic microorganisms. These findings have great practical application in the recent times as infectious diseases are leading cause of death worldwide. Use of antibiotics is the only solution to this problem. Indiscriminate use of antibiotics has led to the development of antimicrobial resistance to drugs. This has forced the search for new antimicrobial substances from various sources [20]. Thus findings of present study may lead to discovery of some potent antimicrobial drugs with some unique characteristics.

Based on the results of present study it was observed that the tuber as well as its other parts is good source of natural bioactive compounds. Moreover n-hexane, acetone, methanol and water are effective solvents for bioactivity evaluation of tuberous plants. Further studies are required to identify the active principles responsible for significant bioactivity and overall screening and isolation of compounds from this plant.

 

Conclusion

It is concluded that Dioscorea alata L. is a plant with rich food and medicinal values. Presence of bioactive compounds such as tannin, saponin, trepenoids, glycosides, reducing sugar in plant parts have shown the potential of biological and pharmacological properties of this tuber crop which is also reflected from antimicrobial activity of tuber extracts. The present study might be useful to supplement scientific information with regards to its medicinal values, bioactive compounds and pharmacological values. Further, more scientific studies are needed to evaluate the pharmacological properties of this plant leading to new molecules which could be used in pharmacological industries.

 

Conflict of Interest

 

All the authors declared that they have no conflict of interest.

 

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Copyright: © 2017 Patra JK, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.