Morphological and anatomical property, phytochemical screening and antibacterial activity of “Green” wild taro (Colocasia esculenta (L.) schott)

Introduction

The traditional medicine has been successful to cure diseases for the past thousands of years (Kala et al., 2006; Sridharan and Sivaramakrishnan, 2018) [13,36] . Approximately 88% (170 countries) of all Member States across all WHO regions acknowledged the use of traditional and complementary medicines in 2018 (WHO, 2019) [43] . These medicinal plants have provided an important source of herbal remedies since ancient times and have been preserved to the present day. Phytochemicals, chemicals derived from primary or secondary metabolism of plants, are considered as a rich source of various drugs (Kala et al., 2006) [13] . Several phytochemicals have therapeutic properties for various diseases and disorders (Rajkumar et al., 2018) [30] as cardiovascular protection, anticancer activity, anti-nociceptive activity and antiinflammatory effects (Mohammed et al., 2014) [18] . Therefore, medicinal-property plants are determined as an indigenous source of new compounds in drug development (Vines, 2004; Newman et al., 2020) [22] . The bioactive phytochemicals are recognized as alkaloids, phenolics, tannins, terpenes, steroids, flavonoids and glycosides (Rajkumar et al., 2018) [30] . Colocasia esculenta (L.) Schott or taro is a tropical plant belonging to the family Araceae and has its origin from South-East Asia. Taro is rich in carbohydrates, proteins and micronutrients (Temesgen & Retta, 2015; Rashmi et al., 2018) [39, 31] . Taro has various potential for investigating its medicinal and pharmaceutical properties (Dutta et al., 2017; Sharma et al., 2019; Nayak et al., 2019) [21,6,33] . Taro has been shown to have anti-tumor, antibacterial (antibacterial and antifungal), antidiabetic, antitoxic and anticancer biological activities (Sharma et al., 2020). Wild taro, a natural type of Colocasia esculenta (L.) Schott, has little studied and is poorly known (Matthews et al., 2012). Wild taro has been valued as a source of traditional medicine and food for human and animals in many countries. The leaf juice of wild taro is useful in treatment of internal haemorrhages, otalgia, otorrhoea, adenitis and buboes and the corm juice is used to treat somatalgia, alopecia areata, haemorrhoids and congestion of the portal system (Namrata et al., 2011) [34] . Chakraborty et al. (2015) [4] confirms the presence of alkaloids, flavonoids, carbohydrates, tannins, terpenoids in methanol extract of both tuber and leaves extract. According to Ivancic and Lebot (1999), wild taro is separated into three distinct morphotypes: "purple", "green", and "green with purple vein junction on lamina". "Green" morphotype is the tallest height (mean 141,52 cm), the largest leaves (mean 81.32 cm), cylindrical sucks (3-10 cm long and thick 1.7-2.5 cm in diameter) stolons. Each inflorescence of "green" morphotype wild taro is made up of peduncle, spathe and spadix. The objectives of this study were to investigate of morphologically and anatomically vegetative characteristics, determinate chromosome number, preliminary determinate chemical composition, and assess antibacteria activity against E. coli and B. subtilis of "green" morphotype wild taro Colocasia esculenta (L.) Schott. DOI: https://dx.doi.org/10.33545/26649926.2021.v3.i2a.33

Materials and Methods

Collection of Plant Material

"Green" morphotype Colocasia esculenta (L.) Schott was collected at Cantho province, Mekong Delta, VietNam.

Morphogical and Anatomical Study

Vegetative organs were keep intact to investigate morphological and anatomical features. Leaves (including lamina and petiole) and tubers (including root and stem) were sliced horizontally (Vo, 2019). The slices were soaked in sodium hypochlorite, rinsed with distilled water, soaked in 5% acetic acid, double-stained with carmine alum laque-iodin blue dye, and washed again with distilled water. The specimens were observed under light microscope.

Estimation of Chromosome Number

Root tips were treated hypo-osmotic shock by sodium citrate dihydrate 0.1% for 45 minutes, fixed in Carnoy'solution (3 ethanol: 1 acid acetic) for 4 hours, and stainned by acetocarmine 1% for 50 -60 minutes. Chromosome numbers of meristem of root tip at pro-metaphase plate were counted and estimated. P value <0.05 was considered statistically significant different (Vo, 2019).

Moisture Content

Each 5g fresh tuber/leaf was cut into small parts, and place the samples in a hot air oven at 50ºC until the dry weight of the samples become constant. Sample moisture content was determined as the percentage moisture: Moisture (%) = (Fresh weight -Dry weight)/Fresh weight (Krishnapriya and Suganth, 2007).

Qualitative analysis of the phytochemicals Preparation for powder sample

Leaf/ tuber were cut into small pieces and dried in hot air oven at 55 0 C until the dry weight of the samples become constant. Grind samples to a fine powder for phytochemical analysis.

Phytochemical screenning

Second metabolic production of leaf/tuber powder of "green" morphotype wild taro were carried out according to the procedures proposed by of Parekh and Chanda (2007) Tannins: 1 g leaf/tuber powder were gently boiled with 20 ml distilled water in 10 minutes, and filtered. Add reagent (CH3COO) 2 Pb/ FeCl 3 1% drop wise to 1 ml of the filtrate. Slightly yellow precipitate/ Blue-black or dark green precipitate indicated tannin's presence.

Antibacterial screening Ethanol extraction

The 30 g sample powder (leaf/ tuber) were extracted with 450 ml ethanol by maceration method in 48 hours. The solvent was remove by evaporating under reduced pressure by using rotary evaporator. The crude extracts were dissolved in DMSO to a concentration of 200 µg/ml, 400 µg/ml, 800 µg/ml.

Antimicrobial assay

Antibacterial activity of wild taro extractions were detected by the agar well diffusion method. Escherichia coli and Bacillus subtilis were cultured in LB agar at 37ºC in 24 hours and adjusted to 10 6 cfu/ml (compared to 0.5 McFarland turbidity standards). Each 100 μl of prepared bacterial suspension were spread on one LB Agar petri dish and inoculated for 24h. At every dish, 100 μl of leaf/ tuber extraction of three different concentrations (200 µg/ml, 400 µg/ml, 800 µg/ml), and negative control DMSO were added into 6 mm diameter wells. After incubating at 37 o C for 24hrs, all petri dishes were taken out to measure the diameter of the inhibition zone in mm. The experiments were repeated 3 times. Statistical analysis of the data was done by using One-way ANOVA, and p value < 0.05 was considered statistically significant.

Results

Morphological Characteristics

"Green" morphotype wild taro (Colocasia esculenta (L.)

Schott.) was a herbaceous, underground stem that developed into corms. The height of the tree were up to 1.2 m -1.4 m. The false stem grew above where the petiole were arranged around.

Leaves consisted of green leaf blade (lamina) and green petiole without purple vein junction on lamina. Leaf blade was simple, broad, heart-shaped, up to 75 cm long and 65 cm wide with prominent veins. The main venation had a "Y" shape that run along the length of the blade leaf and reached 2 sides of lobes. Along the main venation were 8 to10 pairs of subvenations. Petiole was oblong. Closer to the leaf blade, the petiole was rounder and smaller. Tuber consisted of corm and roots. Corms were bulb-like, grow underground and inedible.

The lower part of corm was rhizomes with different shapes and sizes. One or few yellow inflorescences with yellow spathe grew in one bush. Spadix consisted of four portions: female portion (lowest), a sterile region, a male portion, and an appendix. Fruit was watery, spherical-liked shape, and turned from green to orange or red as mature. Fruits contained many yellow-brown seeds (Figure 1). Vo (2020) described further anatomical characterictics of "green" morphotype wild taro's reproductive organs. Corm: Transverse section of corm's starchy flesh showed polygon-spaped parenchyma cells. The size of these parenchyma cells were smaller than that of root. Fibers were also detected in corm. Calcium oxalate crystals were revealed in forms of free needle-liked crystals, druses and raphide bundles. Biforine or biforine-liked cells, another type of raphide bundle, were observed in corm (Figure 3 A,B). Blade leaf: Epidermis, the outermost layer, was present on the upper and lower surfaces of the vein and lamina of leaf blade. Both upper and lower epidermic layers were composed of contiguous, jar-shaped cells. Cuticle layer was revealed on both epidemic layers, but the lower layer were impregnated with a thicker cuticle. Under the upper epidermis was 2 -3 layers of rectangular-shaped palisade mesophyll cells that were embeded many calcium oxalate crystals. Free needleliked crystals, druses, and raphide bundles (included biforine or biforine liked cells) were investigated on leaf blade. Druse crystals on the upper surface of the leaf blade were more densely distributed than on the lower surface. Spongy mesophyll cells were varied in size and shape, and separated by intercellular spaces. Sclerenchyma fibers were arranged circumferentially in the leaf veins. Vascular bundles with various size were scattered in both leaf venation and lamina (Figure 4A,B). Petiole: Epidermis contained cells laced cuticle. Beneath the epidermis was hypodermal paenchyma that was 2 -3 layers of small, angular collenchymal cells. The sclerenchyma fibres were formed phloem caps, and arranged circularly below epidermis. Parenchyma layers were thin-walled cells with various size and polygonal shape. Dozens of vascular bundles were arranged radially throughout the ground tissue, with the phloem bundles facing the epidermis and the xylem bundles slightly toward the center. Air spaces were revealed with different sizes and shapes, and distributed throughout the petiole (Figure 5 A,B) Many researches have been studied morphlogical and anatomical properties of taro (C. esculenta). Stein et al. (2015) [35] were described a multi-layered palisade, air-filled spongy mesophyll, vascular strand, collenchyma bundle, raphide bundle, stomata, etc. of leaf of taro. Pitoyo et al. (2018) [27] were investigated morphological measurements and anatomical characters of 20 taro accessions, and suggested that stomatal data, epidermis thickness, adaxial epidermis thickness, mesophyll thickness, and palisade thickness could be employed to distinguish these varieties. Ezebra et al.

(2015a, b) compared anatomical features of leaf, root, and petiole of five taro cultivars, and found that morphological and anatomical characters (such as stomata data, CaOx crystals, airspace, thickness of epidermis, mesophyll, and palisade, etc.) were an additional parameter in taro identification. Compared with the above studies, the vegetative organs of "green" wild taro showed common histological and anatomical features of Colocasia. However, the stomata and stomatal chambers were rarely revealed in horizontally sectional studies. This study revealed three types of CaOx crystals such as free needle-liked crystals, druses and raphide bundles compared to druses, raphide bundles, prims and sand crystals found in the above studies on taro. Therefore, more studies are needed to compare the three morphotypes of wild taro or between wild taro and cultivated taro. Many methods have been applied to study the anatomical and histological features with good results. In this study, double staining with carmine alum laque-iodin blue dye on the vegetative organs helped to clearly identify and distinguish the anatomical characteristics of the investigated plants.

Estimation Of Chromosome Number

Chromosome number of "green" morphotype wild taro was estimated 2n = 28, 1 ± 1.125 (p > 0.05) (Figure 6). Ivancic and Lebot (1999) [12] reported that all three wild taro morphotypes were diploids (2n = 2x = 28). However, in China, chromosome number of Colocasia species were found as 2n = 26, 28, 38, 42, and 56 (Wang et al.,2017) [45] . Das et al. (2015) revealed ten Indian cultivars of Colocasia esculenta to be possessed from (2n = 2x = 28) to (3n = 3x = 42).

Fig 6: Chromosome number

Water Content and Yield Extract Of "Green" Morphotype Wild Taro Water content of "green" morphotype wild taro was 94.1 ± 0,91 % at leaf and 90,3 ± 0,58 % at tuber. For taro, its moisture content were 73,1% at tuber and 82,7% at leaf (Gopalan et al., 1989). Water content of taro tuber was 56.8% -57% (Krishnapriya and Suganth, 2017; Nayak et al., 2019) [21] . The moisture content of taro ranges from 60 -83% (Huang and Tanudjaja, 1992) [11] . Moisture content of taro varied depending on their variety, growth condition and harvest time. The extract yield of "green" morphotype wild taro was 12, 33 % at leaf and 10,03 % at tuber. Dutta and Aich (2017) [6] was provided the yield extract of leaf taro was 9.12%.

Qualitative Phytochemical Screening

Based on precipitation, colour reaction, crystallization reaction, foaming index, lactone ring opening reaction, diazotization reaction to determine the presence of phytochemical compounds. The results showed on Table 2. Alkaloids, tannins, glycosides and coumarines were found to be positive for all organic reagents tested. Steroids and saponins were indicated positive for Libermann-Burchard reagent but negative for Salkowsky reagent or foam test. Specially, flavonoids at leaf/tuber extracts were proved positive to all reagents tested but negative for Shinoda at tuber extract. In summary, both leaf and tuber extract of "green" wild taro were indicated the presence of steroids, flavonoids, alkaloids, tannins, glycosides, coumarines, and saponins. [9] , and leaf stalks of wild taro, named Hideung Cultivar, was showed the presence of flavonoid, tannin, saponin, alkaloid, and calcium oxalate (Widhyastini et al., 2019) [44] . Tuber extraction of wild taro at Garhwal Himalaya (India) was indicated the presence of glycosides, flavonoids, phenols, resin and tannins (Subhash et al., 2012) [38] . Keshav et al. (2019) [15] reported that alkaloids, glycosides, flavonoids, terpenoids, saponins, oxalates and phenols in C. esculenta could have lot of medicinal benefits such as reducing headache, treatment of congestive heart failure, prevent oxidative cell damage.

Antibacterial Activity of ethanol Extraction of "Green"

Wild Taro Again E. coli and B. subtilis Both leaf and tuber extraction of "green" wild taro inhibited the growth of gram-negative and gram-positive bacteria. For E. coli, the diameter of inhibition zone were varied from 9.33 mm to 19.00 mm at the concentration of 200 µg/ml, 400 µg/ml and 800 µg/ml. For B. subtilis, the diameters of inhibition zone were varied from 5.67 mm to 19.00 mm at the concentration of 400 µg/ml and 800 µg/ml, and no inhibition zone was observed at 200 µg/ml. At both leaf and tuber extraction, the diameter of inhibition zones against E. coli and B. subtilis was not statistically different between 800 µg/ml and 400 µg/ml (p > 0.05). For E. coli, the diameter of inhibition zones of both tuber and leaf extraction at the concentration of 800 µg/ml and 400 µg/ml were statistically different at 200 µg/ml (p < 0.05). The study also found that at both leaf and tuber extraction, the diameter of inhibition zones against E. coli were statistically different from that of B. subtilis. Aqueous leaf extract of wild taro "Arbi" in Northern India showed antibacterial activity against Streptococcus mutans, B. subtilis, Pseudomonas fragi, and E. coli (Singh et al., 2011). Ethanol leaf extraction of wild taro at Western region India showed antibacterial activity against Salmonella typhi, Klebsiella pneumonia, Pseudomonas aeruginosa, and B. subtilis but non-effective on Salmonella paratyphi, Staphylococcus aureus, Proteus vulgaris, and E. coli (Nakade et al., 2013). Leaf extraction of wild taro "keladi" in Malaysia, was against Staphylococcus aureus and Vibrio parahaemolyticus (Padzil et al., 2021). Antibacterial activity of wild taro suggests the potential of medicinal plant to treat typhoid, pneumonia, otitis media, urinary tract infections and diarrhea (Nakade et al., 2013) [9] . Second metabolic productions of plants play an important role as anti-inflammatory (Mohammed et al., 2014) [18] and antibacterial agents (Compean and Ynalvez, 2014). Alkaloids are used for reducing fever and headache Krishnapriya and Suganthi (2017). Flavonoids were recorded their antifungal, antiviral and antibacterial activity (Panche et al., 2016) [25] . Glycosides are used in treatment of congestive heart failure. Coumarin activities range from antimicrobial and antiviral to anticoagulant and anticancer (Stringlis et al., 2019) [37] . Glycosides isolated from plants demonstrated potent cytotoxic effects against various cancer cell lines (Khan et al., 2019). Terpenoids showed defence activity against environmental stress and help to heal injuries (Prakash, 2017) [28] . Saponins showed anti-inflammatory activity in mice and rats inflammation models (Mohammed, 2014) [18] . Saponins exhibit a medicinal properties such as anti-inflammatory, antibacterial, antifungal, antiviral, insecticidal, anticancer and cholesterol-lowering action in animals and human (Aziz et al., 2019). Steroids possess pharmacological properties such as antidiabetic, analgesic, anti-inflammatory and anthelmintic activities (Rahman et al., 2017) [29] . These biological properties in plants are expressed through organic compounds, which can belong to many different phytochemical structures. Traditional medicine has used wild taro as a medicinal ingredient and its antibacterial properties may be related to the presence of qualitative organic substances.

Conclution

Anatomical and morphological characteristics of tuber and leaf of "green" morphotype wild taro (C. esculenta) were discribed on this research. Chromosome numbers of investigated species were estimated as 2n = 28. Leaf and tuber of "green" wild taro were indicated the presence of phytochemicals such as steroids, flavonoids, alkaloids, saponins, tannins, coumarine and glycosides. The leaf and tuber ethanol extract of wild taro were resistant to both strains of E. coli and B. subtilis. Preliminary results showed that the effect of ethanol extract is stronger against gram-negative bacteria than gram-positive. Therefore, this plant has great potential to be used for further study on phytochemical components and their correlation with pharmacological effects.

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