Introduction

Malnutrition and nutritional problems are current issues of grave concern in third-world nations. Dietary deficiencies of both macro- and  micro-nutrients or in combined cause the aforementioned lethal issues¹. Although malnutrition has decreased steadily in Bangladesh, micronutrient deficiencies and non-communicable diseases (NCD) like cardiovascular disease, atherosclerosis, cancer, constipation, and irritable bowel syndrome (IBS) have increased over the last decade. Furthermore, youngsters are now inclined to processed food, rich in fat and salt but less in fiber resulting in being highly prone to NCDs².This food habit is amiable to produce more reactive oxygen species (ROS) or free radicals that lead to numerous disorders in the body. To prevent this provocative reaction, a sufficient daily amount of antioxidant is required to attenuate the damage in the body by ROS. There is no standard guideline for the daily dietary intake of antioxidants. However, physicians and nutritionists suggest for an adult person about 8000 to 11000 antioxidant units daily³. Even though artificial antioxidants are used to avert the gamble of free radicals, some studies revealed that these possess carcinogenic properties^4,5. In this view, the natural sources of antioxidants are intriguing to recent research and the neglected natural plants are entailed to look into as a source of antioxidants. The WWL and RWL cultivars are abundant and grown as a mixed population in all water bodies of Bangladesh. Some ethnic and village people considered water lilies in their diets without knowing its nutritive and medicinal value. Recently, some studies revealed that water lily leaves are used in the treatment of cancer⁶, controlling blood pressure, and erectile function⁷; seeds are used as dietary supplements for diabetes^8,9.The above research work showed the importance of this aquatic plant in terms of nutritional and medicinal value in daily diet. However, data on water lily stem grown in Bangladesh are limited. Hence, in this present study, we investigated the quantification of nutraceuticals and antioxidant properties of both species of water lily (WL). In addition, phytochemical assays such as total phenol and flavonoid as well as screening of tannin and saponin provides a clear understanding of the health benefits of WL stem. 

Materials and Methods

Sample Preparation

Both types of water lily were collected from the Halishahar and Patiya areas of Chattogram, Bangladesh. Then the stems were thoroughly washed and cut into 4 to 5 inches and dried in a cabinet dryer at 60℃ for around 24 hours. Afterward, fine powder samples were prepared by grinding and sieving through a 2mm mesh sieve.

Proximate Composition Analysis

The proximate composition, notably carbohydrate, fiber, crude protein, fat, ash content, and moisture content of water lily stems was done by following the standard Association of Official Agricultural Chemists (AOAC, 2016) method¹⁰.

Mineral Elements

Inorganic substances were analyzed following a standard method described by Uddin Zim et al. (2021)¹¹. Briefly, a 5g sample was acid digested (HNO₃: HClO₄=3:1) and then heated at 95°C for 10 minutes to facilitate digestion. After cooling, digestate was filtered (0.45 µm membrane paper) and diluted to 100 ml with deionized water. Finally, mineral contents (mg/100g) were assessed using Biochemical analyzer (Humalyzer 3000, Germany) and commercial kit (Randox®, England).

Total phenol content

The Folin-Ciocalteu technique¹² was employed to calculate the total phenol value. Succinctly, 1 g of powder sample was mixed with 10 ml methanol (80%) and then supernatant collected after sonication. Subsequently, 1 ml extract was mixed with 2 ml diluted Folin–Ciocalteu reagent and added 10 ml sodium carbonate (20%), then kept for 24 hours at room temperature. Finally, absorbance of the solution was measured by UV-visible spectroscopy (Shimadzu -2600, Germany) at 765 nm. To quantitate total phenol in sample, standard gallic acid curve (R² = 0.9986) was used, and unit was evinced as mg of gallic acid equivalent (GAE) per gram sample based on dried weight (DW).

Total flavonoid content

To evaluate the total flavonoid content, aluminum chloride colorimetric method was used¹³. In short, 50 g dried sample was extracted three times by ethanol (3 × 200 ml) and then dried. The powder was successively extracted with a mixture solvent (hexane, chloroform, ethyl acetate, and butanol) and then dried. Afterward, 4 mg of extracted powder was mixed with 50 ml of deionized water. Then 0.5 ml solution took into a test tube and added 2.8 mL of distilled water, 1.5 mL of methanol, 0.1 mL of aluminum chloride (10%), 0.1 mL of potassium acetate (1 M), and incubate 30 minutes at room temperature. Flavonoid content in samples was quantified against standard curve of quercetin (R² = 0.9985), and the values were calculated as mg of quercetin equivalent (QE) per gram sample based on DW.

Anti-nutrient Screening

The presence of saponin and tannin in lily stems was carried out by following standard detection methods¹⁴. At first, 1 g of sample was taken in a beaker and then boiled with 10 ml of 50% aqueous methanol solution. After boiling, 3 ml extracted aqueous solution was taken into a test tube. Subsequently, the test tube was shaken by hand, and foam formation was considered existence of saponin in sample. Comparably, 1 g of sample was boiled with 3 ml of butanol-HCl reagent (95: 5= n-butanol: concentrated HCL), and the color of solution change to pink was considered the presence of tannin in sample.

Antioxidant Activity

2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging method was used to determine the antioxidant capability of water lily stems described by Akter et al.(2021)¹⁵. Initially, 10 mg of methanolic extracted powder was mixed with 10 ml of methanol and serial dilution to prepare the same concentrated solution as reference solutions (ascorbic acid solution). Afterward, 1 ml of each diluted solution and 4 ml of DPPH solution took in a test tube, shook vigorously, and allowed to stand at room temperature in the dark for 30 minutes. Finally, the absorbance was measured by UV-visible spectroscopy (Shimadzu -2600, Germany) at 517 nm. DPPH and methanol solution was used as a negative control and blank solution respectively. The DPPH free radicals scavenging capacity was measured by the following equation-

% of inhibition = [(A_c– A_s)/A_c× 100]; where, A_c= absorbance of control; A_s = absorbance of sample. The half inhibition concentration (IC₅₀) value was calculated from % inhibition graph (Figure 1) by following equation-

IC₅₀ = (50-b)/m; where, b = intercept value; m = slope of line

Figure 1: DPPH radical scavenging capability (%) of sample and reference. Click here to view Figure

Data Analysis 

One-Way ANOVA and Tukey’s test was performed to evaluate and compare the data by using Statistical package for social sciences (SPSS) version 25 software. All values are exhibited as mean value ± Standard deviation (SD). Level of significance was considered at P < 0.05. 

Results

Proximate Composition

The nutritive value of water lily stems is depicted in Table 1. RWL stem contains a significantly higher amount of ash content than WWL stem. On the contrary, crude fiber content in WWL was significantly higher compared to RWL. Both species possess an almost similar amount of moisture, protein, crude fat, and carbohydrate.

Table 1: Nutritional composition of water lily.

Minerals Content

The stems of both species carried a rich amount of five essential minerals (Na, K, Ca, Mg, and P) display in Figure 2. The highest amount of macro-mineral content (mg/100 g) was K in both species, 871.21 ± 6.17 in WWL and 980.73 ± 5.35 in RWL respectively. Micro-minerals such as zinc (Zn), iron (Fe), and copper (Cu) were also sufficient amount in both species.

Figure 2: Macro and micro mineral content in water lily cultivars’ stem on dry basis; ** and * indicates the P-value<0.001, and P-value<0.005 respectively; ns = no significance Click here to view Figure

Total Phenolic and Flavonoid Content

WWL contains a slightly higher amount of total phenol content (16.51±0.05 mg GAE/g) than RWL (15.48±0.2 mg GAE/g) represented in Table 2. The results revealed that both types of stems contain amost same amount of total flavonoid.

Table 2: Phenolic and Flavonoid Content of water lily stems.

Anti-nutrients Screening

In this study, we screened both saponin and tannin in stem extract. Both species gave a positive result throughout the test shown in Table 3.

Table 3: Saponin and Tannin screening of water lily.

Antioxidant Activity

Free radical scavenging capacity of samples were assayed against DPPH solution. IC₅₀ value of ascorbic acid (AA), WWL, and RWL was 9.22, 36.67, and 28.48 µg/ml respectively depicted in Table 4.

Table 4: DPPH scavenging activity of water lily stem extracts.

 Discussion

Nutraceutical composition such as proximate composition, minerals, total phenol and flavonoid, and anti-nutrients (saponin and tannin) were measured in this study. The results revealed that both WWL and RWL species are rich in protein 8.16±1.07% and 8.46±0.72% respectively. Previous research work reported about 16.4% crude protein in the stem of Nymphaea (Nymphaea alba)¹⁶. This variation in value is may be due to the species variation, water bodies’ composition, and locations’ variation where water lily grown. In Table 1, the crude fiber content is significantly higher in WWL (19.56±0.68%) compared with RWL stems (16.24±1.05%), which are higher than the Nymphaea alba ranging from 9.39 – 9.50 %, 11.56 – 12.30 %and 11.84 – 15.1 % in the leaves, flowers and leaf stalk respectively¹⁷.This high range of fiber in stems may be due to its buoyancy properties, which require fiber and lignin in tissues of floated aquatic plants¹⁵. In concord with this explanation, it is believed that the stems of WL contain more buoyant structural material. A previous study reported that crude fiber is related to lowering the risk of colon cancer, diabetes, obesity, and heart disease¹⁸.  The ash content of RWL (18.52±1.01%) is significantly higher than WWL (15.44±0.79%). These values are within the ranges reported by Adelakun et al. (2016)¹⁹.The moisture content in both species of WL was an average of 13%, quite higher to compare with Nymphaea lotus species (9%)²⁰. Fat is one of the essential nutrients that can increase palatability of food and also carry out the fat-soluble vitamins. The quantity of fat is the lowest amount in both species about 1.5% on an average, interchangeable to the findings reported earlier by Shah et al. (2010)²¹. The probable reason for lower level of fat content in stem may be for its buoyancy properties and soaking with water. Low-fat foods are recommended for weight-reducing diets and also suggested in diets with high cholesterol levels²². As shown in Table 1 both species of WL are good sources of energy as they contain carbohydrates of about 43%  and the result resembles the findings of Shah et al. (2010)²¹.

Minerals regulate the physiological reactions in the body. However, a number of studies  have indicated  consumption of suboptimal micronutrients may lead to numerous nutrition-related chronic diseases and micronutrient deficiencies (MNDs)^23,24,25. In Bangladesh, children and women of reproductive age are prevalent to micronutrient deficiencies, where 41% of women²⁶ and 51% of children under the age of five suffer  Iron deficiency anemia (IDA); zinc insufficiency affects both women and children at 45% and 51%, respectively²⁷ . From Figure 2 it is vividly observed that both species contain a wide range of macro and trace elements. Among macronutrients Na, K and P were in substantial amounts in both species. The sodium content (mg/100g) was significantly higher in WWL (680.08±4.78) compared to RWW (520.24±3.77); conversely, RWL contained higher amount of K (980.73±5.35 mg/100g) than WWL (871.24±6.17 mg/100g), which is higher than of Nymphaea lotus (742.89 mg/100g)¹⁹. This higher mineral content in stem may be due to water qualities where the WL has grown up as the aquatic plants accumulate the nutrients from water bodies²⁸. Both K and Na are responsible for regulating blood pressure, water balance, muscle contraction, and the nervous system²⁹. The content of calcium and magnesium were satisfactory in both species, three times higher than Nymphaea lotus¹⁹. However, this specie contains a higher amount of phosphorus (635mg/100g) than the findings of the present study where the phosphorus was 320.08±3.15 and 511.58±4.51mg/100g in WWL and RWL respectively. These essential minerals play a crucial role in bone tissues, blood coagulation, neuromuscular irritability, and enzyme activity³⁰.  The water lily stems contain a moderate amount of iron, 15.44±1.33 and 20.72±3.25 mg/100g in white and red species those are higher than some vegetables consumed by rural people as curry such as cauliflower, cabbage, okra, pumpkin, etc. Iron is another essential micronutrient, responsible for iron deficiency anemia (IDA), highly prevalent (41%) in women of Bangladesh²⁶. It is essential for the transportation of oxygen, enzyme activity, regulating cell growth and cell differentiation, and boosting the immune system³¹.The copper content of RWL (2.79±0.43 mg/100g) was about doubled that of WWL (1.62±0.42 mg/100g) and zinc levels were 13.52±1.49 and 10.51±2.17 mg/100g respectively, whereas Adelakun et al. (2016) reported that Nymphaea lotus content 8.16mg/100g of zinc that was lower than our findings¹⁹. Both copper and zinc trace elements are essential for innate immune function, DNA and RNA reproduction, maintenance of cell membrane integrity, and destruction of free radicals³².

Phenolic compounds (PCs) are secondary metabolites of plants. Although PCs are not essential elements for the body, but can help to prevent numerous diseases and improve the human health.  The total phenol content of both species stem is about 16 mg GAE/g which is within the range reported in Nymphaea alba ranging from 5.40 to 19.50 mg GAE/g³³. Thus WL stem is a good source of PCs. It shows bioactive properties such as antioxidant activity due to its hydroxyl groups, and antibacterial activity³⁴. The stems of both species contain approximately 8 mg QE/g flavonoid compounds, which is lower than other parts of water lilies such as black Nymphaea lotus seeds (24.057 ± 0.113 mg EC/g) and Nymphaea micrantha seeds (27.026 ± 0.074 mg EC/g). This is due to the stem staying underwater whereas, flowers stay in the upper part of a plant and fight against UV radiation, cold, heat, and pathogens; thus stressor parts accumulate more flavonoid than other parts of plants³⁵.  It plays a crucial role to prevent cancer, inflammation, and viral infections³⁶.Moreover, the presence of tannin and saponin in WL stem revealed that it can be a good source of anti-peroxidative, anti-inflammatory, anti-carcinogenic, and anti-allergic³⁴.

The antioxidants prevent the oxidation caused by free radicals. During the metabolic process, the body produces free radicals by burning fuels. However, smoking, illness, food adulteration, and stress exacerbate the free radical condition³⁷. Our study, divulged that RWL (IC₅₀ value= 28.48 μg/ml) possesses more antioxidant properties than WWL (36.67 μg/ml), this is due to RWL containing more flavonoid and polyphenol in their stems³⁴. In contrast, according to Baehaki et al. (2015), Nymphaea stellate and Nelumbo nucifera species showed IC₅₀ values of 43.21 and 139.84 μg/ml respectively which are higher than our findings³⁸. It is evident from the above result that both species of water lilies can be used as a natural source of antioxidants in diet as well as in the medicinal purposes.

To the best of our knowledge, the present study is primitive that analyzed the nutraceutical and antioxidant properties (in vitro) of Bangladeshi white and red WL cultivars’ stems. The results of this study showed that the stems of both species are a rich source of protein, crude fiber, and essential macro and micro-minerals. Additionally, these can be incorporated into a normal diet to reduce oxidative stress such as aging, and are potential sources of natural antioxidants. However, further studies are recommended to identify additional phytochemical and bioactive substances in WL stems. Research on how different cooking methods affect the nutritional value of WL is also appreciated. Additionally, extensive in-vitro and in-vivo investigations are encouraged before moving forward with the development of supplements from WL stems.

Acknowledgment

The authors would like to express their gratitude and cordial thanks to the Department of Applied Food Science and Nutrition, and Poultry Research and Training Centre (PRTC), CVASU for providing analytical facilities.

Funding Sources

The authors would like to express their gratitude to the Ministry of Science and Technology, Bangladesh, and Advanced Studies &Research, CVASU, for providing funds for this research work.

Conflict of Interest

All authors declare no conflict of interest.

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