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Detection of Quercetin, Kaempferol and High Value Amino Acids from Seed Endosperm of Nymphaea stellata cv. alba L.

B.R. Jana1,*
1ICAR-National Research Centre for Makhana, Darbhanga-846 005, Bihar, India.

ABSTRACT

Background: In this study, the seed endosperm of Nymphaea stellata cv. alba was subjected to quantitative estimation of various phyto-chemicals, as it is commonly consumed as a nontraditional, non-cereal food in rural India. 

Methods: AOAC techniques were used to determine the approximate composition. UHPLC amino acid and flavonoid analyzers are used to measure the amino acids and flavonoids in seed endosperm, respectively.

Result: From standard analysis, it was evident that Nymphaea seed endosperm had 7.5±0.37% protein, 2.9±0.08% crude fiber and 1.8±0.21% total fat. The essential amino acids were determined in the seed endosperm by UHPLC method using pre-column derivatization method with o-phthalaldehyde- 2-mercaptoethanol.The results shown in the endosperm of  Nymphaea stellata cv. alba  produced the maximum amount of amino acids, aspartic acid (0.293 mg/g), glutamic acid (0.701 mg/g), serine (0.278 mg/g), histidine (0.116 mg/g), glycine (0.140 mg/g), threonine (0.149 mg/g) arginine (0.429 mg/g), alanine (0.239 mg/g), tyrosine (0.096 mg/g), cystine (0.424 mg/g), valine ( 0.50 mg/g), methionine (0.135 mg/g), phenylalanine (0.170 mg/g), isoleucine (0.339 mg/g) and leucine (0.34 mg/g). Furthermore, UHPLC chromatogram of quercetin and kaempferol in water lily seed endosperm revealed that the presence of quercetin 0.340 mg/kg and kaempferol 0.127 mg /kg as pure form without other derivatives of the two flavonoids like their glucoside compounds, esters, etc. The Nymphaea stellata cv. Alba seed endosperm has different beneficial composition including amino acids and flavonoids that may be of great importance to determine food quality as they helps in glucose metabolism and reduce oxidative stress caused by reactive oxygen species (ROS).

INTRODUCTION

Water lily (Nymphaea stellata cv. alba  L.) is wildly grown in wetlands belongs to all types of climate from temperate to tropical condition. It belongs to family Nymphaeaceae. India is native place of different types of Nymphaea. Water lily seed is very small and popped  nut is not popular in India. Poor and rural people primarily enjoy its fried nuts/pop with jaggery during ceremonies such as Poush Sankranti. It develops spontaneously in abandoned wetlands and can even be grown like transplanted rice fields as weed (Saji ​et al., 2023). Nymphaea seed endosperm are highly nutritious (Abelti et al., 2023) like other aquatic nut endosperm, makhana (Jana et al., 2022), but scientific cultivation is more easier than that of makhana (Khadatkar et al., 2014). According to Gopalan et al., (1996) water lily seeds contain 10.0% moisture, 8.3% protein, 1.0% fat, 0.9% minerals, 4.2% fiber and 75.6% carbohydrate.  The 100 g  of pop nut provide 345.0 k cal energy. Among minerals, it contained 20 mg/100 g calcium and 110 mg/100 g phosphorus. So nutritionally, Nymphaea seeds are  as important as makhana and lotus nut. Many scientist reported the lotus nut to have different flavonoids, which are anti-oxidant in nature. Flavonoids have anti-inflammatory and anti-oxidative effects also and thus may reduce against diabetic complications. It has been proven that flavonoid increases insulin uptake by modifying GLUT4 (Cho et al., 2010). Recent studies have also  suggested that flavonoids may have hyperglycemic activity by preventing the activity of enzymes that break down carbohydrates, like amylase, glucosidase and disaccharidases (Tadera et al., 2006 and Pereira​ et al., 2011). The flavonoid, quercetin, is a polyphenolic  compound   and the  most abundant flavonoid  in edible vegetables, fruit, wine and nuts.  It is a major bio-flavonoid in the human diet and may produce anti-proliferative effects resulting from the modulation of either EGFR or estrogen-receptor mediated signal transduction pathways. Quercetin, hereby, preventing the production of pro-inflammatory mediators and also reduces inflammation through the lipo-oxygenase and cyclo-oxygenase pathways. In addition, it is effective against cardiovascular disease, hypercholesterolemia, rheumatic diseases, infections and cancer. 
       
Hyperglycemia by improved insulin stimulated glucose uptake in adipocytes via quercetin and kaempferol was illustrated by Fang et al., (2008). Kaempferol has also beneficial role on diabetes by preventing oxidative damage in pancreatic bcells (Khalid et al., 2015). Seed flour of Nymphaea is generally used in preparation of baby foods, kheer, halwas, murkee and sweets. Flavanol glycosides in Nymphaea keep the head cool by helping blood circulation to the scalp and also to reduce the risk of heart disease.
       
As a result, in the current study, we quantified several phytochemicals such as flavonoids like quercetin and kaempferol, as well as nutrient components from water lily seed endosperm, in order to raise awareness about their use in diabetic and elderly patients. These studies provide a window for further research in this non-traditional and  non-cereal aquatic food for human benefit.

MATERIALS AND METHODS

Preparation of samples
 
Seeds of water lily was collected from field during Nov-Dec. Fruit of Nymphaea  is many seeded berry (approx. 2000 seeds /fruit) usually ripens during December (Plate : 1a,b,c). Seeds are collected and dried in sunlight for 3-5 days.  Seeds are very minute and crashed with hammer and endosperms were separated by sieving.

Plate 1: a. Nymphaea stellata cv. alba plant b. Fruits and c. Broken endosperms.


 
Proximate composition
 
Nutritive properties including moisture, ash, crude fat and crude protein contents were determined by AOAC methods (A.O.A.C., 2016). Crude protein was analyzed using Kelplus Elite Ex Micro Kjeldahl method using conversion factor 6.25. Carbohydrates (by difference) % weight was determined as followed by the method of Gopalan et al., (1998). Total dietary fiber (TDF) were determined by digesting the sample with a-amylase (AOAC method 991.43). All the biochemical analyses were carried out in four replications using fresh and dry Nymphaea kernel powder.

Amino acids
 
Amino acid profile was obtained by the analysis of samples through adopting procedures of Bidlingmeyer et al., (1987). Determination of amino acid in seed endosperm by UHPLC amino acid analyzer. The  method  for  amino  acid  extraction  was  followed  with  modification.  The  samples  were hydrolyzed with HCl 6 M at 110°C for 17 h. After hydrolysis, the acid was removed by rotary evaporation. Sample was resuspended in 25 mL of sodium citrate buffer at pH 2.2. A solution of Amino acids standards from Sigma (AAS-18) solution containing 2.5, 5.0 and 7.5 mg/L (Aspartic acid, glutamic acid, serine, histidine, glycine, L- threonine, Arginine, Alanine, Tyrosine, Cystine, Valline, Methionine, Phenylalanine, Isoleucine Leucine, Lysine, Proline) was prepared for calibration curves.
 
UHPLC condition
 
The amino acid is determined by UHPLC method (Rouba and Ulrich, 2013), Instrument :Nexera UHPLC (Shimadzu) with SIL-30AC autosampler (Shimadzu, 2007), Column: Shimpack GIST (C18), 5.0 mm (250 mmL. x 4.6 mm I.D.), Mobile Phase: A: 25 mmol/L Phosphate Potassium Buffer (pH 6.9) ,B: 45/40/15 Acetonitrile/Methanol/Water, Time Program : B Conc.10% (0.01)→11% (0.35 min),→45% (35.00 min)→ 100% (40.00-45.00 min), →10% (60.00 min), Flow Rate: 1.0 mL/min, Column Temp. : 35°C, Injection Volume: 10 mL, Detection: RF-20Axs Ex. at 350 nm, Em. at 450 nm, → Ex. at 266 nm, Em. at 305 nm (9.0 min), Cell Temp.: 20°C. Flow Cell: Conventional cell.
 
Preparation of derivatization reagents
 
Mercaptopropionic Acid- 3- Mercaptopropionic Acid 10 mL in 0.1 mol/L Borate Buffer (pH 9.2) 10 mL, 2.o- Phthalaldehyde Solution L: o-Phthalaldehyde 10 mg in 0.1 mol/L Borate Buffer (pH 9.2) 5 mL.3. Fluorenyl Methyl Chloro Formate (FMOC) - Acetonitrile Solution 9-Fluorenyl Methyl Chloro Formate 4 mg in Acetonitrile 20 Ml (Einarsson​ et al., 2001).
 
Derivatization procedure
 
Mercaptopropionic Acid, OPA and sample was taken in (mL) ratio of 45:22:7.5 and thoroughly mixed and kept for 1 min. then added 10 mL of FMOC and sample was injected to the UHPLC using Autosampler.
 
Quantification of quercetin
 
Structurally quercetin (Quercetin>95% (HPLC Solids), is a pentahydroxyflavone having the five hydroxy groups placed at the 3-, 3'-, 4'-, 5- and 7-positions. So, it is 3,3',4',5,6-Pentahydroxyflavone, 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one. Chemical formulae of quercetin  is C15H10O7  and molecular weight of 302.24.  We  applied  standard quercetin (Q4951-10G)  from Sigma-Aldrich  company, Bangalore, India.

 
Quantification of kaempferol
 
Kaempferol is a tetrahydroxyflavone in which the four hydroxyl groups are located at positions 3, 5, 7 and 4'. Chemical name is  3,5,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one and 3,4',5,7-Tetrahydroxyflavone., Chemical formula and molecular weight  are C15H10O6 and 286.24, respectively. We purchased standard kaempferol (K0133-10MG) from Sigma-Aldrich company, Bangalore, India.
 
 
 
 
Sample preparation
 
Ten gram of sample was homogenized with 70% methanol. Samples were vortexes for 5 min followed by centrifugation at 4,500 rpm for 10 mins. Filter the supernatant in 0.45 µm syringe filter. Then the samples were injected in HPLC system. Column oven temperature  was 30.0°C. Injected volume was 20 mL and flow rate was 1.0 ml/min. Mobile phase A was 6% acetic acid in 2 mM sodium acetate aqueous solution (v/v, final pH 2.55). On the  other hand, the  mobile phase B was Aceto-nitrile. The time gradient was, 0.01, 45.0, 60.0, 65.0, 70.0,75.0 and 80.0 minutes (stop). Post run time was 5.0 minutes (Tsao and Yang, 2003). Before sample loading we run two standards separately with above mention column condition.
 
Statistical analysis
 
The analysis of variance (ANOVA) was employed to compare means between genotypes/samples through the Fisher protected LSD with a significance level of 0.05%.  Results were expressed as the mean of four (sample) measurements (replications) ± SD. The linearity was established using three concentrations of amino acid standards. The data of peak area vs. amino acid concentration were treated by linear least squares regression analysis. The linearity data obtained should obey the equation:
y = bx + a
 
Where a is zero within the 95% confidence limits and the coefficient of determination (R2) is greater than 0.984 (Armagan et al., 2013 and Perucho et al., 2015). Another linearity standard curves were created for two flavonoids.

RESULTS AND DISCUSSION

Proximate composition
 
Data pertaining to Table 1 revealed that Nymphaea seed endosperm contained 7.5±0.37% protein, 2.9±0.08% crude fiber, 1.8±0.21%  total fat and 74.5±0.46 % carbohydrates. Similar results were also obtained by Gopalan et al., (1998) and Jana et al., (2021). According to Gopalan et al., (1996) water lily seeds contain 10.0% moisture 8.3% protein 1.0% fat 0.9-minerals 4.2% fibre and  carbohydrate 75.6%. Jana et al., (2021) also reported that water lily nut contains 10.19±0.92% moisture,  8.0±0.04% protein and 74.9±0.87% carbohydrates. The appreciable amount of protein and crude fibre and low amount of fat is appropriate for good health.

Table 1: Proximate composition of seed endosperm of Nymphaea stellata cv alba.


 
Amino acids
 
The results shown in the Nymphaea stellata cv. alba  produced the maximum amount of amino acids, aspartic acid (0.293 mg/gm ), glutamic Acid (0.701 mg/gm), serine (0.278 mg/gm), histidine (0.116 mg/gm), glycine ( 0.140 mg/gm), threonine (0.149 mg/gm), arginine (0.429 mg/gm), alanine (0.239 mg/gm). tyrosine (0.096 mg/gm), cystine (0.424 mg/gm), valine (0.50 mg/gm), methionine (0.135 mg/gm), phenylalanine (0.170 mg/gm), isoleucine (0.339 mg/gm) and   leucine ( 0.34 mg/gm) (Table 2)  Similar type of  results regarding different amino acids presence i.e. composition  in makhana  was confirmed by Jana and Idris (2018). This is the first ever amino acid profile developed in Nymphaea  seed endosperm  (Fig 1). Research results  from different experiments have shown that arginine can reduce insulin resistance, That is, insulin sensitivity can be increased (Krishna Mohan and Das, 1998). The amino acid arginine is of great importance for the sensitivity of body cells to insulin. Arginine is a precursor of nitrogen oxide, a transmissible substance that has a direct effect on insulin sensitivity. L-arginine improves peripheral and hepatic insulin sensitivity in type 2 diabetic patients (Piatti et al., 2001). In addition, arginine significantly expands blood vessels and reduces BP.

Table 2: UHPLC validation data for amino acid content in Nymphaea stellata cv. alba.



Fig 1: UHPLC chromatogram of amino of water lily seed endosperm.


 
Flavonoids
 
The flavonoid. quercetin,  is a polyphenolic  compound  and the  most abundant flavonoid  in edible vegetables, fruit, wine and nuts. In the present experiment, flavonoids  quercetin and kaempferol were estimated by using UHPLC chromatography. In the present experiment, the  retention time  for quercetin and kaempferol was 64.5 and 67.9 min., respectively. Furthermore, the peak area was 1565 units  and 895 units,  respectively. The presence of quercetin 0.340 mg/kg and kaempferol 0.127 mg/kg as pure form (Table 3) without other derivatives of the two flavonoids was revealed by a UHPLC chromatogram of  Nymphaea seed endosperm. Other glucoside compounds and esters may be present in the  seed endosperm as many other peaks were visible  in Fig 2. This research is first ot its kind. These are pure flavonoids. The seeds may contain their  other derivatives like  quercetin and kaempferol  glucosides and glucuronides.  esters  compounds. With the help of modification of  GLUT4, flavonoid has been  proven to promote insulin uptake Cho et al., (2010). According to recent research, flavonoid  may exhibit anti-hyperglycemic activity by inhibiting  carbohydrate hydrolyzing enzymes such as amylase, glucosidase and disaccharidases (Tadera et al., 2006 and Pereira​ et al., 2011). From this study, we confirmed first time  that  nymphaea seed endosperm contains  anti-diabetic flavonoids like quercetin and kaempferol in appreciable mount. The confirmation of the results corroborated by Zhu et al., (2017) as another aquatic nut i.e. lotus nut that had similar flavonoids. Nymphaea petal contain significant amount of quercetin and kaempferol compounds (Jambor and Skrzypczak 1991) by previous study. In general, kaempferol can potentially ameliorate glycoprotein abnormalities related to the risk of diabetes mellitus (Fang et al., 2008, Lee et al., 2010).  These two flavonoids increase insulin sensitivity and act as free radical scavengers and the amino acids in seeds are a good health indicator. 

Table 3: UHPLC validation data for flavonoid content in Nymphaea stellata cv alba.



Fig 2: UHPLC chromatogram of quercetin and kaempferol of water lily seed endosperm.

CONCLUSION

Thus, the Nymphaea endosperm is very nutritive food and useful for diabetic patient due to having flavonoids like  quercetin and kaempferol along with high value amino acids Arginine, Histidine, Isoleucine and leucine. This research is first of its kind and may open a new window for further research.

ACKNOWLEDGEMENT

Author is thankful to Director, ICAR Research Complex for Eastern Region, Patna, Bihar for funding and technical guidance for smooth running of experiment.

FUNDING

This work was supported by Institute Research Project and grant was obtained from ICAR-RCER, Patna, India.

CONFLICT OF INTEREST

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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