Phytochemical and Physico-chemical characterization with Amino acid profile of Nypa fruticans, Wurmb. from Kerala

 

Lovly M. S.*, Merlee Teresa M. V.

Department of Botany, St. Teresa’s College, Ernakulam, Kerala, India.

*Corresponding Author E-mail: lovlyms@gmail.com, merl1947@gmail.com

 

ABSTRACT:

The aim of the study was to analyse the physico-chemical characters, mineral composition, phytochemical and amino acids of leaf and stem of Nypa fruticans, Wurmb. Moisture content, ash content, acid insoluble ash, water soluble ash, extractive value in water and alcohol and mineral composition were determined in physico-chemical analysis. In mineral analysis, macronutrients such as N, P and K and micronutrients like Fe, Cu, Zn and Mn were analysed. Mineral analysis revealed that stem of N. fruticans, Wurmb. contained more K (6.21mg/g) than leaves. Potassium obtained in higher amount than Nitrogen and Phosphorous. Fe and Zn were recorded high in the stem. Analysis of amino acids and mineral nutrients could reveal the nutrient and therapeutic potential of the plant. Both primary and secondary metabolites were estimated from the leaf and stem of N. fruticans, Wurmb. Carbohydrates, proteins and lipids were the primary metabolites and phenol, flavonoids, tannins, alkaloids, sterols, terpenoids, coumarins and saponins were the secondary metabolites. Carbohydrates and proteins observed high in the stem of Nypa compared to leaves. Among the secondary metabolites, phenols, flavonoids and alkaloids were found more than the other secondary metabolites. Amino acid content of leaf and stem of N. fruticans, Wurmb. were determined using Pico-Tag method. Amino acids were confirmed in the sample by comparing with the retention time of standard amino acids. Among the 17 amino acids, 16 amino acids were identified in all the samples, except cysteine. Total quantity of amino acids was 15.94 mg/g in the leaves and 21.95 mg/g in the stem. Non essential amino acid alanine obtained in highest quantity (4.89 mg/g) in the stem.

 

KEYWORDS: Nypa fruticans, Wurmb., HPLC, Minerals, Amino acids, Phytochemicals

 

 


 

 

 

 

 

 

 

 

 

INTRODUCTION:

Medicinal value of a plant lies in its chemical composition that can cause correction of physiological abnormalities in humans. Nitrogen is one of the essential elements in both plants and animals and it forms one of the constituent elements of amino acids. Amino acids are essential in the synthesis of proteins. Amino acids are precursors in the formation of secondary metabolites of medicinal value. Cuin (2007)1 reported that amino acids are involved in cell signaling, gene expression and homoeostasis regulation. Amino acids are precursors in the synthesis of hormones and have antioxidant property2. Reports have shown that amino acids in plants are related with drought resistance 3.

 

Lovly and Merlee Teresa (2016)4 recorded the phytochemicals such as carobyhydrates, proteins, lipids, phenols, flavonoids and tannins in the leaf, stem, bract, flower, unripe endosperm and ripe endosperm of Nypa fruticans, Wurmb. Traditionally the plant parts are used by the local people for food and fuel. Preliminary qualitative analysis of fresh and fermented Nypa palm sap showed the presence of α amino acid, carbohydrates, glycosides, phenols, reducing sugars and saponins5. Nypa fruticans, Wurmb. has close resemblance with coconut palm in its long pinnately compound leaves. In kerala, the coconut palm is commercially used in the production of ‘toddy’ and ‘neera’. People in Kerala are unaware of the Nypa palm and its uses in the production of fermented Nypa sap and toddy from inflorescence stalk. Nypa palm is a trunkless palm, that can be cultivated in the coastal areas and mangrove forests of Kerala and more reliable in the collection of sap than coconut palm. The strong leaves of Nypa grow directly from the horizontal stem below the soil thus can protect the coastal belts of Kerala from natural calamities. First report of Nypa was made by Lovly and Merlee Teresa (2016)6 from Muziris Pattanam of Ernakulam district, Kerala.

 

In Asia, variety of products viz. wine, alcohol, arak, sugar and vinegar are prepared from Nypa sap or directly used as fresh juice7. Gibbs (1911)8 reported that the sap contained sucrose, protein, minerals as ash, calcium, phosphorous, iron, copper and vitamins. Non-essential amino acids, glycine, glutamic acid, alanine, proline, tyrosine and essential amino acids; methonine and leucine were detected in the Nypa sap9.

 

 Physico- chemical constants, mineral analysis, phytochemical estimation and amino acid profile of plant parts of Nypa were not reported in the previous studies. Amino acids as well as minerals form constituents that help in the formation of macromolecules such as vitamins, hormones, nucleic acids and secondary metabolites. N. fruticans, Wurmb. is reported to have antioxidant10,11,4 anti-diabetic12,4 and anti-inflammatory properties4. The aim of the study was to analyse physico-chemical parameters and mineral composition, quantification of phytochemicals viz. carbohydrates, proteins, lipids, phenols, flavonoids, tannins, alkaloids, terpenoids, sterols, saponins and coumarins, and the estimation of amino acid profile of leaf and stem.

 

MATERIALS AND METHODS:

Sample collection and processing:

Leaves and stem of N. fruticans, Wurmb. were collected from Muziris Pattanam and cleaned using autoclaved double distilled water and shade dried. Dried leaves and stem were powdered and stored in air tight containers.

 

 

Physico-chemical analysis:

a.     Moisture content (AOAC, 1990)13.

b.     Ash content (AOAC, 1990)13.

Sample was weighed, placed in a previously weighed crucible and heated in a muffle furnace at 525°C until white ash obtained. Ash obtained was weighed along with crucible and calculated the ash content with respect to the known sample.

 

Water soluble ash:

Distilled water added to the total ash and boiled. Insoluble ash is collected in an ash less filter paper and ignited. Water insoluble ash weighed and calculated the water-soluble ash in percentage with respect to the air dried known sample.

 

Acid insoluble ash:

Total ash collected and boiled with dilute HCl. Insoluble ash collected in an ash less filter paper, incinerated and weighed. Percentage of acid insoluble ash calculated with respect to the air-dried sample.

 

c.     Extractive value (Anonymous, 2001):

Extractive values obtained from plant extracts were a reflection of chemical composition of the plant extracts.

 

Alcohol soluble extractive value:

Leaf and stem of selected mangroves were extracted with methanol in a soxhlet apparatus and the extract obtained was concentrated. Dried extract weighed and estimated the percentage of extractive value with respect to the air dried sample.

 

Water soluble extractive value:

 Leaf and stem of selected mangrove plants were extracted with double distilled water in a soxhlet apparatus and the extract was concentrated to powder form. Solid extract weighed and calculated the percentage of extractive value with reference to the air dried sample.

 

d.     Mineral Composition:

1.     Macronutrients:

a.     Determination of Nitrogen by Kjeldhal method 15

b.     Determination of Phosphorous by Vanadate molybdate phosphoric acid yellow colour method 16

c.     Determination of Potassium by Flame photometric method 17

 

2.     Micronutrients16

Phytochemical analysis:

Quantitative estimation of phytochemicals was done as described by Rosenthaler (1930)18, Vogel (1958)19 and Harborne (1998)20.

 

Total carbohydrates:

Carbohydrates were estimated by the method of Dubois et al. (1956)21.

 

Total proteins:

Proteins were estimated by Lowry’s method 22.

 

Total Lipids:

Lipids were quantified by the method of Barnes and Blackstock (1973)23.                                                   

 

Total phenols:

Estimated by Folin- Ciocalteu method described by Singleton and Rossi (1965)24

 

Total flavonoids:

Flavonoids were analysed by Aluminium chloride colorimetric assay as described by Zhishen et al. (1999)25

 

Total tannins:

Tannin was estimated as reported by Ranganna (1986)26.

 

Total alkaloids:

Total alkaloids were estimated by the method, described by Harbone (1998)20.

 

Total terpenoids:

Terpenoids were quantified by the method, described by Ferguson (1956)27.

 

Total Sterols:

Sterols were extracted and quantified by the method of Tomita et al. (1970)28.

 

Total saponins:

Total saponin was quantified by the gravimetric method as described by Shiraiwa et al. (1991)29

 

Total coumarins:

Total coumarins were quantified by the method described by Osario and Martins (2004)30.

 

Determination of amino acids by HPLC:

Amino acid contents of leaf and stem of N. fruticans, Wurmb. were determined using Pico-Tag method as described earlier with suitable modifications31. The sample was hydrolysed for 24h at 110ο C with 6M HCl in sealed glass tubes filled with nitrogen. After treating with redrying reagent (MeOH 95%: water: triethylamine, 2:2:1 v/v/v) pre-column derivatization of hydrolyzable amino acids was performed with phenylisothiocyanate (PITC, or Edman’s reagent) to form phenylthiocarbamyl (PTC) amino acids. The derivatized sample (PTC derivative, 20 μL) was diluted with sample diluent (20 μL, 5 mM sodium phosphate NaHPO4 buffer, pH 7.4: acetonitrile 95:5 v/v) before being injected into reversed-phase binary gradient HPLC (Waters reversed-phase PICO.TAG amino acid analysis system), fitted with a packed column (dimethylocatadecylsilyl- bonded amorphous silica; Nova-Pak C18, 3.9 X 150 mm) maintained at 38±1°C in a column oven to be detected by their UV absorbance (λmax 254 nm; Waters 2487 dual absorbance detector). The mobile phase eluents used were eluents A and B, whereas eluent A comprises sodium acetate trihydrate (0.14 M, 940 ml, pH 6.4) containing triethylamine (0.05%), mixed with acetonitrile (60 ml), and eluent B used was acetonitrile : water (60:40, v/v). A gradient elution program, with increasing eluent B was employed for this purpose. An additional step of 100% eluent B is used to wash the column prior to returning to initial conditions. Standard (PIERS amino acid standard H; Thermoscientific) was run before each sample injection. Samples (PTC amino acid derivatives) were injected in triplicate, and the output was analyzed using BREEZE software. The identification of amino acids was carried out by comparing the sample with the standard amino acid. Quantification was done by external standard method using calibration curve and the results were expressed in mg/g.

 

RESULTS AND DISCUSSION:

Physico-chemical analysis:

The physico-chemical constants can be useful as a source of information and gives suitable standards to establish the quality of the plant material for future study or application. Moisture content is an important parameter that determines the physical and chemical stability of pharmaceutical preparations as water content in the sample may lead to their degradation. Moisture content was less in the leaves (6.32±0.84%). Total ash is reflection of inorganic nutrients in the sample. Total ash content was high in the stem (13.90±0.75%). Water soluble ash determines the exact amount of inorganic nutrients in the sample and was found to be high in the stem (11.85±1.15%). Acid insoluble ash present in the sample is an indication of impurities in the sample such as silicates or earthy matters and contaminations. Acid insoluble ash was recorded very less in both leaf (2.20±0.51) and stem (1.25±1.49). Extractive value is useful to evaluate the chemical constituents present in the crude drug. Extractive value helps to estimate the chemical constituents soluble in a particular solvent. Both alcohol soluble and water-soluble extractive values were high in the leaf (29.25± 1.01 and 27.04± 0.79%) and low in the stem (08.59± 1.05 and 07.68± 0.75%). Comparative analysis of physico-chemical constants are shown in the table: 1.

 

Table: 1. Physico-chemical properties of N. fruticans, Wurmb.

(% ±SD)

Physico-chemical constants

Leaf (%)

Stem (%)

Moisture content

6.32±0.84

9.60±0.72

Total ash

09.35±0.95

13.90±0.75

Water soluble ash

6.15±0.98

11.85±1.15

Acid insoluble ash

2.20±0.51

1.25±1.49

Alcohol soluble extractive value

29.25±1.01

08.59±1.05

Water soluble extractive value

27.04±0.79

07.68±0.75

 

 

 

Mineral composition:

Composition of macronutrients and micronutrients analysed are shown in the figure: 1. Nitrogen and phosphorous was found to be high in the leaves (0.92% and 0.223%), whereas potassium was recorded high in the stem (6.21%). Among the micronutrients, iron and zinc were obtained in high concentration in the stem (0.0816% and 0.0582%). Copper was absent in both leaf and stem. Manganese obtained more in the leaves (0.0297%) than in the stem (0.0073%). Potassium is important in cardiovascular and nerve functions, supplementing potassium can be useful in the treatment of hypertension caused by excess intake of sodium (Elson and Haas, 2011)32. N. fruticans, Wurmb. stem can be used in the treatment of hypertension as the stem of the plant possessed exceptionally high amount of potassium. Similary the elements like zinc and iron obtained more in the stem than in the leaves.

 

 

 

 

Figure: 1. Percentage of macronutrients and micronutrients in N. fruticans, Wurmb.

 

Phytochemical analysis:

Both the carbohydrate and protein content recorded high in the stem (283.00±0.87 mg/g and 86.151±0.79 mg/g). Total lipid was observed high in the leaves (87.91±0.89 mg/g). Traditionally parts of Nypa plant are used as food by the local people. Being rich in carbohydrates and proteins and poor in lipids the stem of N. fruticans, Wurmb. can be consumed as a healthy food. Among the secondary metabolites, flavonoids and phenols were obtained high in the leaves (99.50±0.54 mg/g and 79.91±0.96 mg/g) and less in the stem (11.34±0.52 and 33.19±0.79 mg/g). Leaves are rich in phenols and flavonoids and are related with the high antioxidant property. Antioxidant property of the Nypa fruticans has reported by Aziz and Jack (2015)11. Secondary metabolites were less in the stem compared to leaves. Alkaloids and saponins were reported in high quantity in the leaves (58.28±0.88 and 49.52±0.41mg/g) compared to tannins (16.66± 0.25 mg/g), terpenoids (3.4±0.12 mg/g) and coumarins (8.714±1.18 mg/g), that were less in amount. Sterol content in the leaves and stem were 15.71±0.40mg/g and 14.69±0.25mg/g respectively). Terpenoids and coumarins were very less in the stem (1.45±0.35mg/g and 1.918±0.32 mg/g). A comparative analysis of primary and secondary metabolites is given in the figure: 2

 

Figure: 2 Primary and secondary metabolites of N. fruticans, Wurmb.

 

HPLC analysis of amino acids:

Amino acids were identified by comparing the peaks of chromatograms obtained for the amino acids in the sample and the standard amino acids. Retention time of peaks obtained for the amino acids in the sample compared with retention time of peaks of standard amino acids and confirmed the amino acids in the sample. HPLC chromatograms are shown in the figure: 3.A-C. Area percentage obtained for each amino acid is given in the table: 2. Quantification in mg/g was estimated by external standard method using calibration curves and showed in the table:2.

 

Both the essential and nonessential amino acids were recorded in the leaves and stem of N. fruticans, Wurmb. Essential amino acids such as valine and leucine were obtained more in both the leaves (1.09 and 1.27mg/g) and stem (1.72 and 1.40mg/g) than other amino acids. Essential amino acids such as threonine (1.22mg/g), methionine (0.36mg/g) and lysine (0.93mg/g) were found higher in the stem than in the leaves. Non essential amino acids except cysteine were observed both in the leaf and stem. Alanine recorded highest amount in the stem (4.89 mg/g) than other amino acids.

 

Nypa palm is a mangrove palm and can grow under stress related conditions. According to Nirjhar et al. (2015)33 amino acids such as aspartic acid, alanine, leucine and proline can maintain water potential and provide adaptation under substrate salinity. Arginine, cysteine, glutamine, leucine and proline regulate chief metabolic pathways, and necessary for maintenance, growth, reproduction and immunity are called functional amino acids. All these functional amino acids were present in N. fruticans, Wurmb., except cysteine. Amino acids can be considered in the category of metabolites with antioxidant34, anti-diabetic 35 and anti-inflammatory potential36,37. Glutamic acid is recorded both in the leaves (1.50 mg/g) and stem (2.21 mg/g). Glutathione is an antioxidant, its synthesis takes place from glutamic acid. So antioxidant property reported in Nypa may be related with amino acids like glutamic acid. In vivo antidiabetic potential12 and antioxidant property of N. fruticans, Wurmb.10 have been recorded and may be related with the presence of amino acids. Leucine is obtained in moderate quantity in both the leaf (1.27 mg/g) and stem (1.40 mg/g). Anti-diabetic property of Nypa is related with amino acid like leucine. Leucine (Leu) stimulates insulin secretion by the change of Leu to ∞-ketoisocaproate, and enhances insulin release through distinct mechanisms38.

 

Table: 2. Amino acids in percentage area and mg/g in the leaf and stem of N. fruticans, Wurmb.

Test Amino acids

Amino acid analysis in N. fruticans, Wurmb.

 

Amino acids in % Area

Amino acids in mg/g

 

Leaf

Stem

Leaf

Stem

Aspartic acid

5.06

4.65

1.22

1.52

Glutamic acid

6.90

7.46

1.50

2.21

Serine

2.60

2.37

0.58

0.72

Glycine

12.42

8.33

1.84

1.68

Histidine

2.50

1.83

0.45

0.45

Arginine

4.30

5.12

0.75

1.22

Threonine

3.52

4.55

0.69

1.22

Alanine

10.87

18.58

2.10

4.89

Proline

12.51

7.44

1.71

1.38

Tyrosine

3.05

2.79

0.43

0.54

Valine

6.58

7.60

1.09

1.72

Methionine

0.06

1.56

0.01

0.36

Cysteine

0.16

0.01

0.02

0.00

Isoleucine

5.14

4.61

0.81

1.00

Leucine

8.25

6.70

1.27

1.40

Phenylalanine

5.16

3.47

0.77

0.71

Lysine

7.96

7.76

0.70

0.93

Total amino acids

97.04

94.83

15.94

21.95

 


 

 

 

 

 

Figure: 3. HPLC Chromatogram A. Standard amino acids, B. Amino acid profile of N. fruticans, Wurmb. leaf, C. Amino acid profile of N. fruticans, Wurmb. stem

 


CONCLUSION:

Physico- chemical parameters tested in the leaves and stem showed that the moisture content were less in the leaves. Acid soluble ash value found less in both samples indicated negligible amount of impurities. Both the alcohol and water extractive values of the samples were high, will help in the estimation of chemical constituents. High extractive value also denoted the solubility of variety of phytochemicals in the solvent. All the tested primary and secondary metabolites were obtained from the plant. Isolation and purification of different classes of phytochemicals and their characterization will be benefitted in the future research. Mineral analysis showed the highest potassium content in the stem of N. fruticans, Wurmb. Both essential and non-essential amino acids were obtained in the leaves and stem of N. fruticans, Wurmb. Presence of amino acid in the plant reflected the antioxidant, anti-diabetic, anti-inflammatory and antioxidant properties of the plant. Some amino acids act as a precursor of phytochemicals and hormones.

 

ACKNOWLEDGEMENT:

Authors sincerely extend their gratitude to Dr. Vijayagopal, Dr. Kaladharan and Mrs. Shylaja of CMFRI for their cooperation and support in doing amino acid analysis. Authors are also thankful to Department of Botany, St. Teresa’s College, Ernakulam for their cooperation and support in providing laboratory facilities for the analysis of the plant.

 

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

 

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Received on 17.06.2018          Modified on 15.09.2018

Accepted on 19.10.2018  ©A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2018; 10(4): 304-310.

DOI: 10.5958/0975-4385.2018.00049.3