Estimation of Phenolic acid and Flavonoids in leaves of Amaranthus viridis and Amaranthus spinosus

 

Swarnakumari S*, Sasikala M, Mohan S, Divyanand Maharaj U, Kavipriya G

Karpagam College of Pharmacy, Coimbatore, Tamil Nadu, India.

*Corresponding Author E-mail: swarnapremanand@gmail.com

 

ABSTRACT:

Objective: To explore physiochemical and phytochemical standardization of leaves of Amaranthus viridis and Amaranthus spinosus. Method: As per WHO guidelines, physical standardization parameters with various solvents system were evaluated. The preliminary phytochemical screenings were adopted for disclosing the existence of phytochemicals in the discrete extracts. Thin layer chromatography and HPTLC were employed for methanolic extract of leaves of Amaranthus viridis and Amaranthus spinosus. Result: Preliminary phytochemical screening with various extracts reveals phytoconstituents. HPTLC fingerprint were executed for leaves of Amaranthus viridis and Amaranthus spinosus using selected solvent system. HPTLC analysis implies the presence of phenolic compounds and flavonoids in both the plant Amaranthus viridis and Amaranthus spinosus. Conclusion: Quercetin and Gallic acid was raise to be more in Amaranthus viridis compared to Amaranthus spinosus, however rutin was reported only in Amaranthus spinosus. The outcome of the research leads for isolation, purification and utilization in herbal industries.

 

KEYWORDS: HPTLC, Methanolic extract, Amaranthus viridis, Amaranthus spinosus, Quercetin, Gallic acid, Rutin.

 

 

INTRODUCTION:

In the recent decades, the necessity of herbal medicines is increased in traditional and modern medicine. Herbs, from starting raw material to finished products are considered as Herbal medicine. Since raw materials influences over the quality, efficacy and safety of finished herbal preparations, standardization of raw materials is required1,2. Plants are considered as biochemical factory, since they have capacity to biosynthesis many natural products (primary and secondary metabolites) within themselves. The production of secondary metabolites will be varied based on the precursor (primary metabolite) engaged in metabolic pathway which occurs within the plant cell. Secondary metabolites act as basis for drug development and therapeutic purposes3.

 

Amaranthus viridis belongs to the family Amaranthaceae, commonly known as slender amaranth.

 

It is an edible herb in southern parts of India and also used in ayurvedic formulation due it’s numerous pharmacological efficiencies4. Amaranthus spinosus, thorny amaranth belongs to the same family native to America. It is used as a folk medicine and traditionally reported to possess more activities5. The presence of various phytochemicals (natural products)6,7 were reported in Amaranthus viridis and Amaranthus spinosus and their quantification may leads to new drug development.

 

MATERIAL AND METHODS:

Plant material:

The leaves of A. viridis and A. spinosus were collected in Coimbatore District, Tamil Nadu, India and authenticated by Dr. C. Murugan, Scientist ‘D’ and Head of office, T.N.A.U, Coimbatore, Tamil Nadu, India. Authentication number BSI/SRC/5/23/2018/Tech-2897. The leaves of each plant were segregated from the whole plant, dried in shade with ambient temperature, grounded and stored well.

 

Physiochemical evaluation:

The crude powdered drugs were subjected to various physiochemical investigations.

Total ash:

2g of powdered drugs of leaves of Amaranthus viridis and Amaranthus spinosus were tarred in silica crucible at 450°C. The residues were cooled in suitable desiccator. The total ash was determined8.

 

Water- soluble ash:

In crucible, boil ash with H2O for 5 minutes. Using ash less filter paper, the insoluble contents are collected, ignited in silica crucible and weighed after cooling. On subtraction of insoluble ash weight from total ash, the water soluble content is calculated8.

 

Acid-insoluble ash:

25ml of Dilute Hydrochloric acid is mixed with total ash and boiled for 5minutes. On filtering the content acid insoluble matter were collected and also neutralized by washing with hot water. Dried filter paper along with insoluble matter were incinerated in silica crucible and cooled in desiccator8.

 

Determination of Extractive value:

Based on Extractive value of medicinal plants, the quantity of active constituents can be determined. Solubility of phytochemicals is variable and so different solvents are employed from polar to non-polar9. 5g of accurately weighed powdered drug is extracted with alcohol (90%) in stopper flask for 6 hours with frequent shaking. Later for about 18 hours, the content is extracted on rotary shaker. Filtered and the filtrate is evaporated. The alcohol (90%) soluble extractive is figured8,9. The same procedure was repeated with disparate solvents like chloroform, Benzene, Ethanol, Ethyl acetate and water. The above procedures have been conducted for Amaranthus spinosus and also for Amaranthus viridis and their extractive valves were determined.

 

Preliminary Phytochemical evaluation:

To detect the existence of phytoconstituents, various solvent extracts of Amaranthus viridis and Amaranthus spinosus were employed9,10,11,12.

 

Total phenolic content (Folin-Ciocalteuassay):

Total phenol present in extract of leaves of Amaranthus viridis and Amaranthus spinosus were determined by UV spectrophotometer (Shimadzu UV-1800). In 25ml of volumetric flask, 5ml of Folin-Ciocalteu phenol reagent was mixed with 1ml of extract (1g/100ml) by shaking and 4 ml of Sodium carbonate (75g/l) solution is mixed and volume made up to 25ml. The standard solutions were prepared using gallic acid with various concentrations (20-100µg/ml). Incubate and measure absorbance at 550nm. Total phenol content was determined13.

Thin layer chromatography (TLC) of Amaranthus viridis and Amaranthus spinosus:

The ME of Amaranthus viridis and Amaranthus spinosus was subjected to Thin Layer Chromatography. In 500ml beaker, Chloroform: ethyl acetate: Acetic acid (9: 0.5: 0.5) were saturated before chromatogram development. Using capillary tube sample is placed in stationary phase (Silica gel G) and placed in beaker with mobile phase for development. After 3/4th development of chromatogram, plate will be removed, dried and detected by UV florescence chamber at 254nm and 365nm. The retention factors for active compounds eluted were calculated14.

 

HPTLC of ME of Amaranthus viridis and Amaranthus spinosus:

HPTLC were executed to endorse the existence of Phytoconstituents by comparing with the standards and also for quantification. To obtain best separation and peak, various sequences of mobile phase were pursued by trial and error method. The adequate resolution obtained in Toluene: Ethyl Acetate: Formic Acid: Methanol (3: 6: 1.6: 0.4)

 

Quercetin, Rutin and Gallic acid are standard for HPTLC analysis. 5μl and 10μl extract of A. viridis were loaded in track 1 and track 2 respectively. 5μl and 10μl of A. spinosus were in track 3 and track 4 respectively. 5μl of Quercetin, Rutin and Gallic acid were loaded in track 5,6 and 7 respectively on pre coated sheets. WIN CATS software was programmed for application of spot using Hamilton syringe attached to Camag HPTLC system. Develop plates in Twin trough glass chamber. The air-dried plates were observed using CAMAG Visualizer. The chromatograms were scanned by a CAMAG TLC Scanner after drying at 60° in a hot air oven15,16,17,18

 

RESULTS AND DISCUSSION:

Physiochemical evaluation:

The physiochemical characters are analysed and reported. As per procedure described various ash values and extractive values were analysed and reported in the Table 1.

 

Table 1: Physiochemical values for the leaves of Amaranthus viridis and Amaranthus spinosus

Contents

 

Amaranthus viridis

Amaranthus spinosus

Ash value

Total ash

26%

29.5%

H2O soluble

8.5%

3.5%

Acid insoluble

14.5%

12.2%

Extractive value (%) leaves powder

Benzene

4.7

0.8.

Methanol

17

5.2

Chloroform

12.9

4.4

Pet Ether

5.0

0.8

Ethyl Acetate

5.7

2.0

Water

16

13.6

 

Preliminary Phytochemical evaluation:

Phytochemical screening of Amaranthus viridis and Amaranthus spinosus reported in table 2.

 

Table 2: Preliminary Phytochemical Studies for leaf extract of Amaranthus viridis and Amaranthus spinosus

 

 

Petroleum ether

Chloroform

Methanol

Ethyl acetate

Water

Amaranthus viridis

Alkaloid

-

+

+

-

+

Carbohydrate

+

-

+

+

+

Glycosides

-

-

+

-

+

Sterol

+

 

 

 

 

Saponins

-

-

+

-

-

Tannins

+

+

-

+

+

Proteins

-

-

+

-

+

Flavonoids

-

-

+

+

+

Phenolic compounds

-

-

+

-

-

Amaranthus spinosus

Alkaloid

-

+

+

-

+

Carbohydrate

+

-

+

+

+

Glycosides

-

-

+

-

+

Sterol

+

 

 

 

 

Saponins

-

-

+

-

-

Tannins

+

+

-

+

+

Proteins

-

-

+

-

+

Flavonoids

-

-

+

+

+

Phenolic compounds

-

-

+

-

-

+ indicates presence of the phytochemical, – indicates absence of the phytochemical

 

Total phenolic content by UV spectrophotometer:

Based on the Folin-Ciocalteu assay, phenol content in Amaranthus viridis was found to be 57.25 mg GAE/100g and in Amaranthus spinosus was52.7 mg GAE/100g. Total phenol content in Amaranthus viridis is more when compared to Amaranthus spinosus.

 

Thin layer chromatography (TLC) of ME of Amaranthus viridis and Amaranthus spinosus:

The extracts A. viridis and A. spinosus were spotted in stationary phase the developed chromatogram of Amaranthus viridis is viewed under 365nm and 254nm noted in the Figure 1. Five spots were observed under 365nm - Rf0.1, 0.18, 0.28, 0.88 and 0.96 whereas three were observed under 254nm with Rf values 0.1, 0.18 and 0.28.

 

When the chromatogram of Amaranthus spinosus observed under 365nm, five spots were observed - Rfvalues- 0.16, 0.17, 0.337, 0.475 and 0.812. Three spots in 254nm, with Rf values 0.16, 0.337 and 0.475.

           

Figure 1: TLC of Amaranthus viridis and Amaranthus spinosus

 

HPTLC of ME of Amaranthus viridis and Amaranthus spinosus:

HPTLC fingerprint of Amaranthus viridis and Amaranthus spinosus were done using selected solvent system toluene: ethyl acetate: formic acid: methanol (3: 6: 1.6: 0.4) for methanolic extract. Then chromatogram visualized under 254nm and 3-dimensional view of chromatogram were recorded in Figure 2. The methanolic leaf extract of Amaranthus viridis in Track 1 with spot volume 5µl exerts 3 bands and Track 2 with spot volume 10µl exerts 4 bands and the methanolic leaf extracts of Amaranthus spinosusin Track 3 and Track 4 with spot volume 10µl exhibited the presence of 7 bands and 4 bands respectively in figure 3.

 

Rf value of the peaks with 0.85 and 0.76 of Track 1 and 0.85 and 0.72 of Track 2 coincides with Quercetin and Gallic acid respectively reported, Figure 4. Revalue of Rutin does not coincides with any of the bands in Track 1 and Track 2 and so found to be absent in the extract.

 

Rf value of the peaks with 0.82, 0.70 and 0.08 of Track 3 and 0.80, 0.69 and 0.09 of Track 4 coincides with Rf value of Quercetin, gallic acid and Rutin respectively in Figure 5.

 

Figure 2: 3-Dimensinal View of Chromatogram of Sample of Amaranthus viridis and Amaranthus spinosus with Standard Quercitin, Rutin and Gallic Acid

 

HPTLC finger print profile for ME extracts of leaf of Amaranthus viridis

 

HPTLC finger print profile for ME extracts of leaf of Amaranthus spinosus

 

Figure 3: ME of A. viridis and A. spinosus Track 1, 2, 3 and 4

 

Figure 4: Comparision of HPTLC Finger Print Sample from leaves of Amaranthus viridis (d) and (e) with Standard Quercitin (a), Rutin (b) and Gallic acid (c).

 

Figure 5: Comparision of HPTLC Finger Print Sample from leaves of Amaranthus spinosus (d) and (e) with Standard Quercitin (a), Rutin (b) and Gallic acid (c).

 

Based on the HPTLC chromatogram, the presence of Quercetin, Gallic acid and Rutin were qualitatively and quantitatively analyzed. Amaranthus viridis chromatogram exhibit presence of Quercetin and Gallic acid whereas chromatogram of Amaranthus spinosus exerts presence of Quercetin, Gallic acid and Rutin. Amaranthus viridis has more amounts of Quercetin and Gallic acid when compared to Amaranthus spinosus (Table 3 and 4).

 

Table 3: Quantity of Quercetin, Gallic acid and Rutin present in methanolic extract of Amaranthus viridis

S. No.

Name of the Standard

Conc. of the Standard (µg)

Volume of the Spot (µl)

Conc. of Sample (µg)

1

Gallic acid

5

5

0.1105

10

0.360

2

Quercetin

5

5

0.046

10

0.618

3

Rutin

5

5

-

10

-

 

Table 4: Quantity of Quercetin, Gallic acid and Rutin present in methanolic extract of Amaranthus spinosus

S. No.

Name of the Standard

Conc. of the Standard

(µg)

Volume of the Spot (µL)

Conc. of the Sample (µg)

1

Gallic acid

5

5

0.0923

10

0.149

2

Quercetin

5

5

0.049

10

0.264

3

Rutin

5

5

0.186

10

0.298

 

CONCLUSION:

The results of the present study provided a valuable information can be utilized for the isolation, purification and utilization of phenolic compounds. Since both the species of Amaranth us are edible and easy available, the phytochemicals can be isolated and utilized in drug development.

 

ACKNOWLEDGEMENT:

The authors thank the management of the college for encouraging and providing the research facilities. The authors also thank Dr. C. Murugan for identification of the plant and Dr. M. Sasikala for her enormous support during the completion of work.

 

CONTRIBUTION OF AUTHORS:

All authors have contributed equally to carry out the work.

 

CONFLICTS OF INTERESTS:

No conflicts of interest.

 

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Received on 22.06.2020           Modified on 18.08.2020

Accepted on 11.09.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(7):3889-3895.

DOI: 10.52711/0974-360X.2021.00675