Phytochemical screening and column chromatography studies of

Aervalanata

 

A.Anita, A. Malar Retna*, J. Joseph

Department of Chemistry and Research Centre, Scott Christian College (Autonomous), Nagercoil - 629 003

*CorrespondingAuthorE-mail:iammalar80@gmail.com

Medicinal plants have bioactive compounds which are used for curing various human diseases. These phytochemicals are chemicals which are present naturally in plants and occur as combination of various types of bioactive compounds with different polarities. The present study evaluates the phytochemical screening of various aerial and root extracts of Aervalanata by standard methods. The results showed the presence of phytochemicals such as alkaloids, saponins, phytosterols, triterpenes, proteins, glycosides, coumarins, flavonoids, phenols, carbohydrates, tannins and steroids. Since the plant extracts usually occurs as combination of various phytochemicals   their separation still remains as a challenge in the process of identification and characterization. In present study the isolation of phytochemicals from methanol extracts of Aervalanata were carried by column chromatography. The methanol aerial extract of   Aervalanata  yield 2-bromo ethyl 2-( chloro methyl) but 3- enoate, in 85% chloroform : 15% methanol. Similarly the methanol root extract of  Aervalanata yield 6- furan-2-yl-2-oxo-1,2-dihydro-pyridine-3- carboxylic acid 2- bromo- ethyl ester in 85% chloroform: 15% methanol which were confirmed by spectroscopic studies. Thus the isolated compounds may be useful for the preparation of plant based medicine which may be helpful for further pharmaceutical industries for preparing new medicines which is used to treat diseases without any side effects.

 

 

 

1. INTRODUCTION:

Medicinal plants constitute the main source of pharmaceuticals and health care products¹. Medicinal plants have bioactive compounds which are used for curing various human diseases and they also play an important role in healing². Phytochemicals are chemicals that are present naturally in plants and cure diseases without causing any harm to human beings. Hence, they are called ‘man- friendly medicine’³. Terpenoids exhibit various important pharmacological activities like anti-inflammatory, antiviral, anti-bacterial activities⁴. Terpenoids are very important in attracting useful mites and consume the herbivorous insects⁵.

 

Plants having alkaloids are used in medicines for reducing headache and fever. The plant extracts usually occur as a combination of various types of bioactive compounds or phytochemicals with different polarities, their separation still remains as a big challenge in the process of identification and characterization of them. For the isolation of these bioactive compounds a variety of separation techniques such as thin layer chromatography (TLC), column chromatography, gas chromatography, should be used to obtain pure compounds⁶.

 

In the present investigation , the various aerial  and root extracts of Aervalanata were subjected to the preliminary phytochemical screening studies and finally the methanol aerial and root extracts of Aervalanata were subjected to chromatographic studies.

 

2.MATERIALS AND METHODS:

2.1 Collection and Identification of plants:

The medicinal plant Aervalanata belonging to the family Amaranthaceaehas been collected in and around Nagercoil area, Kanyakumari district. The collected plants were identified using standard keys and voucher specimen was submitted at the herbarium of Scott Christian College (Autonomous), Nagercoil. The voucher number was SCCN 3267.

 

2.2 Preliminary Phytochemical screening tests:

The shade dried parts ofaerial and root parts of  Aervalanatawere extracted in a soxhlet apparatus for 24 hours using different solvents in the order of polarity such as hexane, chloroform, ethyl acetate, acetone, methanol and water. The extracted solvents were distilled by vaccum distillation under reduced pressure in order to separate the solvents from the extracts. The crude extracts obtained from soxhlet extraction were subjected to preliminary phytochemical screening.

 

Phytochemical screening involves testing the different extracts of Aervalanata for various phytochemicals by qualitative analysis, which gives a general idea regarding the nature of constituents present in the crude extracts⁷. The qualitative phytochemical test carried for various extracts were given below.

 

a)Determination of Alkaloids:

About 3 ml of concentrated extract was taken in a test tube and 1 ml   of HCl was added to the mixture which is heated gently for 20 minutes and is then cooled and filtered. The filtrate was used for the following tests.

 

i. Wagner’s test:

The filtrate was treated with Wagner’s reagent (solution of iodine in potassium iodide). Formation of brown reddish precipitate indicates the presence of alkaloid⁸.

 

ii. Mayer’s Test:

One ml of the filtrate is treated with 4-5 drops of Mayer’s reagent (Potassium mercuric iodide).  Formation of a yellow or white coloured precipitate or turbidity indicates the presence of alkaloids⁹.

 

b)Test for Saponins:

i) Froth test:

Exactly 0.5 g of the extract was dissolved in 2ml distilled water in a test tube. Frothing which persisted on warming was taken as preliminary evidence for saponins⁹.

 

ii) Foam test:

5 ml of the extracts were mixed with 20 ml of distilled water and then agitated in a graduated cylinder for about 15 minutes. Formation of foam indicates the presence of saponins¹⁰.

c) Test for Phytosterols:

Libermann Burchard’s test:

Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of acetic anhydride, boiled and cooled. Concentrated sulphuric acid was added. The formation of brown ring at the junction indicates the presence of phytosterols¹¹.

 

d) Test for Proteins:

Xanthoprotein Test:

The extracts were treated with few drops of concentrated nitric acid. Formation of yellow colour indicates the presence of proteins¹¹.

 

e) Test for Triterpenes:

Salkowski test:

Extracts were treated with chloroform solution and is then shaken with one or two drops of concentrated sulphuric acid and then it is allowed to stand. Appearance of golden yellow colour indicates the presence of triterpenes¹².

 

f) Test for Diterpenes:

Copper acetate test:

The extract was dissolved in water and treated with 10 drops of copper acetate solution. Formation of emerald green colour indicates the presence of diterpenes¹³.

 

g) Test for Glycosides:

i) Glycoside test:

0.5 mg of the extract was dissolved in 1 ml of water and then the aqueous NaOH solution was added to the extract. Formation of yellow colour indicates the presence of glycosides¹⁴.

 

ii) Concentrated H₂SO₄Test:

To 5ml extract, add 2ml of glacial acetic acid, one drop 5% FeCl₃and concentrated H₂SO₄. Appearance of brown ring indicates the presence of glycosides¹⁵.

 

h) Test for Tannins:

Lead acetate test:

To the extracts add few drops of 10% lead acetate solution were added. Formation of precipitate indicates the presence of tannins¹⁶.

 

i) Test for steroids:

Salkowski’s test:

5 ml of the extract was dissolved in 2 ml of chloroform and equal volume of concentrated sulphuric acid was added along the sides of the test tube. The upper layer turns red and lower layer turns yellow with green fluorescence, indicating the presence of steroids¹⁷.

 

j) Test for coumarins:

10% NaOH (1ml) was added to 1 ml of the plant extracts. Formation of yellow colour indicates the presence of coumarins¹⁸.

k) Test for Flavonoids:

i) Pew’s tests:

To two ml of the extract zinc powder was added  in a test tube, followed by dropwise  addition of concentrated HCl. Formation of purple red or cherry colour indicates the presence of flavonoids¹⁹.

 

ii) NaOH Tests:

To two ml of the extract sodium hydroxide were added in a test tube. Formation of intense yellow colour that becomes colourless on addition of few drops of dilute HCl indicates the presence of flavonoids¹⁹.

 

l)  Test for Phenols:

Ferric chloride test:

Extracts were treated with four drops of alcoholic Fecl₃  solution.  Formation of bluish black colour indicates the presence of phenols²⁰

 

m) Test for carbohydrates:

Molisch's test:

To two or three ml of the aqueous extract two drops of alpha napthol solution in alcohol is added and shaken well. Then add concentrated sulphuric acid from the sides of the test tube. Violet ring formation   indicates the presence of carbohydrates²¹.

 

2.3 Column Chromatography:

The active constituents present in methanol extract of aerial and root parts ofAervalanatawas separated by column chromatography. The column was packed with a solution of silica gel (60-120 micron) with chloroform by wet slurry method²¹. This involves preparing a solution of silica gel, with chloroform in a beaker and subsequently adding to the column till three- fourths of the column is filled. This method was used to prevent the trapping of air bubbles. Here silica gel acts as stationary phase. The solvents chloroform and methanol act as mobile phase. The high pressure is given by using nitrogen gas through the tube which is connected at the top side of the column. The packed material gets separated inside the column by using the stationary phase and the mobile phase.

 

2.3.1 Characterization of purified compounds:

FTIR: 

The Fourier transform infrared spectrum was recorded in the range of   400- 4000 cm^-1 using the instrument of model Thermo Nicolet, Avatar 370. 

 

NMR:

¹H- NMR of purified compounds was recorded at 1-12 ppm using the instrument model Bruker  Avance III, 400MHz.

 

 

LC- MS:

LC-MS spectrum was recorded using the single quadeanalyser and using ESI as ion source.

 

3. RESULTS AND DISCUSSION:

3.1 Preliminary phytochemical screening Analysis:

i)Phytochemical screening of aerial extracts of Aervalanata:

Qualitative phytochemical screenings was done in various aerial extracts of Aervalanata and are presented in the Table.1.1. The phytochemical screening of aerial extracts of Aervalanata indicates the presence of alkaloids, saponins, phytosterols, triterpenes, proteins, glycosides, coumarins and flavonoids. Saponin is found only in chloroform extract. The phytochemicals coumarin, protein, glycosides were found almost in all extracts. The maximum numbers of phytochemical constituents are found in chloroform, ethyl acetate and water extracts. The minimum number of phytochemical constituents  are found in acetone extract.

 

Table 1.1 Phytochemical screening of aerial extracts of Aervalanata

Chemical category	Name of  the  test	H	C	E	A	M	W
Alkaloids	Mayer’s test	+	-	-	-	-	+
	Wagner’s test	+	-	-	-	-	+
Saponins	Foam test	-	+	-	-	-	-
	Froth test	-	+	-	-	-	-
Phytosterols	Liberman’s Burchard’s test	-	+	+	-	-	-
Triterpenes	Salkowski test	-	-	+	+	-	+
Proteins	Xanthoprotein test	+	-	+	-	+	+
Glycosides	Glycoside test	-	+	-	+	+	+
	Concentrated sulphuric acid test	-	+	-	+	+	+
Coumarins	10% NaOH+1ml plant extract	+	+	+	-	-	+
Flavonoids	Pew’s test	-	+	+	-	+	-
	NaOH test	-	+	+	-	+	-

H - Hexane extract,         E -   Ethyl acetate extract,  

M -   Methanol extract, C - Chloroform extract,  

A -   Acetone extract, W -   Water extract, + present,  -  absent.

 

ii) Phytochemical screening of root extracts of Aervalanata:

Qualitative phytochemical screening was done in various root extracts ofAervalanataandare presented in theTable.1.2. The phytochemical screening of root extracts of Aervalanataindicates the presence of alkaloids, saponins, phenols, coumarins, steroids, proteins, triterpenes, glycosides, flavonoids, carbohydrates, tannins and phytosterols. The  phytochemical coumarin is observed only in the acetone extract. The carbohydrates and tannins are found in methanol and water extracts. Glycosides are present in all the extracts taken for the study. The  phytochemical phenols were found in methanol and water extracts. The phytochemical proteins were found in all extracts except hexane. The maximum amount of phytochemical constituents is found in methanol extract and the minimum number of phytochemical constituents is found in hexane and ethyl acetate extracts.

 

3.2 Isolation of bioactive compound from Aervalanataby column chromatography:

3.2.1 Isolation of bioactive compound from methanol aerial extract of Aervalanata:

5 g of methanol aerial extract of Aervalanata   was loaded in a column packed with a solution of silica gel (60-120 micron) with chloroform by the wet slurry method. The two fractions have been eluted in the composition of 2% methanol and 98 % chloroform.  Finally the product is eluted from the second fraction in composition 15% methanol and 85% chloroform. The isolated compound is subjected to FT-IR, ¹H-  NMR and  LC-MS spectroscopic studies to elucidate its structure.

 

i) FT- IR spectroscopic analysis of pure methanol aerial extract of Aervalanata:

The FT-IR analysis of pure methanol aerial extract of ofAervalanata was shown in Fig. 1.1 and the data is presented in Table.1.1. Based on the vibrational frequencies the following information was obtained. The -C-O stretching vibration is observed at 1129.92 cm^-1. A strong - CH stretching was observed at 2945.10  cm^-1_. The resonance peak at  680 cm^-1   corresponds to –C-Br^. The resonance peak at 610  cm^-1 corresponds to –C-Cl stretch.   Thus, from above datas the presence of  -CO, -C-Br stretch, - CH stretching , -C-Cl stretch  were confirmed.

 

 

 

Table 1.2 Phytochemical screening of root extracts of Aervalanata

H - Hexane extract,   C - Chloroform extract,   E - Ethyl acetate extract,

A - Acetone extract,   M -  Methanol extract,    W - Water extract,

+    present,                 -      absent.

 

 

wave numbers   cm^-1

Fig. 1.1 FT-IR analysis of pure methanol aerial extract of Aervalanata

 

Fig. 1.2 Proton NMR spectroscopic analysis of pure methanol aerial extract of Aervalanata

 

 

Table. 1.1 IR spectral data of pure methanol aerial extract of Aervalanata

Vibrational frequencies	Functional groups present
680 cm-1	-C-Br stretch
610 cm-1	-C- Cl stretch
2945.10 cm-1	strong - CH aromatic stretching
1129.92cm-1	_ CO stretch

 

ii) Proton NMR spectroscopic analysis of pure methanol aerial extract of Aervalanata:

The Proton NMR spectroscopic analysis of pure methanol aerial extract of Aervalanata was shown in the Fig. 1.2. The resonance peak is found in the region at 2ppm, 2.5 ppm,  3.1 ppm, 3.4 ppm corresponds to - CH₂ protons. The above data shows the presence of methylene protons.

 

iii) LC- MS spectrum of pure methanol aerial extract of Aervalanata:

The LC- MS spectrum of pure methanol aerial extract of Aervalanata was shown in the Fig.1.3. The exact mass of the compound I is 234. The mass spectrum shows m/z values at 235 including the isotopes.

 

Fig.1.3 LC-MS spectrum of pure methanol aerial extract of Aervalanata

 

The peak at 116 corresponds to

 

Hence the probable structure of the isolated compound II is shown in Fig.1.4. The molecular formula of isolated compound I is C₇H₁₀O₂ClBr_. The IUPAC name of the above compound is 2‐bromoethyl 2‐ (chloro methyl) but‐3‐enoate.

 

2‐bromoethyl 2‐ (chloromethyl) but‐3‐enoate

 

Fig.1.4 Molecular structure of purified 2‐bromoethyl 2‐(chloromethyl) but‐3‐enoate

3.2.2 Isolation of bioactive compound from methanol root extract of Aervalanata

5 g of methanol root extract of Aervalanata was loaded in a column packed with a solution of silica gel (60-120 micron) with chloroform using wet slurry method. Two fractions have been eluted in the composition of 2% methanol and 98% chloroform. Finally the product is eluted from the second fraction in the composition 15% methanol and 85 % chloroform. The isolated compound is subjected to FT-IR, ¹H- NMR and LC- MS spectroscopic studies to elucidate its structure.

 

i) FT- IR spectroscopic analysis of pure methanol root extract of Aervalanata

The FT-IR analysis of pure methanol root extract of Aervalanata was shown in Fig. 1.5 and the data is presented in Table. 1.2. Based on the vibrational frequencies the following information was obtained. The vibrational frequency at 3422 cm^-1, 3382 cm^-1, 3363 cm^-1, 2854 cm^-1 shows the presence of -NH group. The aromatic -C-H stretch was observed at 2925cm^-1. A weak -C–H stretch was observed in the vibrational frequency at 1733 cm^-1. The -C=C stretching vibration is found at 808 cm¹and 709 cm^-1. The vibrational frequency at 615 cm^-1 and 464 cm^-1 shows the presence of-C-Br. The -C-O stretching vibration was found at 1116 cm^-1  and 1053 cm^-1and the -C=O stretching vibration was observed at 1633 cm^-1and 1195 cm^-1. 

 

Fig.1.5 FT-IR analysis of pure methanol root extract of Aervalanata

 

Fig.1.6 Proton NMR spectroscopic analysis of pure methanol root extract of Aervalanata

 

Fig.1.7 LC- MS spectrum of pure methanol root extract of Aervalanata

 

 

Table. 1.2I.R spectral data of pure methanol root extract of Aervalanata

Vibrational frequencies	Functional groups present
3442 cm-1,  3382 cm-1,  3363 cm-1, 2854 cm-1	-NH
1116 cm-1, 1053 cm-1, 1384 cm-1	-C-O stretching
1195 cm-1,1633 cm-1	-C=O stretching
808 cm-1,  709 cm-1	-C=C
615 cm-1,  464 cm-1	-C-Br
2925 cm-1	aromatic C-H stretching
1733 cm-1	weak CH stretching

ii) Proton NMR spectroscopic analysis of pure methanol root extract of  Aervalanata:

The Proton NMR spectroscopic analysis of pure methanol root extract of Aervalanata was shown in the Fig. 1.6. The resonance peak value found in the region at  8.3 ppm corresponds to the –NH protons. The resonance peak value found in the region at 2.5 ppm, 3.4 ppm  corresponds to methylene protons. The resonance peak found  in the region around   0.8 - 1.3 ppm   which shows the presence of alkane - C- H bond. Thus  from above datas the presence of -NH- proton, alkane -C-H bond and methylene protons were confirmed .

 

iii) LC- MS spectrum of pure methanol root extract of Aervalanata

The LC- MS spectrum of pure methanolroot extract of Aervalanata was shown in the Fig. 1.7. The exact mass of the compound II is 314. The mass spectrum shows m/z values at 316.8 including the isotopes.

 

The structure of isolated compound III is shown in the Fig.1.8. The molecular formula of the above compound is C₁₂H₁₀NBrO_4. The IUPAC name of the above compound is 6-furan-2-yl-2-oxo-1, 2-dihydro-pyridine-3-carboxylic acid 2-bromo-ethyl ester.

 

Fig.1.8 Molecular structure of purified 6-furan-2-yl-2-oxo-1, 2- dihydro-pyridine- 3-carboxylic acid 2-bromo-ethyl ester

 

4. CONCLUSION:

Phytochemical analysis showed the presence of active phytoconstituents present in the extracts of each plant taken for the study. Phytochemical analysis of the plant is very important commercially and has greatly involved in pharmaceutical companies for the production of new drugs for curing various diseases. The preliminary phytochemical screening reveals the useful findings about the chemical constituents present in the plants. The chromatographic technique is most valuable in the identification of phytochemicals from Aervalanata. The active components separated were confirmed by by FT- IR, ¹H- NMR and LC- MS spectroscopic studies. From the above spectroscopic studies the structure of isolated compounds was determined. Thus the isolated compounds may be useful for the preparation of plant based medicine for pharmaceutical industries for preparing new medicines which is used to treat diseases without any side effects.

 

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Received on 08.11.2017         Modified on 13.12.2017

Accepted on 24.12.2017         © AJRC All right reserved