1. INTRODUCTION:

Natural products are the components or substances that are produced from natural sources such as plants. These products which occur naturally have an impact on human life and therefore, used as Medicines, dietary supplements, and cosmetics. The components of plants that have medicinal value and healing properties are called as Bioactive compounds and they undergo a series of the process called Extraction, Isolation, and Characterization^1. Column Chromatography procedure for plant extracts is one such technique for isolating and identifying the Bioactive compound using a stationary phase such as Silica gel or Alumina. Before Isolation and extraction, identification of the bioactive compound is done based on a method called Fingerprint which defines the character and gives complete information about a specific plant.

Fingerprint analysis gives an accurate identification with similar peaks and is helpful in determining the intrinsic quality of the bioactive compound².

The Development of analytical techniques has to lead to a qualitative as well as quantitative analysis of Isolation of bioactive compounds from Plants which are herbs with a complicated system of mixtures formed through various pathways. Chromatography is one such analytical technique used for the authentication and identification of bioactive compounds in a plant and is readily available. High-Performance Liquid Chromatography (HPLC) is the most extensively used application in Pharmaceutical industries for the isolation and purification of bioactive compounds from medicinal plants. This application requires larger stainless steel Columns packed with normal Phase Silica which not only isolates but also gives information about the sample containing new synthetic products. This is a very helpful technique for the pharmaceutical industry to introduce a new product into the market within less timeframe³.

Many more feasible techniques are emerging to control the quality of the bioactive compounds by using Solid phases such as Silica Gel with less particle size and shorter Column size. Ultra High-Performance Chromatography (UHPLC) is such a technique that is gaining momentum because of its enhanced selectivity and decreased analysis time, in isolating bioactive compounds from medicinal plants⁴. Therefore, the main objective of this study was to provide step-by-step visual demonstration of fractionation and isolation of biologically active Lantana camara leaves of secondary metabolites using column-chromatographic techniques.

2. MATERIALS AND EQUIPMENTS:

2.1. Plant material collected for the study:

Lantana Camara leaves were collected from Koodapattu, Tirupattur, Tamil Nadu and India. The collected plant leaves were washed thoroughly in tap water, shade dried and finely powdered. The plant authentication (Reg. no of Certificate no: 4133, Plate - 1) Institute of Herbal Botany, Plant Anatomy Research Centre, Presidency college, Chennai.

Lanatana camara leaves

Plate – I

2.2. Methods of Extraction:

The 10g of Lantana Camara leaves powder were used for extraction method by Soxhlet extraction using methanol (100ml). Extraction was considered to be completed when the Leaves materials become exhausted of their constituents that were confirmed and filtrates obtained are dried at temperature of 40±2°C to have gummy concentrate of the crude extract. The extract is kept in a suitable container with proper labeling and then stored for further use.

2.3. Bioactive compounds from Lantana camara leaves by Column Chromatographic Techniques:

These following apparatus and chemicals used for column chromatography studies. Cylindrical chromatographic column, Separating funnel, Silica gel 60, Toluene (need for packing of column), Cotton or silica sand, Toluene (need for packing of column), Ethyl acetate, Ethanol, Iodine chamber, UV light detector (254 nm), TLC plate developing chamber, Lead pencil, Capillary tubes, Spatula, Preparative TLC plates (20cm x 20cm), Kim wipe tissue and Solvent mixer.

2.4. Preparation of Reagents:

2.4.1. Test sample:

Air dried sample powder was extracted using suitable solvent and vacuum dried before loading into column. Amount of the sample should be measured in order to define the yield of desired extract. Gradient solvent system: Gradient solvent system such as non-polar to high polar solvent provides best elution and separation of various organic compounds from any plant-based organic extract. However, volume of solvent can be dependent on the amount of the sample to be purified.

2.4.2. Method:

A cylinder shaped glass column containing stationary phase (Silica gel) is encountered slowly from with a liquid solvent Mobile phase that flows down the column with the help gravity or external pressure applied. This technique is used for the purification of compounds from a mixture. Once the column is ready, the sample is loaded inside the top of the column. The mobile solvent is then allowed to flow down through the column. The compounds in mixture have different interactions ability with stationary phase and mobile phase, thereby will flow along the mobile phase at different time intervals or degrees. In this way, the separation of compounds from the mixture is achieved. The individual compounds are collected as fractions and analyzed further for structure elucidation.

2.5. Procedure:

Isolation and purification of bioactive compounds from plant samples

· A suitable size long cylindrical glass column (based on the amount of the sample) should be stand firm on a column-chromatography stand.

· Completely dried plant extract sample should be mixed with silica gel to make a fine powdered form for easy distribution of sample in already packed silica gel column.

· Sample powdered mass should be placed on the top of the pre-packed silica column and sample should be covered with a layer of cotton.

· Then solvents of different polarities were passed through column at uniform rate under gravity to fractionate the sample extract.

· Each fraction was collected separately in a test tube and numbered consecutively for further analysis on thin layer chromatography (Figure 1).


(i) Packed Column	(ii) Movement of mobile	(iii) Collection of fractions

Figure 1: Steps of Column chromatography techniques

2.5.1. Thin Layer Chromatography (TLC):

· Thin layer chromatography (TLC) provides partial separation of both organic and inorganic materials using thin-layered chromatographic plates especially useful for checking the purity of fractions.

· Each fraction is applied on activated TLC plates with the help of capillary tube at a 1/2 inch apart from the lower edge of TLC plate, and plate is kept in a developing chamber containing suitable solvent system for specific time until the developing solvent reaches top of the upper edge of TLC plate.

· Plate is taken out from developing chamber, dried and solvent front is marked by lead pencil. Compound bands/spots visualized on TLC chromatoplate can be detected by visual detection, under UV light (254nm), in iodine chamber and by using spray reagent (vanillin-sulfuric acid) for the presence of specific compounds.

· The visualized spots of the components in the chromatoplate are marked and the Rf value of each spot is calculated by the formula: Rf = distance travelled by the sample (cm)/distance travelled by the solvent (cm).

· TLC plate showing number of bands (compounds) for each fraction can be further purified using high performance liquid chromatography (HPLC).

· Based on the nature of the compounds, further spectral analyses such as Infrared (IR), Gas Chromatography and Mass spectrometry (GC-MS) and Nuclear magnetic resonance (NMR) can be performed to elucidate the chemical structure of target compounds⁵.

3. RESULTS AND DISCUSSION:

3.1. Preliminary column chromatography studies of Lantana camara leaves:

The gradient solvent system to be used in the column-chromatography for the isolation of bioactive molecules from any test samples Table 1summarizes the ratio of gradient solvent to be used in column chromatography.

3.2. Preliminary Column Chromatography:

Column chromatography involves ion exchange, molecular sieves and adsorption phenomenon. The flushing in conventional chromatography greatly dilutes the material and the fractions usually require another step for concentration. A newer method called displacement chromatography elute with some compounds that has great affinity for the adsorbent. Fractions of elute materials can be more concentrated than the original solution applied to column. The Table 1 shows the gradient solvent system to be used in the column-chromatography for the isolation of bioactive compounds.

Table 1: Gradient Solvent System of Column Chromatography

S. No.	Solvent Polarity	Solvent Mixture/ Ratio (ml)	Fractions	Fraction Color
1	0.29	Toluene	F1, F3, F4, F5	Colorless
2	0.40	Toluene + Ethyl acetate (9:6)	F6 - F10	Pale green
3	0.50	Toluene Ethyl acetate (3.75:11.25)	F11 – F15	Colorless
4	0.58	Ethyl acetate	F16 – F20	Colorless
5	0.70	Ethyl acetate + Ethanol (9:6)	F21 – F25	Pale green
6	0.80	Ethyl acetate + Ethanol (3.6:11.4)	F26 – F30	Colorless
7	0.88	Ethanol	F31 – F35	Colorless

3.3. TLC Profile of Lantana camara leaves:

Thin layer chromatography (TLC) The first practical application of thin layer chromatography was given by Stahl 1965. Compared to paper chromatography, the special advantage of TLC is the versatility, speedy and sensitive. TLC is an adsorption chromatography where samples are separated based on the interaction between a thin layer of adsorbent attached on the plate. The technique mostly employed for the separation of low molecular weight compounds (Table 2 and Figure 2).

Table 2: Thin Layer Chromatography Profile of Lantana camara leaves

S. No.

Solvent system

Column purified fractions

R_f value

P-anisaldehyde Stains

Expected compound

Toluene +

Ethyl acetate (9:6)

F6 – F10

0.71

Aldehydes, Ketones and Alcohols

Ethyl acetate +

Ethanol (9:6)

F21 – F30

0.70

Figure 2: Demonstration of isolation pattern of pure compounds on TLC plate

3.4. The Phytochemical screening of column fractions of Lantana camara leaves:

The Table 3 shows the secondary metabolites of column fractions of Lantana camara leaves. The column fractions of Lantana camara leaves sample was subjected to various qualitative phytochemical tests namely alkaloids, flavonoids, phenols, tannins, saponins, carbohydrates, amino acids, steroids, terpenoids and protein as per standard procedure were respectively.

Table 3: Phytochemical Analysis of Column Fractions of Lantana camara leaves

S. No.	Phytochemical Constituents	*Lantana camara* leaves
1.	Alkaloids	+
2.	Flavonoids	+
3.	Terpenoids	+
4.	Phenol	+
5.	Saponins	+
6.	Tannins	+
7.	Carbohydrates	+
8.	Amino acids	+
9.	Protein	+
10.	Steroids	+

Indicates + - Present: - = Absence

It was obvious that the fractions from F6 –F10 (0.40) and F21 – F30 (0.70) showed functional groups such as aldehydes, ketones and alcohols, that functional groups may be bioactive compounds like alkaloids, terpenoids and flavonoids. There is a vital must to search for unconventional therapies as naturally happening biologically active phytochemicals. Assured phytoconsutitents act in many methods on different types of disease and may be possibly contribute in the field of pharmacology as natural supplements to control various infectious diseases in future. Numerous synthetic drugs cause unadorned side effects that were not acceptable excluding as treatments of last resort for terminal diseases such as cancer⁶.

Considering the harmful effects of synthetic antibiotics, isolation, purification and characterization of novel types of plant secondary metabolites could be a safer alternative to synthetic compounds. Subsequently, bioactive compounds stirring in plant materials consist of multi-component combinations, their extraction, isolation, identification and determination still makes problems. Practically most of them have to be purified by the combination of several chromatographic techniques and many other purification methods to isolate bioactive compounds were respectively⁷.

4. CONCLUSION:

It can be concluded that the various chromatographic techniques have been used for successful fractionation and purification of biologically active compounds from variety of sample. Column-chromatography is one of the most popular and widely used separation techniques to characterize both organic and inorganic materials suggesting is potential usefulness in chemical analysis of complex extract material. This research visualized successful application of column-chromatographic techniques for the isolation of biologically active secondary metabolites from plant extract. However, ongoing investigation of toxic or irritant properties is imperative, especially when considering any new biologically active compound for human use, by them medicinal or otherwise. No conclusion can be drawn concerning these Lantana camara leaves extract, unless the isolates are exposed to Fourier Transform Infra-Red (FT- IR), Nuclear magnetic resonance (NMR)and Gas chromatography–mass spectrometry (GC MS) study for establishing the structure prediction.

5. ACKNOWLEDGEMENT:

This work was supported by Sacred Heart College, Tirupattur-635601, Tirupattur District, Tamilnadu, India, through Don Bosco Research Grant. We would like to show our gratitude to the Management of Sacred Heart College, Tirupattur-635601, Tirupattur District Tamilnadu, India. We are also thank Dr. E. S. Karthy, Director, Awe Care Analytical and Research Laboratories, Erode for supporting their research by granting Don Bosco Research Grant.

6. CONFLICT OF INTEREST:

The authors report that they have no conflicts of interest.

7. REFERENCES:

1. Abdul, K.K., Palwasha. A., Ayeesha. M., Safdar Ali. K. and Rasool. B.T. (2019) Response of plant parts and age on the distribution of secondary metabolites on plants found in Quetta. Pakistan Journal of Botany. 41(5): 2129-2135.

2. Mahesh Chand Meena and Vidya Patni. (2018) Isolation and identification of flavonoid “Quercetin” from Citrulluscolocynthis (Linn.) Schrad. Asian Journal Experimental Science. 22(1): 137-142.

3. El-Shemy HA, Aboul-Enein AM, Aboul-Enein KM, Fujita K. (2017) Willow leaves’ extracts contain antitumor agents effective against three cell types. PLoS One 2: e178.

4. Bajpai, V.K. and Kang, S.C. (2018) Isolation and characterization of biologically active secondary metabolites from Metasequoiaglyptostroboides Miki ex Hu. J Food Safety. 31: 276-83.

5. Lin, B.F. and Chao, W.W. (2010) Isolation and identification of bioactive compounds in Andrographis paniculata (Chuanxinlian), Chinese Medicine, 5, 2010, 17.

6. Rajkumar, V., Gunjan, G., R. and Ashok, K. (2012) Isolation and bioactivity evaluation of two metabolites from the methanolic extract of Oroxylum indicum stem bark, Asian Pacific Journal of Tropical Biomedicine, 2 (1), 2012, 7-11.

7. Choudhury Pradeep Kumar and Jadhav Sachin (2013) Extraction and Isolation of Bioactive Compounds from Ficus racemosa Bark and Cissampelos pareira Root by Chromatographic Technique. International Journal of Pharmaceutical Sciences Review and Research, 20(2), 101-106.

Received on 29.03.2020 Modified on 27.05.2020

Research J. Pharm. and Tech 2021; 14(3):1607-1611.

DOI: 10.5958/0974-360X.2021.00285.7