ISSN: 2320-0189
K Karthika1*, R Shyam Praveen1, S Vimal Priya2
1Department of Territory and Agro Forestry System (TESAF), University of Padova, Padova, Italy
2Department of Botany, University of Kongunadu Arts and Science College (Autonomous), Coimbatore, Tamil Nadu, India
Received: 04-Apr-2022, Manuscript No. JBS-22-57013; Editor assigned: 06-Apr-2022, Pre QC No. JBS-22-57013 (PQ); Reviewed: 20-Apr-2022, QC No. JBS-22-57013; Revised: 02-Jun-2022, Manuscript No. JBS-22-57013 (R); Published: 14-Jun-2022, DOI: 10.4172/2320-0189.11.6.008.
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Tylophora subramanii, a climbing herb belongs to Family Asclepiadaceae. This is endemic Species habitat along the southern Western Ghats in India. It is used as traditional and folklore medicine for treating various disease like fever, cold, cough, diarrhoea, ulcer, external tumour, cut wounds and headache. The present study, primarily aims to carry out a preliminary phytochemical screening so as to detect the major class of compounds present in T.subramanii leaf and stem to perform Thin Layer Chromatography (TLC) profiling of all sequential extracts. Phytochemical analysis of the different solvent extracted samples suggested the presence or absence of various metabolites including, alkaloid, saponin, flavonoid, carbohydrate in varying concentration. TLC profiling of the T.subramanii was constituted different coloured phytochemical compound with different RF values. The present study provides evidence that solvent extracts if Tylophora subramanii contains medicinally important bioactive compounds and this justifies the use of plant species as traditional medicine for treatment of various diseases.
Tylophora subramanii; Phytochemicals; TLC; Rf value; Alkaloid
Genus Tylophora R.Br. is a pantropical genus distributed in tropical and subtropical Asia, Africa, and India to Australia about 60 species [1]. In India it is reported to have 21 species, of which 14 species occur in Tamil Nadu. Tylophora subramanii is a native plant of southern India commonly found in evergreen forest areas of Theni, Tirunelveli and Kanyakumari districts of Tamil Nādu up to 1200 m elevation [2]. It is a slender branched climber with smooth pubescent bark. Leaves, watery latex and root part of the plant has been used for treating various local health care systems. The genus Tylophora has been used for treating various diseases like asthma, leukorrhea, dysentery, fever and headache. Root of this genus is acrid and is said to be emetic [3]. The plant is used to cure nervous disorders among Kani tribe community of Agastiyamalai hills in Tamil Nadu. The plant is having watery latex in all over the body to have a number of secondary metabolites and high hydrocarbon content. The fruits and leaves of the plant have possessed the antioxidant capacity due to the presence of secondary metabolites. Tylophora subramanii plant stem and leaf was selected for this study is based on its traditional medicinal use [4]. The purpose of the present study is to investigate the phytochemical analysis and TLC profile of different extracts of T.subramanii stem and leaf. The obtained result provides a support for the use of this plant in traditional medicine and its further investigation and isolation of phytoconstituents.
Collection of plant material
Plant was selected for this study is based on its traditional medicinal use. Fresh stem and leaf were collected from Megamalai Wildlife Sanctuary of Theni district, Tamil Nadu (Figure 1). In India, in the month of September 2021. The plant voucher specimen’s identification was done with the help of Dr. Ravichandran. Senior preservation Assistant, Botanical Survey of India, Southern Regional Centre, Coimbatore. The Identified plant was arranged alphabetically with their botanical name with author citation, family and habit referring to Flowering plants of Kerala Flora of Presidency of Madras and Flora of Presidency of Tamil Nadu [5].The voucher specimen is deposited in the herbarium of the Department of Botany, Kongunadu Arts and Science College, Coimbatore, Tamil Nadu.
Preparation of plant extract
After collection of the plant material the stem and leaf sample were dried at room temperature until they were free from moisture. The plant material was subjected size reduction to get coarse powder, 200 gm of shade dried plant material were grinded to powder then stored in clean ait tight container [6]. It was separately subjected to Soxhlet extraction with the following solvent Hexane, Chloroform, Acetone, Ethanol, and Water in order of their increasing polarity. The solvents were then evaporated under reduced pressure and died using a rotary evaporator at 55°C. Dried extracts were stored in labelled sterile flasks at 5°C in the refrigerator, until when required for use.
Phytochemical analysis
Plants are the resource of primary and secondary metabolites namely alkaloids, terpinoids, flavanoids, saponins, coumarins, glycosides, phenolics, carboxylic acids, amino acids, sugars, proteins etc. these phytochemicals have significant biological functions and also which contribute specific characteristic and property of the plant [7]. Here Preliminary qualitative phytochemical screening was carried out with the following methods.
Test for alkaloids
Test for flavonoids
Test for glycosides
Test for saponins
Test for steroids
Test for tannins
Test for terpenoids
Test for Triterpenoids
Test for Phenols
Test for Coumarins
Test for fixed oils and fats
Test for gums and mucilage’s
Test for volatile oil
Test for protein and amino acid
Test for carbohydrates
Thin Layer Chromatography (TLC Profile)
TLC can be used to identify compound present in a given substance and is less time consuming, low cost and can be performed with less complicated technique [15]. It has a wide application and significantly used in pharmaceutical purposes [16]. The mobile phase should be prepared freshly at the time of experiment (Figure 2 and 3).
The purity of the solvents and quality of the solvent’s mixture should be strictly followed. Thin layer chromatography was carried out with the various extract of stem and leaf of Tylophora subramanii and maximum spots been separated on precoated silica gel G TLC plate with trial-and-error methods.
For TLC analysis plate with Silica gel 60 F254 TLC (Merck, Germany), 15X9 cm was cut with ordinary household scissors. Plate markings were made with soft pencil. Glass capillaries were used to spot the sample for TLC applied sample volume 1-μl of sample by using capillary at distance of 1 cm at 5 tracks, by the different solvent system Hexane: Acetic acid (9:1) solvent system-1 and In the solvent system-2, Ethyl acetate: Methanol: Glacial acetic acid: water (9:2:1:2) used.
After pre saturation with mobile phase for 20 min for development were used. Freshly prepared iodine spray reagents were used to detect the bands on the TLC plates. The movement of the analyte was expressed by its Retention Factor (Rf), values were calculated for different samples.
RF = Distance travelled by the compound / Distance travelled by solvent front
Percentage of yield extract
The yield of sequential extracts (%) is shown in Table 1.
Table 1. Extractive value of different extracts of Tylophora subramanii stem and leaf.
Name of the solvent used | Stem | Leaf | ||
---|---|---|---|---|
Colour of extract | Percentage yield (%w/w) | Colour of extract | Percentage yield (%w/w) | |
Hexane | Light green | 4.04% | Green | 9.68% |
Chloroform | Green | 2.44% | Dark green | 3.77% |
Acetone | Dark green | 1.96% | Dark green | 3.095% |
Ethanol | Dark yellowish Brown | 7.36% | Yellowish brown | 14.74% |
Water | Brown | 3.57% | Light brown | 5.01% |
Phytochemical analysis
Phytochemical screening of the sequential extract of Tylophora subramanii revealed the presence of various bioactive components of which phenolics, saponins, alkaloids, tannin, Glycosides, Proteins, Carbohydrates, and Amino acids are the most prominent components and the result of phytochemical test given in the Table 2. Among these phytochemical tests, Alkaloids were present in all solvent extracts. Whereas most of the active compounds are alkaloid, flavonoid, Glycosides, Tannin, Phenols are present in the ethanolic extract of plant material [17].
Table 2. Quanlitative phytochemical analysis of the different extracts of Tylophora subramanii stem and leaf.
S. No | Phytochemicals | Stem extract | Leaf extract | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
TEST | Hex | Chl | Ace | Eth | Wat | Hex | Chl | Ace | Eth | Wat | ||
1. | Alkaloid | + | ++ | ++ | +++ | + | - | + | + | +++ | + | |
2. | Flavanoid | - | + | + | +++ | + | - | - | - | ++ | + | |
3. | Steroid | - | - | - | + | + | - | - | - | - | - | |
4. | Terpenoid | ++ | - | + | - | - | + | - | - | + | - | |
5. | Triterpenoid | + | - | - | - | - | + | - | - | - | - | |
6. | Tannin | - | ++ | ++ | +++ | ++ | - | + | + | + | - | |
7. | Phenol | - | + | + | ++ | + | - | + | + | +++ | ++ | |
8. | Coumarin | - | - | - | - | - | - | + | - | - | - | |
9. | Glycosides | + | + | - | +++ | + | + | + | + | +++ | + | |
10. | Saponin | - | - | - | - | + | - | - | - | ++ | + | |
11. | Gums and mucilage | +++ | + | - | - | - | +++ | + | - | - | - | |
12. | Volatile oil | + | + | - | - | - | - | - | + | ++ | + | |
13. | Fixed oil | + | - | - | - | - | + | + | - | - | - | |
14. | Carbohydrate | + | + | + | ++ | ++ | - | - | + | ++ | + | |
15. | Protein | - | - | + | ++ | + | - | - | - | + | + | |
16. | Amino acid | - | + | + | ++ | ++ | - | - | + | ++ | + |
(+++ abundant; ++ moderately present: +weakly present; -----absent, Hex-Hexane, Chl-Chloroform, Ace-Acetone, Eth-Ethenol, Wat-Water).
Thin Layer Chromatography (TLC profile)
Thin layer chromatography was performed with various crude extracts such as such as hexane, chloroform, acetone, ethanol and water of Tylophora subramani stem and leaf. For each extract, two different solvent systems were used to analysed the presence of secondary metabolites. The retention factor (Rf) for each of the extracts of Tylophora subramani in different solvent system were observed (Table 3 and Figures 2 and 3). This result will help us to select solvent system for further isolation of bioactive compounds from this plant [18].
Table 3. TLC profile of various extract of stem and leaf.
TLC profile of different solvent extract | Solvent system 1 Hexane: Acetic acid (9:1) | Solvent system 2 Ethyl acetate: Methanol: Acetic acid: water (9:2:1:2) | ||
---|---|---|---|---|
Rf value of stem | Rf value of leaf | Rf value of stem | Rf value of leaf | |
Hexane | 0.13,0.37 | 0.39,0.73 | 0.22, 0.44 | 0.81 |
Chloroform | 0.26,0.46 | 0.62 | 0.52, 0.60 | 0.74,0.81 |
Acetone | 0.69 | 0.39,0.87 | 0.61 | 0.55, 0.72,0.84 |
Ethanol | 0.62 | 0.74 | 0.52 | 0.56, 0.72,0.81, |
Water | 0.52 | 0.51,0.80 | 0.71 | 0.75,0.92 |
TLC profile of stem and leaf solvent system 1 (a-stem under visible light, b-stem under UV visible light, c-leaf under visible light, d-leaf under UV visible light.
TLC profile of stem and leaf solvent system-2 (e-stem under visible light, f-stem under UV visible light, g-leaf under visible light, h-leaf under UV visible light).
Researchers have spent tremendous amount of time and resource to find the importance of medicinal plants. Each and every plants contain special compounds which helpful in various pharmacological purposes. The investigation of our present study is a preliminary screening of Tylophora subramanii as a rich source of secondary metabolites. These findings suggested that Tylophora subramanii could be a potential source of natural antioxidant having great importance as therapeutic agent and preventing oxidative stress related degenerative diseases. The stem and leaf portion of the T.subramanii can provide lead molecules which could be useful substrate for the synthesis of new broad spectrum antibiotics for the treatment of infections caused by the organisms. Further purification, identification and characterization of the active compounds would be our priority in future studies.
Our sincere thanks to Kongunadu Arts and Science College, Coimbatore for supporting our project. We thank to Department of Botany and Biotechnology for providing Lab facilities and necessary support.
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