INTRODUCTION:

Ayurveda is derived from word ‘Ayur’ (Life) and ‘Veda’ (Science), hence means the science of life. In India the sacred Vedas, dating back 3500B.C – 800B.C give much reference of medicinal plants. Today, people around the globe are giving preference to alternative medicines such as Ayurveda, Naturopathy, Homeopathy and herbal medicine. Herbal medicine is cost effective and less expensive than allopathic medicines.

According to WHO, herbal medicines serve the health needs of about 80% of the world’s population. One of the important and second largest plant genus was Ipomoea which had great medicinal values.

Figure 01: Ipomoea obscura (L.) Ker Gawl.

Ipomoea obscura commonly known as “Lakshmana” in Ayurveda, belongs to the morning glory family Convolvulaceae. It is slender, twinning perennial herb are usually found in tropical Asia, tropical east Africa, subtropical regions. In India it is found in grasslands, hedges and waste lands at an altitude of 3000ft. It contains anti-inflammatory and anti-cancer properties. It is therapeutically used as stomachic, aperients, aphathae and antiseptic.^[1-3]

Extraction is the separation of medicinally active portions of plant using selective solvent through standard procedures. The purpose of extraction is to separate the soluble plant metabolites, leaving behind the insoluble cellular marc (residue). The initial crude using these methods contain complex mixtures of many plant metabolites such as alkaloids, glycosides, steroids, triterpenoids and flavonoids. During extraction, solvents diffuse into the solid plant material and solubilise compounds with similar polarity. The quality of an extract is majorly depended on the solvent which is used for extraction procedure that is followed for extraction and which part of the plant is used as starting material.

Major extraction methods are percolation, digestion, infusion, decoction and maceration. In maceration (for fluid extract), whole or coarsely powdered plant drug is kept in contact with the solvent in a stoppered container for a defined period with frequent agitation until soluble matter is dissolved.

Phytochemicals are the biologically active compounds present in plants. These phytochemicals are derived from various part of plant such as leaves, root, flowers, seed, stem, barks and pulps. The biologically active compounds present in plants. The preliminary phytochemical screening tests are useful in detection of the bioactive principles and subsequently may lead to the drug discovery and developments. These methods have been developed for estimation and separation of chemical constituents effectively from herbal extract.

High Performance Thin Layer Chromatographic Analysis is one of the best analytical methods. TLC is done in UV fluorescence method employed for the detection of separated spots on the TLC plates at 365nm. Antioxidant activity is done in UV/Visible spectrophotometer at 517nm.^[4,5,11]

EXPERIMENTAL:

MATERIALS:

The intended plant contents are given in the table 01. The instruments effective for the research are described in the table 02.^[1,4,5]

Table 01: Plant details

S. No.	Parameters	Subject
1.	Plant Name	Obscure morning glory
2.	Botanical Name	Ipomoea obscura (L.) Ker Gawl
3.	Family	Convolvulaceae
4.	Location	Krishnagiri
5.	Part of the plant	Stem
6.	Authentication No.	BSI/SRC/5123/2019/Tech/3003
7.	Place of Authentication	BSI, Coimbatore-641003, Tamil Nadu, India

Table 02: Instruments used

S. No.	Name of the Instrument	Model name
1.	Precision Balance	Wesner
2.	Hot Plate	Cintex
3.	UV cabinet	CAMAG and Deep vision
4.	HPTLC	CAMAG

METHODS:

Plant Collection, drying and powdering:

The plant was collected from the Krishnagiri district of Tamil Nadu. The plant were dried and taken into laboratory for data analysis. Various morphological species of ipomoea species were noted along with flower colour, odour, habitat, host species etc. Dried Ipomoea obscura stem part powdered. It was extracted with the help of hydroalcoholic solvents i.e water and ethanol (50:50%v/v). The reason for selecting the solvent was due to cost efficiency and higher alcohol produce high toxicity. 16.0g of powdered plant were subjected to hydroalcoholic extraction. The mixture of extraction solvent was prepared by water and ethanol in the ratio of 50:50 and kept in round bottom flask for 7 days with frequent shaking to achieve complete extraction. The round bottom flask was covered with aluminium foil sheet.^[4,5]

The extraction mixture was taken and filtered by using muslin cloth. The filtered liquid was taken in a china dish, the retained volume after extraction was about 1440.0ml. It was kept on hot plate at below 40℃ for evaporation. Finally semisolid extract is obtained and calculate the yield.

· Weight of empty china dish (A+B+C)= 95.0gm + 93.5gm + 92.0gm

· Weight of china dish + liquid extract (A+B+C)= 160.0gm + 166.5gm + 168.5gm

· Weight of china dish + final extract after evaporation(A+B+C)= 98.0gm + 95.0gm + 93.5gm

· Weight of final extract = 6.30gm

· Percentage yield = 39.37% w/w

Preliminary Phytochemicals Evaluation:

Phytochemical examinations were carried out for all the extracts as per the standard methods.

Detection of Alkaloids:

Mayer’s Test:

Filtrates were treated with Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow coloured precipitate indicates the presence of alkaloids.

Wagner’s Test:

Filtrates were treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids.

Dragendroff’s Test:

Filtrates were treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids.

Hager’s Test:

Filtrates were treated with Hager’s reagent (saturated picric acid solution). Presence of alkaloids confirmed by the formation of yellow coloured precipitate.

Detection of Carbohydrates:

Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates were used to test for the presence of carbohydrates.

Molisch’s Test: Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube. Formation of the violet ring at the junction indicates the presence of carbohydrates.

Benedict’s Test: Filtrates were treated with Benedict’s reagent and heated gently. Orange red precipitates indicates the presence of reducing sugars.

Iodine test: Filtrate were treated with iodine solution. Formation of deep blue colour indicate the presence of starch.

Barfoeds test: Filtrate were treated with Barfoeds solution. Boiled and waited for few minutes. Appearance of brick red precipitate indicates the presence of monosaccharides.

Seliwanoffs test: Filtrate were treated with seliwanoffs solution and boiled. Formation of deep red colour was due to the existence of ketoses.

Bials test for pentoses: Filtrate were mixed with bial’s reagent and warm slowly. Formation of green colour of precipitate indicates the presence of pentoses.

Fehling’s test: Filtrate were treated with Fehling’s solution AandB. It was kept for boiling. Formation of yellow to red precipitate indicates the presence of carbohydrates.

Osazone test: Filtrate were treated with phenyl hydrazine hydrochloride and sodium acetate. Glacial acetic acid was added to that boiled in a water bath and cooled slowly.

Needle- shaped yellow osazone crystals: Glucose, Fructose and Mannose.

Mushroom shaped crystals: Lactose.

Flower- shaped crystals: Maltose

Detection of Glycosides: Extracts were hydrolysed with dil. HCl, and then subjected to test for glycosides.

General Test:

TEST A: Dissolve 200mg of drug with sulphuric acid and then add 5%NaOH solution for neutralisation. Add fehling’s solution A and B to above mixture. Red colour is produced.

TEST B: Dissolve 200mg drug with sufficient amount of water. Add further water to dilute the solution. The solution is tested with fehling’s solution A and B. Red colour is produced from the reducing sugar present in the drug.

If the colour of test A is more intense than test B; glycosides present confirmed.

Legal’s Test: Extracts were treated with sodium nitroprusside in pyridine and sodium hydroxide. Formation of pink to blood red colour indicates the presence of cardiac glycosides.

Detection of Saponins:

Froth Test: Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.

Foam Test: 0.5gm of extract was shaken with 2ml of water. If foam produced persists for ten minutes it indicates the presence of saponins.

Detection of Phytosterols:

Salkowski’s Test: Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of golden colour indicates the presence of triterpenes.

Libermann Burchard’s Test: Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of acetic anhydride, boiled and cooled. Conc. Sulphuric acid was added. Formation of brown ring at the junction indicates the presence of phytosterols.

Detection of Phenols:

Ferric Chloride Test: Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols.

Detection of Flavonoids:

Shinoda Test: Filtrate were treated with Magnesium powder and a few drops of concentrated Hydrochloric acid or Sulphuric acid. Formation of orange, pink, red or purple colour indicates the presence of flavones, flavanols and xanthones. Formation of week pink to magenta colour/no colour indicates the presence of flavonones and flavonols.

Sulphuric acid Test: Filtrate were treated with sulphuric acid. Formation of deep yellow colour indicates the presence of flavones and flavonols. Formation of red or bluish red-bluish colour indicates the presence of chalcones and aurones. Formation of orange to red colour indicates the presence of flavonones.

Zinc hydrochloride Test: Filtrate were treated with the mixture of zinc dust and concentrated hydrochloric acid. It should give red colour after few minutes.

Alkaline Reagent Test: Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids.

Lead acetate Test: Extracts were treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates the presence of flavonoids.

Detection of Proteins and Amino acids:

Xanthoproteic Test: The extracts were treated with few drops of conc. Nitric acid. Formation of yellow colour indicates the presence of proteins.

Ninhydrin Test: To the extract, 0.25% w/v Ninhydrin reagent was added and boiled for few minutes. Formation of blue colour indicates the presence of amino acid.

Detection of Diterpenes:

Copper acetate Test: Extracts were dissolved in water and treated with 3-4 drops of copper acetate solution. Formation of emerald green colour indicates the presence of diterpenes.^[4-10]

THIN LAYER CHROMATOGRAPHIC: ANALYSIS:

The existing constituents were separated by using proper mobile phase which was selected based on trial and error method.

TLC Chromatographic Parameters^[6,9]:

Stationary phase selection:

Principle: Adsorption

Support material: Glass plate

Dimension of the plate: 8.0 × 3.0cm

Adsorbent: Silica gel G

Method of thin layer preparation: Pouring method

Layer thickness: 1.0mm

Plate activation temperature: at 105.0°C for one hour

Mobile phase selection:

Chamber: 250ml glass beaker with petri dish lid

Chamber dimension: 8.0 × 3.0cm

Chamber saturation time: 20 minutes

Fluorescence detection: UV chamber at 365.0nm (Longer wavelength)

Mobile phase selected for separation:

Flavonoids:

· Chloroform: Methanol: Formic acid (8.5:1.0:0.5v/v/v)

· Isobutanol: Acetic acid: Water (4.0:1.0:5.0v/v/v)

· Benzene: Methanol: Acetic acid(11.25: 2.0:1.0v/v/v)

· Conc. HCl: Acetic acid: water(1.0:10.0:3.33v/v/v)

· Toluene: Ethyl acetate: Formic acid: Methanol(3.0:6.0:1.6:0.4v/v/v)

· Ethyl acetate: Formic acid: Glacial acetic acid: Water(10.0:1.1:1.1:2.6v/v/v)

Calculation of R_f value

Ideal R_f value: 0.1-0.9

High-Performance Thin Layer Chromatographic Parameters:

Plate size: 10.0×10.0cm

Material: HPTLC silica gel 60F₂₅₄

Manufacture: E. MERCK KGaA

Pre-washing: No

Modification: No

Sample Application:

Instrument: CAMAG Linomat 5 “ Linomat 5-170147

Linomat 5 Application Parameters:

Spray gas: Inert gas

Sample solvent: Water

Dosage speed: 50nl/s

Predosage volume:0.2µ

Sequence:

Syringe size: 100µl

No. of tracks: 2

Band length: 8.0nm

Development: Glass tank:

Chamber type: Twin Trough Chamber 20.0×10.0cm

Mobile phase: Chloroform-8.5ml: Methanol-1.0ml: Formic acid-0.5ml

Solvent front position: 70.0mm

Volume: 10.0ml

Drying: Hair dryer

Temperature: 60°C

Time: 5minutes

Detection:

Detector: CAMAG TLC Scanner

Measurement Table:

Wavelength: 254nm

Lamp: D₂and W

Measure type: Remission

Measure mode: Absorption

Detector mode: Automatic

DPPH RADICAL SCAVENGING ASSAY^[11-17]:

1,1 Diphenyl 2- Picryl Hydrazyl is a stable (in powder form) free radical with red color which may turns yellow when scavenged. The DPPH assay uses this character to show free radical scavenging activity. The scavenging reaction between (DPPH) and an antioxidant (H-A) can be written as,

(DPPH) + (H-A)→ DPPH-H + (A)

Antioxidants react with DPPH and reduce it to DPPH-H and as consequence the absorbance decreases. The degree of discolouration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability.

Reagent preparation:

0.1mM DPPH solution was prepared by dissolving 4mg of DPPH in 100ml of ethanol.

Working procedure:

Different volumes of plant extracts were made up with ethanol and DPPH solution was added. The reaction mixture was incubated in dark condition at room temperature for 20minutes. After 20minutes, the absorbance of the mixture was read at 517nm. The percentage radical scavenging activity of the plant extract was calculated using the following formula,

RESULTS AND DISCUSSION:

Alkaloids Ninhydrin Molisch Phenolic Lead acetate:

Figure 02: Test for primary metabolite and secondary metabolite

Table 03: Preliminary Phytochemicals Evaluation

S. No	Phytochemical	Name of test	Result
1	Alkaloids	Mayer’s test	+
		Hager’s test	+
		Dragendroff’s test	+
		Wagner’s test	+
2	Carbohydrates	Molisch’ test	+
		Benedict’s test	-
		Fehling’s test	-
3	Glycosides	General test	-
4	Saponins	Foam test	-
4	Saponins	Froth test	-
5	Steroids and Triterpinoids	Libermann Burchard’s test	-
5	Steroids and Triterpinoids	Salkowski’s test	-
6	Phenols	Ferric chloride test	+
7	Flavonoids	Lead acetate test	+
		Shinoda test	+
		Alkali test	+
		Sulphuric acid test	+
8	Proteins and Aminoacids	Ninhydrin test	+

Note (+): Sign which indicates presence of phytochemical

(-): Sign which indicates absence of phytochemical

TLC Analysis:

The TLC mechanisms are interpreted in the fig 03and table 04. HPTLC graph are interpreted in figure 04.

Fig. 03: TLC Profile of Ethanolic extract of Ipomoea obscura

Table 04: Thin Layer Chromatographic Analysis

S. No.	Sample of interest	Mobile phase	Development Time (min)	Fluorescence spot colour at 365nm	Distance travelled by the solute (cm)	Solvent front (cm)	R_f value
1.	Flavonoids	Chloroform: Methanol: Formic acid (8.5:1.0:0.5v/v/v)	13	Bluish white	4.6	5.0	0.92
2.		Isobutanol: Acetic acid: Water (4.0:1.0:5.0v/v/v)	15	Bluish white	4.2	5.0	0.84
3.		Benzene: Methanol: Acetic acid (11.25: 2.0:1.0v/v/v)	16	Bluish white	3.7	5.5	0.67
4.		Conc. HCl: Acetic acid: water (1.0:10.0:3.33v/v/v)	18	Bluish white	3.1	5.5	0.56
5.		Toluene: Ethyl acetate: Formic acid: Methanol (3.0:6.0:1.6:0.4v/v/v)	17	Bluish white	3.0	5.0	0.60
6.		Ethyl acetate: Formic acid: Glacial acetic acid: Water (10.0:1.1:1.1:2.6v/v/v)	15	Bluish white	3.2	5.5	0.58

The Bluish white fluorescence at the longer wavelength were observed. The flavonoid compound could be quantified, isolated and characterized by using suitable pharmaceutical analytical technique.

HPTLC Analysis

Figure 04: HPTLC Chromatogram of Ethanolic extract and standard Quercetin

Table 05: Percentage of inhibition of Ethanolic extract

S. No	Concentration	Absorbance of Ascorbic acid	Absorbance of Ethanolic extract	Percentage of inhibition of Ethanolic extract
1	25	0.662	0.303	52.05%
2	50	0.704	0.363	42.56%
3	75	0.762	0.379	40.03%
4	100	0.784	0.403	36.23%
5	150	0.874	0.420	33.54%
6	200	0.920	0.475	24.84%

DPPH ASSAY:

Figure 05: Percentage of Inhibition of Ipomoea obscura

CONCLUSION:

The maceration process is an official and convenient method compared to other process and it is suitable for thermo labile substance. Hydroalcoholic extract was selected due to cost efficient and alcohol acts as preservative in hydroalcoholic extract. Ethanol is used for extraction of various polar and non-polar compounds. Various compounds can be detected using ethanol. Among all the alcohols, ethanol has low boiling point of just 78°C. In Indian traditional healing medicinal Ipomoea obscura (L.) Ker Gawl is used in the treatment of aphathae, open sores, stomachic and aperients. The biologically active compounds detected through TLC are Alkaloids, Flavonoids, Carbohydrates, Proteins and Amino acids. Flavonoids have antioxidant activity, alkaloids have anti-inflammatory activity. Hence, Flavonoids are selected for HPTLC due to presence of antioxidant activity.

ACKNOWLEDGEMENT:

This research was defended by the Management and Principal of Karpagam Educational Institutions, Coimbatore. We express gratitude to our colleagues from Karpagam College of Pharmacy and Faculty of Pharmacy, Karpagam Academy of Higher Education, Coimbatore who provided wisdom and expertise that incredibly assisted the study.

CONFLICT OF INTEREST:

It is declared none.

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Received on 24.09.2019 Modified on 23.11.2019

Research J. Pharm. and Tech. 2020; 13(10):4661-4667.

DOI: 10.5958/0974-360X.2020.00820.3