Under the family of Apocynaceae, Allamanda cathartica L., often known as Golden Trumpet, is a 6 m tall woody climbing green shrub. It is cultivated in India for its eye-catching Karnataka flowers and is close to Brazil and Central America. Using phytochemical research, a class of chemicals found in plant extracts can be identified as plants for therapy. The identification of the active ingredients found in plants is the primary goal of phytochemical research. This plant reproduces vegetatively through cuttings and sexually through seeds. Around the world, A. cathartica is commonly grown as an ornamental in tropical and subtropical climates1. Antimicrobial substances can still be found in large quantities in medicinal plants. Globally, there has been a rise in interest in the medicinal properties of natural goods. It is said that the world's flora, nature's medicine, has the solution for any crippling human ailment. Human health has been significantly maintained by phytochemicals. Allamanda Cathartica has been chosen for the current investigation. This plant is said to have several therapeutic uses, including being an effective purgative and an antidote for poisoning, inflammation, constipation, and ascites2.  It has been utilized to treat malaria-related jaundice and enlarged spleen. It is effective on human nasopharyngeal cancer both in vitro and in vivo in mice. Both the watery extract and the alcohol have high blood pressure. It has antimicrobial and perhaps anticancer properties, and it is cathartic (milky sap). Plant alkaloids are known to have significant biological activity. The leaf of A. cathartic contains alkaloids, sterols, and flavonoids.  Strong anti-oxidative cell damage and robust anti-cancer activity are exhibited by flavonoids, which are water-soluble antioxidants and free radical scavengers. Flavonoids have been shown to reduce blood pressure and enhance blood circulation. The flowers of Allamanda cathartica were extracted using acetone, petroleum ether, chloroform, ethanol, and water. Various phytoconstituents, including alkaloids, phenolic substances, saponins, flavonoids, and glycosides, terpenoids, steroids, coumarins, quinones, phytosterols, proteins, and carbohydrates, were found in these extracts. Allamanda cathartica extracts in petroleum ether and chloroform showed encouraging antifungal activity3.

(Golden Trumpet)

PLANT PROFILE5:

       
           
       
    Table no. 2: Taxonomical classification of plant.

MATERIAL AND METHODS4:

Collection of plant material:

From the surrounding Wardha areas, we gathered the leaves, stems, and flowers of a cathartic plant. The leaves were dried after being cleaned of pollutants using tap water. We gathered fresh stems and let them dry in the sun. The maceration method involved the use of dried flowers. Afterward, the dried stems were ground into a fine powder and, depending on the polarity of the solvents, were extracted using a Soxhlet with petroleum ether and hydroalcoholic (ethanol).

Instruments and Glassware used:

Measuring cylinder, beakers, desiccator, water bath, Petri dishes for weighing balance, test tubes, pipette, evaporating dish, silica crucible, muffle furnace, and funnel for the Soxhlet apparatus.

Reagents and solvents Use:

The concentrated hydrochloric acid solution, sodium hydroxide, dilute hydrochloric acid, chloroform, ethanol, ethyl acetate, methanol, petroleum ether phloroglucinol, safranin, glacial acetic acid, concentrated sulphuric acid, anhydrous acetic anhydride, molisch reagent, Fehling’s solution, flavonoid test, tannins test, Hagar’sreagent, dragendroff’s reagent and Mayer’s reagent, sulphuric acid.

Extraction6,7,8:

The Allamanda Cathartic flower is taken in a maceration bottle and filled with petroleum ether for a 7-day maceration process. Later we dry the flower After this, process we take another solvent, Hydroalcoholic (80:20 ethanol: distilled water). For the extraction of the Allamanda cathartica L. stem, we used petroleum ether solvent in soxhlet assembly for 3 days (16 cycles). Afterward, we dry the crude drug (stem) and change the solvent, Hydroalcoholic for 4 days (18 cycles).

       
           
       
    Fig 2: Soxhlet assembly

       
           
       
    Fig 3: Maceration

Microscopical analysis9:

1. Transverse section of leaves: A cut-through leaves showing their internal structure.
2. Transverse section of stem: Cut through a stem showing a single-layered epidermis with trichomes.
3. Longitudinal section of stem: A cut along the length of a stem.
4. Transverse section of the ovary: Cut through an ovary showing its structure.
5. Powder Characteristic: Evaluation of medicinal plants using staining reagents.
6. Palisade ratio: Average number of palisade cells under one epidermal cell in leaves.
7. Stomatal number & Stomatal Index: Number of stomata per sq mm and ratio of stomata to epidermal cells.
8. I = S / E + S, where I is a stomatal index.
9. Vein islet & vein termination number: Number of vein islets and veinlet terminations per sq mm of leaf surface.

These techniques are used to study and identify plant structures for various purposes, such as research, quality control, and taxonomy.

1. Total Ash Value:

Total ash is a measure of the mineral oxide content of activated carbon on a weight basis. It is determined by converting the mineral constituents to the respective oxides at 800°C. The ash primarily consists of silica and aluminum, and its amount depends on the base raw material used for production.

2. Acid Insoluble Ash:

Acid insoluble ash is determined by dissolving ash in dilute hydrochloric acid (10% m/m), filtering the liquid through ashless filter paper, and then washing it with hot water. The filter paper is ignited, cooled, and weighed.

3. Water-soluble Ash:

This is the part of the total ash that dissolves in water under specified conditions.

4. Sulphated Ash10:

Sulphated ash, like crude ash, can indicate the level of known metal-containing additives or impurities in organic material. The procedure involves heating the ash with sulfuric acid, which can be hazardous and corrosive.

5. Loss on Drying (LOD):

This test determines the moisture content of a sample by measuring the loss of volatile matter. It does not usually refer to molecularly bound water or water of crystallization.

6. Foaming Index:

The foaming index is a measure of the ability of a substance to produce foam. It is calculated based on the volume of plant material decoction in a test tube that produces a 1 cm foam height. The procedure involves boiling the material, filtering, and then shaking the filtrate in test tubes to measure foam height.

Foaming index = 1000/a.

        a =volume in ml of the decoction in the test tube showing 1 cm height.

7. Fibre Content:

Fibres in pharmacognosy are defined as elongated thick-walled cells with cellulose walls, which may or may not contain lignin. In the context of surgical dressings, fibre includes both natural and artificial fibres.

       
           
       
    Fig 12: Total ash

       
           
       
    Fig 13: Muffle furnace

       
           
       
    Fig 14: Acid insoluble ash

       
           
       
    Fig 15: Sulphated ash

       
           
       
    Fig 16: Loss on drying

       
           
       
    Fig 17: Fiber content

TLC is a chromatographic technique that uses a solid fixed phase (silica gel) and a liquid mobile phase (chloroform: methanol = 8:2) to separate compounds in plant extracts based on their polarity. Silica gel plates are activated and used as the stationary phase. Different solvent systems are applied to identify active principles in plant extracts. Visualization is done under UV light, and specific spray reagents are used for enhancement. TLC is rapid, cost-effective, and provides good resolution and sensitivity, making it ideal for phytochemical investigation.

Stationary Phase 

Silica gel G, particle size 10 – 40 µm applied as a thin layer on a clean glass plate support and activated (1100C for 30 minutes) just before use.

Mobile Phase

Quantity – 50 ml

The mobile phase was –

1. Chloroform: methanol = 2:8
2. Ethyle acetate: methanol=6:4

Development Method

One dimensional ascending method by using a standard protocol as per IP was followed.

Visualization

After the development of the TLC plate, initially, three spots were visualized in the UV chamber (365 nm).

Flavonoid Estimation14:

Total flavonoid content (TFC) determination. The amount of flavonoids present in the extracts overall was ascertained using the aluminum-chloride spectroscopic assay. In a 10 mL volumetric flask, 1 ml of the extract (1 mg/mL) was combined with 4 mL of methanol. The flask received 0.80 mL of 5% sodium acetate added to it. One milliliter of 10% AlCl3.6H2O solution was added to the mixture after five minutes. Using the same process as for the extracts, a series of standard quercetin solutions (200,180,160,140,120,100, 80, 60, 40, and 20 µg/ml) were made. Using a UV/Visible spectrophotometer, the absorbances of the extracts and standard solutions were measured at 415 nm in relation to the reagent blank. The calibration curve was used to calculate the total flavonoid concentration, which was then represented as milligrams of quercetin equivalent (QE) per gram of extracts. Duplicate measurements of the total flavonoids in the extracts and standards were made.

       
           
       
    Fig 21: UV spectrophotometer

       
           
       
    Fig 22: Dilutions

       
           
       
    Fig 23: Micropipette

RESULTS AND DISCUSSION:

Physicochemical analysis:

This analysis performed for isolation, purification, and identification of active constituents is a chemical method of evaluation.

Phytochemical screening is used to find out the active constituent of plant extract (crude drug).

       
           
       
    Table no. 5: Observations of phytochemical screening tests.

Observation in different solvent systems.

Fig  24: TLC plates

Flavonoid estimation:

Standard: Quercetin