INTRODUCTION

Amaranthus dubius (Amaranthaceae) is an important medicinal plant much used in ayurveda and other traditional medicines in many parts of the world. Synonym: Spleen amaranth. Vernacular Names: English-Green spinach, Malayalam-cheera, Tamil-Arai keerai. Biological source: The drug consists of dried, mature whole plant of Amaranthus dubius. Family: Amaranthaceae¹

Fig No: 1 Amaranthus dubius

Geographical source:

Annual herb native to South America, Mexico, West Indies.

Table No 1. Taxonomical classification

Kingdom	Plantae
Order	Caryophyllales
Family	Amaranthaceae
Genus	Amaranthus
Species	Dubius
Binomial name	Amaranthus dubius

Habitat:

Amaranthus dubius is an annual herb native to South America, Mexico and the West Indies. This species has been widely introduced as a green vegetable for human consumption and as a medicinal herb.

Description of the plant:

It is typically are annuals or short-lived perennials. The stems often are greenish in color and sometimes are armed with spines; they bear simple alternately arranged leaves having petiole 2-9 cm. The plant produce small green flowers and seed is lenticular, around 1mm in diameter, dark brown to black in color and shiny.

CHEMICAL CONSTITUENTS²:

Proteins	Phenolic compound
Lysine	Vitamins
Niacin	Calcium
Vanillin	Irons
Caffeic acid	Carbohydrates
Beta sitosterol	Quercetins
Trilinolein	Spina sterol

TRADITIONAL USES³:

It is a good with medicinal properties for young children, lactating mothers and for patients with fever, hemorrhage, anemia, constipation or kidney complaints. Amaranth really shines when it comes to skincare working to hydrate, detoxify soothe, improve elasticity and reduce wrinkles. It is used to produce medicinal products against atherosclerosis, stomach ulcers, and tuberculosis. It is used as antiseptic, antifungal and anti-inflammatory preparations. Promote hair growth.

Pharmacognostical Studies:

Macroscopical Evaluation:

Macroscopic or organoleptic characters like appearance, colour, odour and taste were evaluated.

Microscopical Evaluation:

Microscopical evaluation includes:

1. Qualitative Microscopic Studies⁴:

a) Transverse Section:

The sections of leaf were cleared with chloral hydrate solution and then stained with phloroglucinol and hydrochloric acid (1:1), mounted in glycerin and observed under the microscope.

b) Powder Microscopy:

The powdered drug treated with phloroglucinol-HCl solution and glycerin to determine the presence of trichomes, epidermal cells vessels etc.

2. Quantitative Microscopical Studies⁵:

A) Leaf constants:

a) Stomatal Numbers and Stomatal index:

The upper epidermis of the leaf between midrib and lamina was peeled and transparent area was cleared with clearing solution and mounted on glass slide. The stomata and epidermal cells were traced on black sheet between 0.8 mm square using prism type camera lucida under high power (45x). The number of epidermal cells and stomata were counted as per rule from each drawn square. The experiment was repeated and stomatal number was directly calculated.

Stomatal index was calculated using formula:

Stomatal index (SI) = S / E + S×100

b) Vein islet number and Vein termination number:

The leaf portion between midrib and margin was macerated in concentrated chloral hydrate solution for 24 h and decolorized with bleaching solution (5% calcium chloro-hypochlorite). The cleared lamina portion was mounted on glass slide and vein islet and vein terminations were traced on black sheet between 0.2 mm square using low power (45x). The process was repeated and values were determined per sq.mm of leaf area between midrib and margin.

Physico-Chemical Evaluation⁶:

Determination of Ash Value:

a. Total ash value:

This is usually done to measure the mineral content and other organic matter of the crude drug. Weigh accurately about 2 gm of the powdered drug in silica crucible. Incinerate the powdered drug by increasing the heat gradually until the sample was free from carbon and cool it keep it in a desiccators. Weigh the ash and calculate the percentage of total ash in contrast to the air-dried sample.

Total Ash Value = (weight of the ash) ÷ (weight of the crude drug) ×100

b. Acid Insoluble Ash Value:

Boil the total ash obtained as the above procedure for 5 minutes and mix 25 ml of dilute hydrochloric acid. Filter and collect the insoluble matter on an ash less filter paper, after that wash the filter paper with hot water, ignite in tared crucible, cool and keep in desiccator. Weigh the obtained residue and calculate acid-insoluble ash of the crude drug.

Acid Insoluble ash value = (weight of acid insoluble ash) ÷ (weight of the crude drug) X 100

c. Water Soluble Ash Value:

Boil the total ash for 5 minutes with 25 ml of water; collect the insoluble matter in a Gooch crucible or on an ash less filter paper, wash with hot water, and ignite to constant weight at a low temperature.

Water Soluble Ash Value = (weight of water-soluble Ash) ÷ (weight of the crude drug) X 100

Determination of Crude Fiber Content:

Weigh 2g of powdered drug in a beaker. Add 50ml of 10%v/v nitric acid. Heat to boil with constant stirring (30 sec). Filter through Buchner funnel; give washing to the residue with boiling water. Transfer the residue from the cloth to beaker. Add 50ml of 2.5%v/v sodium hydroxide solution. Heat to boil (30 sec), stain and wash with hot water. Transfer the residue in a clean and dried crucible by the residue and determine % crude content.

% Crude fiber content = Final weight ÷ Drug weight × 100

Determination of Length and Width of Trichomes:

Calibrate the eyepiece micrometer using the stage micrometer and calculate the factor. Stain the powder with one drop each of phloroglucinol and mount a little of the stained powder in dilute glycerin and observe the slide under the low power (10x). Measure the length and width of the stained trichomes by focusing the lines. Calculate the value for 10 trichomes and multiply each value by obtained factor and calculate the average value.

Fluorescence Analysis:

Two to three drops of freshly prepared reagents are mixed with each extract. Observe under day light, shorter and longer wavelength in UV cabinet.

Determination Of Extractive Value⁷:

Extractive value helps to determine the number of soluble constituents in a given amount of medicinal plant material, when extracted with solvents.

a) Ether soluble extractive value procedure:

Macerate about 5g accurately weighed powder with 100ml of pet. ether in a stoppered flask for 24 hours shaking frequently during 6 hours. Filter through filter paper. Evaporate 25ml of extract to dryness in a tared flat bottomed shallow dish. Dry at 105˚ C and weigh. Keep it in desiccator.

b) Alcohol soluble extractive value Procedure:

Macerate about 5g accurately weighed powder with 100ml of 90% Ethanol in a stoppered flask for 24 hours shaking frequently during 6 hours. Filter through filter paper. Evaporate 25ml of ethanolic extract to dryness in a tared flat bottomed shallow dish. Dry at 105˚ C and weigh. Keep it in desiccator.

c) Water soluble extractive value Procedure:

Macerate about 5g accurately weighed powder with 100ml of chloroform water in a stoppered flask for 24 hours shaking frequently during 6 hours. Filter through filter paper. Evaporate 25ml of extract to dryness in a tared flat bottomed shallow dish. Dry at 105˚ C and weigh. Keep it in desiccators.

Loss on Drying:

The loss in the weight was recorded as moisture content.

Place the 2g of the substance to be examined in a Petri dish and dry at 105˚C. The drying was continued until two successive reading. Matched each other or the difference between two successive is not more than 0.25% of constant weight. 24 hours shaking frequently during 6 hours. Filter through filter paper. Evaporate 25ml of extract to dryness in a tared flat bottomed shallow dish. Dry at 105˚ C and weigh. Keep it in desiccator.

Phytochemical Analysis⁸:

Test for Carbohydrates:

Molisch’s test:

To the test solution, add 1ml of Conc.H₂SO₄and Molisch reagent. It produces red or violet ring is appeared b/w two junction.

Test for Glycosides:

Balget’s test: Sodium picrate was added to the test solution. It produces yellow to orange color.

Keller killani test: To 2ml of extract, add glacial acetic acid,one drop of 5% Fecl₃ and Conc. H₂SO₄.Reddish brown color appear at junction of two layer and upper layer shows green color.

Test for Saponin:

Foam or Froth test: The extract was vigorously shaken with water. Produce persistent foams.

Test for Phenolic compounds:

Ferric Chloride test: The extract is dissolved in fecl₃ solution. Blue or black precipitate is produced.

Lead nitrate test: The extract is treated with lead nitrate solution. Produce white precipitate.

Test for Alkaloids:

Dragendorff’s test: 2ml of Dragendorff’s reagent was added to few ml of extract. Prominentredcolour willbe obtained.

Wagner’stest: Few drops of Wagner’s reagent were added to few ml of the extract. Formation of reddish-brown precipitate.

Mayer’stest: Few drops of Mayer’s reagent were added to 1ml of extract.A yellowish or white precipitate was formed.

Hager’stest: The extract was treated with few drops of Hager’s reagent. A yellow precipitate was formed.

Tannic acid test: The extract was treated with few drops of tannic acid reagent. A buff-coloured precipitate is formed.

Test for proteins:

Biuret Test: To test solution 4%NaOH and 1%CuSO4 is added. Violet or pink colour appears.

Millions Test: 3ml of test solution and millions reagent is added to it. It produces white precipitate.

Test for phytosterols:

Liebermann Burchard test: The extract was dissolved in chloroform and added 3 ml of acetic anhydride.3 ml of glacial acetic acid were warmed and cooled. To this a few drops of concentrated sulphuric acid were added along the sides of the tube. Red colour is obtained.

Formulation⁹:

Herbal formulation means a dosage form consisting of one or more herbs or processed herbs in specific quantities. Herbal formulation can be given to the patient in various forms like solid, semisolid, liquid. Different types of herbal formulation in market which includes Gel, Tablet, Powders, Liquid orals, Paste, Ointment, and Creams etc.

Gels are semisolid system consisting of dispersions of small or large molecule in an aqueous liquid vehicle rendered jelly like by the addition of a gelling agent.

Preparation of Amaranthus dubius herbal gel:

Excipients¹⁰:

These are the substance other than active pharmaceutical component and packaging elements, often influencing the quality of final product, sometime forming almost complete formation. It includes:

TableNo 2: Excipients

INGREDIENTS	QUANTITY (Gm)
Ethanolic extract of Amaranthus dubius	0.2
Carbopol 934 (Polymer)	2
Propylene glycol (Emollient)	10ml
Methyl paraben (Preservative)	2
Propyl paraben (Preservative)	2
Purified water (Vehicle)	20ml
Triethanolamine (Neutralizer)	Q.S

Accurately weighed Carbopol 934 was taken in a beaker and dispersed in 10 ml of distilled water. Kept the beaker aside to swell the Carbopol for half an hour and then stirring should be done using mechanical/lab stirrer at 1200 rpm for 30 min. Take 2ml of propylene glycol and required quantity of Extract. Take 10 ml propylene glycol in another beaker and add weighed quantity of propyl paraben and methyl paraben to it and stirred properly. After all, Carbopol dispersed, 0.2 gm extract and preservatives solutions were added with constant stirring. Finally, volume made up to 20 gm by adding remaining distilled water and Triethanolamine was added drop wise to the formulations for adjustment of required skin pH (6.8-7) and to obtain the gel at required consistency. Mixing of Gel: Mechanical stirrer is employed for the mixing of the ingredients

Fig No: 2 Amarathus leaf

Macroscopical Evaluation:¹¹

Type: Simple, Shape: Oval, Venation: Reticulate, Arrangement: Simple and Alternative, Petiole: 8.5 cm. Color: Green, Odour: Grassy smell, Apex: Round, Margin: Entire, Texture: Smooth, Base: Wedged.

Fig No: 2 Amarathus leaf transverse section

Microscopical Evaluation:¹²

Lamina:

Upper epidermis– Rectangular cells. Ground tissue consists of angular collenchyma and thin parenchyma. Lower epidermis – rectangular cells present.

Mesophyll:

Thin parenchyma– Cells filled with chlorophyll. Trichomes: Uniseriate multicellular trichomes present.

Midrib:

Epidermis is uniseriate ground tissue consists of angular collenchyma below the epidermis followed by thin parenchyma. Ground tissue present. Oval parenchyma cells. Prismatic crystals of calcium oxalate present.

Vascular Bundles:

Vascular bundles separated with round shape. Xylem and Phloem present. Pith: Parenchymatous anomalous cambic ring.

Quantitative microscopic studies¹³:

Table No: 3 Quantitative microscopy: Leaf constants

Parameters	Observation
Stomatal number	26
Stomatal index	34.57
Vein islet number	120
Vein termination number	56

Physiochemical evaluation

TableNo 4 Determination of Ash Value ¹⁴

Ash Value	Result
Total ash	21%w/w
Acid insoluble ash	3.5%w/w
Water soluble ash	7%w/w
Sulphated ash	13%w/w

Determination of Crude Fiber Content¹⁵

% crude Fiber content= (0. 28÷2) ×100 = 14%

Determination of Length and Width of Trichomes ¹⁶

Length: 114.416 micron-231.41micron - 357.55micron width: 14.802micron-22.84micron-42.90micron

Table No: 6 Determination of Extractive Value ¹⁷

Extractive Value	Result
Water soluble extractive value	7.2%w/w
Alcohol soluble extractive value	6.4%w/w
Ether soluble extractive value	5.6%w/w
Chloroform extractive value	1.6%w/w

Loss on Drying:

Moisture content = 7.5%w/w

Table No: 7 Phytochemical Screening¹⁸

Phytoconstituents	Results
Carbohydrates	-ve	Phenolic compounds	-ve
Glycosides	+ve	Alkaloids	+ve
Saponin	-ve	Proteins	-ve
		Phytosterols	+ve

Table No: 8 Thin Layer Chromatography of Amaranthus dubius¹⁹

Secondary Metabolites	Mobile Phase	Detecting Agent	Rf Value
Glycosides	Toluene: Methanol: Glacial acetic acid: Water (7:4:3:1)	UV chamber UV chamber	0.62 0.87
Alkaloids	Butanol: Acetic acid: Water (4:1:3)	Iodine chamber	0.57
Phytosterols	Hexane: Acetone (9:1)	UV chamber	0.21

Table No: 9 Evaluation of formulation ²⁰

Parameter	Result
Physical appearance
· colour	Olive green
· odour	Odourless
· consistency	Non sticky
Grittiness	No gritty particles
pH	6.7
Spread ability	0.87g/sec
Homogeneity	No aggregates

CONCLUSION:

This plant has a wide range of medicinal value due to their variety of chemical constituents and can be further investigated on toxicological and other parameters to obtain a valuable marketed product²¹. The parameters studied were macroscopy, microscopy, and powder microscopy and physio chemical properties. The colour, odour, taste and appearance of leaves were studied through macroscopical evaluation. The microscopical studies revealed that the leaf is dorsiventral in nature.²² Midrib contains many vascular bundles Xylem and phloem, calcium oxalate crystals present. Thin parenchyma present with intercellular spaces. Epidermis is uniseriate ground tissue consists of angular collenchyma below the epidermis followed by thin parenchyma. The powder microscopy shows the presence of prismatic type of calcium oxalate crystals and multicellular trichomes. The leaf constants were also determined.²³

Various physiochemical constants were evaluated for the leaf such as ash values and extractive values. This helps in conforming the identity and purity of these plant and to detect adulterants and its nature and will useful for future researches. In physio chemical analysis, extraction is the first step involved. This coarse powder was extracted by petroleum ether, ethanol and chloroform by successive solvent extraction. The preliminary phytochemical screening of various extract has revealed the presents of constituents like Carbohydrates, Proteins, Alkaloid, Glycosides, Phytosterols, Fixed oils and fats etc. TLC analysis of Pet.ether, ethanolic and chloroform extract was carried out to detect identify phytoconstituent present. Formulation of Amaranthus dubius gel was performed and standardization was carried out on the gel of colour, odour, consistency, grittiness, spread ability, pH and homogeneity.

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Received on 23.01.2024 Modified on 30.03.2024

Asian J. Research Chem. 2024; 17(3):139-144.

DOI: 10.52711/0974-4150.2024.00026

SL.NO	Powder + Reagent	Fluorescence In Day Light	Fluorescence Under UV (365 nm)	Fluorescence Under UV (254 Nm)
1	Drug + Picric acid	Green	Green	Green
2	Drug + 50% Acetic acid	Brown	Greenish brown	No change
3	Drug + Al. KOH	Light green	Dark green	Light green
4	Drug+ 1%HCL in water	Brown	Dark brown	Brown
5	Drug + N/10 iodine	Greenish yellow	Dark green	No change
6	Drug + N/10 iodine	Light brown	Greenish brown	Light brown