INTRODUCTION:

Herbal drugs play an important role in health care programs especially in developing countries. Ancient Indian literature incorporates a remarkably broad definition of medicinal plants and considers ‘all’ plant parts to be potential sources of medicinal substances¹. Achyranthes aspera Linn. is an erect or procumbent, annual or perennial herb, 1-2m in height, often with a woody base, commonly found as a weed of waysides, on roadsides. Achyranthes aspera Linn has been mentioned in the manuscripts of Ayurveda and Chinese medicine. It is described in “Nighantus” as purgative, pungent, digestive, a remedy for inflammation of the internal organs, piles, itch, abdominal enlargements and enlarged cervical glands.

A decoction of the plant is used as diuretic in renal dropsy and general anasarca. In Philippines the plant is used to relieve toothache, dysentery and bowel complaint³. Decoction of root is useful in pneumonia, cough, and kidney stones and in large doses it acts as ecbolic. The ash of whole plant is used in haemorroids⁴. The plant is used as expectorant, revulsive, anodyne, depurative, anthelmintic, sudorific, stomachic and to treat asthma, dyspepsia, bronchitis, and flatulence and menstrual disorders⁵. In the Chittoor district of Andhra the tribals use this plant in the treatment of epilepsy and the Payasam or Kheer made of its seeds in milk is used as a good remedy for diseased brain.⁶

The plant is a good source of trace elements and each element has its individual impact in the structural and functional integrity of the living cells and organisms⁷. The work is an effort to establish microscopic and chemical standardization of Achyranthes aspera Linn. Leaves. However no scientific standard parameters are available to determine the quality and genuineness of the drug. The present work will provide a beneficial information towards the quality of the drug and also standardization of the drug.

MATERIALS AND METHODS:

Material:

Fresh sample of Plant of Achyranthes aspera Linn., were collected from Shirdi Taluka Rahata District Ahmednagar (Maharashtra), and dried in the shade at room temperature. The plant was authenticated by Mrs. Priyanka A. Ingle, Scientist ‘C’ by comparing morphological features. (Ref. No. BSI/WRC/100-1/Tech11/2019/46 Dated on 22^thOctober 2019).

Figure No. 1: Authentication letter of Achyranthes aspera Linn

Methods:

1. Pharmacognostic Study:

Macroscopic:

The macroscopic characters such as size, shape, margin, nature, texture, apex, surface, colour, odour, taste were studied for morphological investigation.

Microscopy:

For microscopy, free hand section of root was cut and with different microchemical reagents like phloroglucinol: Hydrochloric acid (1:1), Sudan Red III, dilute Hydrochloric acid, observed under microscope at 10X^8,9.

Physicochemical Evaluation:

The extractive values, ash values and loss on drying were performed according to the official methods prescribed in Ayurvedic Pharmacopoeia of India and WHO guidelines on quality control for medicinal plant materials¹⁰.

Phytochemical screening:

The preliminary phytochemical screening for stem, leaves and root were carried out as per WHO Guidelines on quality control for medicinal plant materials¹⁰.

Powder microscopy:

Powder microscopy were performed the powdered characteristics of leaves shows lignified cells, pericyclic fibers, stone cells, cuticles, calcium oxalate crystals¹¹.

1. Phytochemical Study:

Extraction:

Dried and powdered root of the plant Achyranthes Aspera was extracted successively with various solvents viz. ethanol, Petroleum ether and Water ethanol in Soxhlet extractor. The marc left will extract using suitable solvents. Extract were concentrated by vacuum distillation and then dried in open air to produce respective extract

Preliminary phytochemical screening of various extracts was carried out using the standard procedures

1. Test for Carbohydrates:

A. Molish test: Two ml of extracts solution was treated with few drops of 15 percent ethanolic a- napthol solution in a test tube and 2ml of concentrated sulphuric acid was added carefully along the side of tubes. The formation of reddish violet ring at the junction of two layers indicates the presence of carbohydrates.

B. Fehling’s test: Five ml of extract solution was mixed with 5ml Fehling’s solution (equal mixture of Fehling’s solution A and B) and boiled. Development of brick red precipitate indicates the presence of reducing sugars.

2. Test for Proteins:

A. Biuret test: The extract was treated with 1ml of 10 percent sodium hydroxide solution and heated. A drop of 0.7 percent copper sulphate solution was added to the above mixture. The formation of purple violet color indicates the presence of proteins.

B. Millon’s test: The extract was treated with 2ml of Millon’s reagent. Formation of white precipitate indicates the presence of proteins and amino acids.

3. Test for Amino acids

Ninhydrin test: The extract was treated with Ninhydrin reagent at pH range of 4-8 and boiled. Formation of purple color indicates the presence of amino acid

4. Test for Steroids:

A. Salkowski test: One ml of concentrated sulphuric acid was added to 10mg of extract dissolved in 1ml of chloroform. A reddish brown color exhibited by chloroform layer and green fluorescence by the acid layer suggests the presence of steroids.

B. Liebermann-buchard test: 10mg extract was dissolved in 1ml of chloroform and 1ml of acetic anhydride was added following the addition of 2ml of concentrated sulphuric acid from the side of the test tube. Formation of reddish violet color at the junction indicates the presence of steroids.

C. Liebermann’s test: To 2ml of the residue a few ml of acetic anhydride was added and gentle heated. The content of the test tube was cooled and 2ml of concentrated sulphuric acid was added from the side of the test tube. Development of blue color gave the evidence for presence of steroids.

5 Test for Terpenoid:

2ml of the organic extract was dissolved in 2ml of chloroform and evaporated to dryness. 2ml of concentrated sulphuric acid was then added and heated for about 2 min. A greyish colour indicates the presence of terpenoids.

6. Test for Glycosides:

A. Borntrager’s test: To 3ml extract dilute sulphuric acid was added, boiled and filtered. To the cold filtrate, equal volume of benzene was added and shaked well. Organic layer was separated and to that ammonia solution was added. The ammonical layer turns pink or red.

B. Cardiac glycoside:

Keller-killani test: To 2ml of extract, glacial acetic acid, one drop 5% Ferric chloride and conc. Sulphuric acid was added. Presence of cardiac glycosides is indicated by formation of reddish brown color at the junction of the two liquid layers and upper layer appeared bluish green.

7. Test for Saponins:

Foam formation test: One ml solution of the extract was diluted with distilled water to 20ml and shaken in a graduated cylinder for 15minutes. The development of stable foam indicates the presence of Saponins.

8. Test for Alkaloids:

A. Dragendroff’s test: 0.1ml dilute hydrochloric acid and 0.1ml Dragendroff’s reagent was added in 2ml of extracts in a test tube. Formation of orange brown precipitate indicates the presence of alkaloids.

B. Mayer’s test: To 2ml of extracts, 0.2ml of dilute hydrochloric acid and 0.1ml of Mayer’s reagent was added. Formation of yellowish buff precipitate indicates the presence of alkaloids.

9. Test for Tannins and Phenolic compounds:

A. Ferric chloride test: Five ml of extract solution was allowed to react with 1ml of 5 percent ferric chloride solution. Greenish black coloration indicates the presence of tannins.

B. Dilute nitric acid test: Two ml of extract solution was allowed to react with few drops of dilute HNO₃solution. Formation of reddish to yellow color indicates the presence of tannins.

10. Test for Flavonoids:

A. Shinoda test: To the extract, 5ml (95%) ethanol, few drops of con. HCl and 0.5g of magnesium turnings was added to give pink color.

B. Lead acetate test: Few drops of 10 percent lead acetate were added to the extract. Development of yellow colored precipitate confirms the presence of flavonoids.

C. Sodium Hydroxide test: To the extract increasing amount of Sodium Hydroxide was added. Yellow color was produced which disappeared after addition of acid.

Thin Layer Chromatography:

All extracts of root subjected for TLC with suitable solvent system for better separation of the components. The R_f values of the separated components were recorded.

RESULT AND DISCUSSION:

1. Pharmacognostic Studies:

Macroscopy study:

The macroscopic study of roots of Achyrenthes Aspera Linn is givenin following table:

Table No.: 1 Macroscopic study of roots of Achyrenthes Aspera Linn

Sr. No	Organoleptic Character	Observation
1.	Colour	Yellowish brown in colour
2.	Odour	Slight
3.	Taste	Slight sweet and mucilaginous
4.	Size	Main root is Long cylindrical think Secondary and tertiary root is slightly ribbed
5.	Shape

Microscopic Study:

Transverse section of root shows lignified cells, stone cells, calcium oxalate crystal and suberised cork cells upto 10 layers. Cortex consists of 4-6 rows of conjunctive parenchymatous tissue. Vessels having simple and bordered pits with helical thickening are present. Tracheids are also present with tapering end walls. Pith is absent.

Powder Characteristics:

Table No.: 3 Microscopic studies of roots of Achyrenthes Aspera Linn

Powder	White colour
Phloem Fibers	Lignified with Tapering ends, Narrow lumen
Starch Grains	Granules form
Calcium Oxalate Crystals	Acicular raphides in Medullary Rays and Phloem Parenchyma
Oil Cells	Big and Isolated

Physicochemical evaluation:

The loss on Drying (LOD), Ash Values likes (Total Ash, Acid insoluble ash, Water soluble ash) water soluble extractive, Alcohol soluble extractive and Swelling Index of powder are given in table 4.

Preliminary Phytochemical tests:

The preliminary phytochemical tests of different extracts were performed and identified using specific reagents and results shows presence of carbohydrate, Glycosides, alkaloids, flavonoids, tannins and Steriods in Ethanol, petroleum ether and Water: Ethanol extract. (Table 5)

Table No.: 4 Physicochemical evaluation of roots of Achyrenthes Aspera Linn

Sr. no	Physical constant	Value
1	Foreign organic matter	10 (% w/w)
2	Determination of moisture content	8 % (loss on drying)
3	Determination of Total ash	4.1 (% w/w)
4	Determination of Water- soluble ash	2.6 (% w/w)
5	Determination of Acid -insoluble ash	1.7 (% w/w)
6	Determination of water-soluble extractive value	9.6 (% w/w)
7	Determination of Alcohol-soluble extractive value	6.3 (% w/w)

Table No.: 5. Preliminary Phytochemical tests

Sr. No.	Chemical constituent	Chemical Tests	Observations
Sr. No.	Chemical constituent	Chemical Tests	Petroleum Ether extract	Ethanol extract	Hydro-Alcoholic Extract
1.	Tests for carbohydrates	Molish Test	+	+	+
		Fehling Test	+	+	+
		Benedict Test	+	+	+
2.	Test for Proteins	Biuret test	+	+	-
2.	Test for Proteins	Millions test	-	+	+
3	Test for amino acid	Ninhydrin test	-	-	-
4.	Tests for Steroids	Salkowaski test	-	+	+
4.	Tests for Steroids	Libermann Burchard test	+	+	+
5.	Tests for Terpenoids	-	+	+	+
6.	Test for Glycosides	Borntrager’s Test	+	-	+
6.	Test for Glycosides	Killer- Killani Test	+	-	+
7.	Test for Saponin	Foam test	+	-	+
8.	Tests for Flavonoids	Shinoda test	+	-	+
		Lead acetate Test	+	+	-
		Sod-hydroxide Test	+	+	+
9.	Tests for Alkaloids	Mayers Test	+	+	+
		Wagner’s Test	+	+	+
		Hager’s Test	+	+	+
		Dragendorff Test	+	+	+
10	Test for Tannins and Phenolic compounds	FeCl3	+	+	+
10	Test for Tannins and Phenolic compounds	Lead acetate	+	+	+

Sr. no.	Chemical constituent	Mobile Phase	Visualization Spraying reagent	Color of spot	Rf- value
1.	Alkaloids	Toluene: Ethyl acetate: Formic acid (50:40:10)	10% H₂SO₄ in ethanol	Violet	Ethanol: 0.86 Hydroalcoholic: 0.75
2.	Glycoside	Ethyl acetate: Methanol: Water (100: 16.5: 13.5)	Under UV -365	Brown	Petroleum ether :0.82 Ethanol: 0.70 Hydroalcoholic :0.62
3.	Flavonoid	Toluene: Ethyl acetate: Glacial acetic acid: Water (100:11:11:26)	Anisaldehyde – Sulfuric acid.	Yellowish green	Petroleum ether :0.86 Ethanol: 0.82 Hydroalcoholic: 0.89
4.	Tannin	Ethyl acetate: Formic acid: Acetic acid: Water (100:11:11:26)	5 % FeCl₃in 0.1N HCl	Black	Petroleum ether :0.73 Ethanol: 0.6
5.	Steroids	Ethyl acetate: Methanol: Acetic acid (70: 20: 10)	Vanillin – Sulfuric acid.	Pink	Petroleum ether :0.89 Ethanol: 0.82 Hydroalcoholic :0.78
6	Sugar	Benzene: Glacial Acetic Acid: Methanol (20:20:60)	Anisaldehyde – Sulfuric acid	Brown	Ethanol: 0.84 Hydroalcoholic :0.80

Thin layer chromatography:

The R_f value and color helps in ascertaining the number of similar type of compounds present in the extracts. It results presence of Alkoloids carbohydrates, steroids, glycosides, flavonoids and Tannins (table-6).

CONCLUSION:

Achyranthes aspera has numerous uses in traditional system of medicine to treat several ailments like asthma, pneumonia, liver complaints, rheumatism, scabies, menstrual disorders, gastric disorders and skin diseases. Due to its wide significance it becomes necessary to standardize it for use as a drug. No pharmacognostical work on this plant has been reported so far. The present study is undertaken to lay down these standards. The phytochemical investigation showed the presence of sterols, proteins, flavanoids tannins, carbohydrates, alkaloids, glycosides, were identified. The work reveals standardization profile of drug Achyranthes aspera which would be very useful in botanical identification and authentication of plant drug and may help in preventing its adulteration

REFERENCE:

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Received on 22.01.2024 Modified on 17.02.2024

Asian J. Pharm. Tech. 2024; 14(2):103-107.

DOI: 10.52711/2231-5713.2024.00018

Sr. No.	Test	Observation	Inference
1.	T. S. + Phloroglucinol + Conc.HCl (1:1)	Pink Colour	Lignified Cells , Pericyclic Fibers, Stone Cells
2.	T.S. + Dil. Ruthenium red	Blue Colour	Starch Grains
3.	T.S. + Dil. Hydrochloric acid	Soluble	Calcium Oxalate Crystals
4.	T.S. + Dil. Acetic Acid	Insoluble	Calcium Oxalate Crystals

Qualitative Assessment of Achyranthes aspera Linn bark extract