Pharmacognostic Characterization and Phytochemical Screening of Achyranthes Aspera

Achyranthes aspera Linn. is a well-known plant drug in Ayurvedic, Unani-Tibbi, Siddha, Allopathic, Homeopathic, Naturopathic & Home Remedies. The present communication deals with the pharmacognostic evaluation on the different parts of Achyranthes aspera Linn. (Amaranthaceae). Macroscopic and microscopic examinations of the different organ, observations and differential microchemical test has been carried out for the authentication of the samples. Physiochemical values such as the moisture contents, percentage of total ash, acid insoluble ash, acid soluble ash, extractive values like petroleum ether-soluble extractives, ethanol-soluble extractives, methanol-soluble extractives and water-soluble extractives were calculated as well as colour reactions of powder and extract with different chemicals were performed. The extracts were subjected to qualitative screening test for various constituents. This revealed the presence protein, glycosides, alkaloids, tannins and phenolic compound, steroid reducing sugars and saponin glycosides. These observations will help in the Pharmacognostical identification and standardization of the drug in the crude form and also to distinguish the drug from its adulteration.

Achyranthes aspera is widely used for asthmatic cough, snakebite, hydrophobia, urinary calculi, rabies, influenza, piles, bronchitis, diarrhoea, renal dropsy, gonorrhoea and abdominal pain [3][4][5][6][7] .A powder of dried leaf mixed with honey is useful in the early stages of asthma 8 .One of the drugs from Siddha system of medicine, Naayuruvi kuzhi thailum has A. aspera as the primary constituent is reported to be quite effective in the management of asthma 9 .
Traditionally, the plant is used in asthma and cough.It is pungent, antiphlegmatic, antiperiodic, diuretic, purgative and laxative, useful in oedema, dropsy and piles, boils and eruptions of skin etc. Crushed plant is boiled in water and is used in pneumonia.Infusion of the root is a mild astringent in bowel complaints.The flowering spikes or seeds, ground and made into a paste with water, are used as external application for bites of poisonous snakes and reptiles, used in night blindness and cutaneous diseases 10 .

MATERIALS AND METHODS
The fresh, healthy, mature plants were collected from farm of Dhule away from pollution.The plant materials were identified using the Flora of Dhule and Nandurbar District 23 , Department of Botany, SSVP Sansthas, L.K.Dr.P.R. Ghogrey Science College, Deopur, Dhule-(M.S) India and herbariums were also preserved.The leaves, stem and roots were washed and used for the present study.The macroscopic observations of the mature plants, leaves, inflorescences and other parts were noted down.For microscopic studies, some plant material preserved in 70% alcohol.Leaf epidermal studies were carried out on fresh specimens.Peels were removed mechanically using some chemicals.They were stained in 1% safranin mounted in glycerin and made semi-permanent by ringing with DPX solution.Stomatal index (SI), stomal frequency, vein-islet, vein termination number and palisade ratio were calculated as defined by Salisbury 24,25 .

S SI E S = × +
Where 'S' = number of stomata per unit area and 'E' = number of epidermal cells in the same area.Stomatal index (SI), stomal frequency, vein-islet, vein termination number and palisade ratio have been calculated out of an average of 10 readings.
Transections of leaf, petiole, stem and root were taken by free hand.Fresh and preserved materials were used.Sections were stained in safranin (1 %), light green (1 %) and mounted in DPX after the customary dehydration.Some hand sections were also examined in glycerine.Microphotographs of leaf, petiole, stem and root sections were taken by using DIGI-EYE High resolution Cameras affixed to microscope Olymphus CH 20I.
The fresh, healthy plants were rooted out and washed with water.They were separated as root, stem and leaves and shade dried.The dried plant materials were pulverized into fine powder using a grinder (mixer).About 1 kg of powdered material was prepared.After that powder were kept into air tight bags.Physiochemical values such as the percentage of total ash, acid insoluble ash, acid soluble ash, extractive values like petroleum ethersoluble extractives, etahnol-soluble extractives, methanol-soluble extractives, and watersoluble extractives were calculated according to the methods described in the Indian pharmacopoeia 26,27 .
Phytochemical studies such as qualitative examination were done on the dried powdered material for this about 5gm of powdered material was extracted in soxhlet extraction apparatus with 250 ml of each of the following solvents; ethanol, methanol, diethyl ether, and chloroform 28 .The respected solvents were evaporated (at 40ºC) with the help of heating mantle.Some of the extracts of each solvent were used for the qualitative phytochemical screening for the identification of the various classes of active chemical constituents, using standard prescribed methods [29][30][31][32] .The positive tests were noted as present (+) and absent (-).
Physiochemical values such as the percentage of total ash, acid insoluble ash, acid soluble ash, extractive values like petroleum ethersoluble extractives, alcohol-soluble extractives and water-soluble extractives were calculated according to the methods described in the Indian pharmacopoeia 33,34 .

RESULTS AND DISCUSSIONS
The first step towards ensuring quality of starting material is authentication.Thus, in recent years there has been a rapid increase in the standardization of selected medicinal plants of potential therapeutic significance 35,36 .Despite the modern techniques, identification of plant drugs by pharmacognostic studies is more reliable.According to the World Health Organization, the macroscopic and microscopic description of a medicinal plant is the first step towards establishing the identity and the degree of purity of such materials and should be carried out before any tests are undertaken.The result of this study as follows:

Macroscopic characters
Plants are erect, woody, tall herb, leaves elliptical, ovet, elliptic lanceolate, 3-6 x 2-3.5cm.membranous, acut or rounded at base silky beneath, flower pale green in terminal, Simple and branched spike reaching 30cm long in fruit, utricles at apex, rounded at base, brown.The dried leaves had a characteristics odour and were slightly mucilaginous in taste.

Microscopic Characters Epidermal features
The epidermis cells are more or less straight and wavy walled with thick cuticle.The anticlinal epidermal cell walls are straight, arched or sinuous.The adaxial epidermal cells are large with thin walls as compared to the abaxial epidermal cells.The cells of lower epidermis are wavy walled single layered.Nonglandular trichomes and fglandular trichomes are observed on the surfaces but lower surface possess greater number of trichomes.The Glandular trichomes are with uni to tricellular stalk and unicellular head.The cells on the veins are generally elongated.The cells in the intercostals region are variously oriented.Stomata are present on the adaxial and abaxial surface of the leaf and are densely covered with trichomes.The shape of stomata is generally circular, oval or elliptical in outline.The stomata are anisocytic and anomocytic.

T. S. of Petiole
The petiole is crescent shaped in out line.
Single layered epidermis composed of thin-walled cubical to oval cells covered with thick cuticle.Stomata are few.Glandular and non-glandular trichomes are common.Epidermis is followed by multilayered hypodermis of collenchyma cells.Hypodermis is followed by wide zone of thin walled parenchymatous cortex containing calcium oxalate crystals.The three vascular bundle has found situated to mid region.Phloem consists of sieve tubes, companion cells and phloem parenchyma and xylem consists of vessels trachids and xylem parenchyma.

T. S. of Leaf
The leaf is dorsiventral and hypostomatic.The lamina shows a single layered epidermis on the upper side composed of cubical cells.The upper epidermis shows mostly uni, bi and multicellular hairs.Glandular hairs are rare.The epidermis is followed by a layer of hypodermis which is usually 3-5 layers of cells thick and is interrupted at places by the palisade layer.The cells of lower epidermis are cubical in shape mostly with uni -tricellular trichomes.Three vascular bundles are scattered in ground tissue consisting of thin layered parenchymatous cells.Vascular bundle consists of xylem vessels, trachids and xylem parenchyma, Phloem consists of sieve tubes, companion cells and phloem parenchyma and pericycle.The pericycle is made of 2-3 layers of thick-walled, nonlignified cells.

T. S. of Stem
A transverse section of stem have wavy cylindrical in outline and shows the presence of both glandular and non-glandular trichomes.Single layered epidermis is covered by thick cuticle; following the epidermis is a band of collenchyma of 4-8 layers of cells.Thin walled cork cells; pericycle a discontinuous ring of lignified fibres.The cortical zone is wide with uniform width, vascular tissues show anomalous secondary

T. S. root
In sectional view it is circular.Epidermis has small cells compactly arranged.Periderm formation is noted.Cork is 3-5 layered made up of rectangular cells compactly arranged in radial rows.Below it a few layered phelloderm.Ground tissue has phloem fibres, stone cells, oxalate crystals and starch grains.Secondary growth is more.Xylem developed in large amount.Medullary rays of 1-3 rows of cells.

Quantitative microscopy
The leaf microscopic characters like stomatal frequency, stomatal index, vein islet number, vein termination and palisade ratio were determined (Table 1).

Qualitative Phytochemical screening
The extracts and powder drug were subjected to phytochemical screening for the presences of type of phytoconstituets.The extracts and powder were found to contain protein, glycosides, alkaloids, tannins and phenolic compound, steroid reducing sugars and saponin glycosides (Table 2).

Powder Drug with Chemical Reagents
The powder drug with different chemical reagents show different colour when seen on naked eye.(Table 3)

Physical constants
Results of moisture contents, ash analysis and extractive values of the dried stem and root have been presented in Table 4.

CONCLUSION
The present study shows the major pharmacongonstic characters in the selected parts of species A. aspera.The extractive values are useful to evaluate the chemical constituents present in the crude drug and also help in estimation of specific constituents soluble in a particular solvent 37 .In the present investigation we observed the high extractive values in ethanol compared to other solvents.The pharmacognostical characters reported in this work can serve as a valuable source of information and provide suitable diagnostic tool for the standardization as well as identification of adulterants in future investigation or application.It will also be immense using carrying out further research and revalidation of its use.The microscopic features could help in laying down micro morphological standards as per WHO guidelines for authentication of the original drug.