preliminary phytochemical and pharmacognostical screening of the ayurvedic drug hygrophila...

O R I G I N A L A R T I C L EP H C O G J .

28 Pharmacognosy Journal | July 2011 | Vol 3 | Issue 23

*Address for correspondence:Mobile no: +91-9889902496E-mail: [email protected]

DOI: 10.5530/pj.2011.23.5

Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata

(K. Schum) Heine

Mohammed Sarfaraj Hussain*1, Sheeba Fareed1, Mohammed Ali2

1Faculty of Pharmacy, Integral University, Lucknow. 226026. India. 2Department of Pharmacognosy & Phytochemistry,Faculty of Pharmacy Jamia Hamdard, New Delhi 110062. India.

A B S T R A C T

The plant Hygrophila auriculata (K.Schum) Heine, (Acanthaceae), has been traditionally used for the treatment of

study of leaf, stem and root of Hygrophila auriculata

Hygrophila auriculata

crude drugs.

Key words: Hygrophila auriculata (K. Schum) Heine; Preliminary phytochemical screening; pharmacognostical

INTRODUCTION

Search for eternal health and longevity and to seek remedy to relieve discomfort prompted man to develop diverse ways and means of health care. The early man explored his immediate natural surroundings, tried many things like plants, animals and minerals and developed a variety of therapeutic agents. The knowledge gathered by generations was either documented or passed on to the posterity and this practice was generally termed as “Traditional Medicine”. The World

Health Organization (WHO) estimates that about 80% of the population living in the developing countries relies almost exclusively on traditional medicine for their primary health care needs. In almost all the traditional medicines, the medicinal plants play a major role and constitute the backbone of the traditional medicines. The modern (Allopathic) system of medicine is based on drastic cures through synthetic drugs and chemical compounds. It is noteworthy that since last twenty- ve years or so, a notable drop in the popularity of this system is noticeable. This trend can be attributed to the increasingly harmful side effects induced by some of these synthetic drugs. There is a famous line that says, “One man’s Aspirin is another man’s peptic ulcer”.[1] It is now known that chronic ailments, which require long-term treatments, are not always cured by allopathic drugs. Moreover, the synthetic drugs and intermediatery chemicals

Pharmacognosy Journal | July 2011 | Vol 3 | Issue 23 29

Hussain, et al.: Preliminary Phytochemical and Pharmacognostical Screening of the Ayurvedic Drug Hygrophila auriculata (K. Schum) Heine

and impotence and as an aphrodisiac.[4] The plant Hygrophila auriculata (K. Schum) Heine has been reported to contain

avonoids (apigenin 7-O- glucuronide, apigenin 7-O- glucoside),[5] alkaloids (asteracanthine and asteracanthicine),[6] aliphatic esters (25- oxo - hentricontyl acetate, methyl -8- hexyltetracosanoate),[7] minerals (Fe, Cu, Co),[8] sterols (Stimagsterol),[9] triterpenes (lupeol, hentricotane, betulin, luteolin, luteolin -7-O- rutinosides)[10,11,7] and essential oils. [6] Earlier scienti c investigation of Hygrophila auriculata (K. Schum) Heine showed that the crude extract has anti-nociceptive,[12] antitumor,[13,14] antibacterial,[15,16] antioxidant,[17,18] hepatoprotective,[19,20,21] hypoglycemic,[22] haematinic,[23] diuretic,[24] anabolic and androgenic activities,[25] anthelmintic activity,[26] anti-in ammatory and antipyretic activity.[27] Hence, the present study was designed to study the detail pharmacognostic study of the leaves, stem and roots including macroscopy, microscopy, quantitative microscopy, determination of various physicochemical parameters,

uorescence characters of powder, phytochemical screening and TLC and HPTLC pro le of extracts, and determination of total phenolic contents etc.

MATERIALS AND METHODS

Description of Hygrophila auriculata

(K. Schum) Heine

The plant is a sub shrub, usually growing in marshy places along water courses. The stem is reddish brown and the shoot has 8 leaves and six thorns at each node (Figure 1, 2).

are extremely expensive. For these and other reasons synthetic drugs are being widely replaced with the medicinal plants which are non-polluting renewable resources and are the only hope for sustainable supplies of cheaper medicines for the world’s growing population. Plants are also appreciated in pharmaceutical research as the major resource for new medicine and a growing body of medical literature supports the clinical ef cacy of herbal treatments. Today, about 40% doctors, especially in India and in China (the Mystic Orient) have reverted to increasing use of indigenous drugs and natural medicines. Steadily, a sizeable section of scientists in biological, biochemical and biomedicinal discipline have embarked on research on medicinal plants, which are the staple sources of many indigenous drugs.

Hygrophila auriculata (K. Schum)Heine (synonym: Asteracantha longifolia Nees, Barleria auriculata schum, Barleria longifolia linn) Acanthaceae, is a wild herb commonly found in moist places on the banks of rivers, ditches and paddy elds throughout India, Sri lanka, Burma, Malaysia, and Nepal. The plants are described in the ayurvedic literature as Ikshura, Ikshagandha and Kokilasha having eyes like the kokila or the Indian cuckoo. It is classi ed in the ayurvedic system of medicine as Seethaveryam, mathuravipaka and is used for the treatment of a number of conditions including Premeham (diabetes) and athisaram (dysentery).[2,3] A survey of the ethnobotanical literature shows that the roots, seeds, and aerial parts of the plant are widely used in the traditional system of medicine for the treatment of jaundice, hepatic obstruction, rheumatism, in ammation, pain, urinary infection, edema, gout, malaria,

Figure 1, 2: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Exomorphic features of the plant-Shoots showing axillary flowers, thorns, and cluster leaves.



Photomicrographs

Microscopic descriptions of tissues are supplemented with micrographs wherever necessary. Photomicrographs of different magni cations were taken with Nikon Labphot 2 microscopic unit. For normal observation, a bright eld microscopy was used and for the study of crystals, starch grains and ligni ed cells, a polarized light was employed. However, since these structures have birefringent property, under polarized light they tend to appear bright against the dark background. Magni cations of the gures are indicated by the scale-bars. Descriptive terms of the anatomical features are as per the standard anatomy books.[31]

Extraction and Fractionation

The air-dried plants of Hygrophila auriculata (K. Schum) Heine were prepared as a coarse powder and 500 g of this powdered material was macerated with distilled water and 95% w/v alcohol separately for 24 hrs and 72 hrs, respectively. Then, the individual extracts were ltered through a muslin cloth and the resultant ltrates were concentrated under reduced pressure and vacuum dried. The yield of aqueous extract was 43% w/w and that of the alcoholic extract was 29% w/w. The alcoholic extract was taken and suspended in water and successively fractionated with petroleum ether, chloroform, ethyl acetate and n-butanol.[32] The percentage yield of the petroleum ether, chloroform, ethyl acetate and n-butanol fractions was 5.78%, 3.93%, 2.09% and 1.19% w/w, respectively.

Quantitative investigation

Quantitative leaf microscopy to determine palisade ratio, stomata number, stomata index, Vein-islet number and vein-let termination number were carried out on epidermal strips. [31] Other parameters determined for the powdered drug of Hygrophila auriculata (K.Schum) Heine were uorescent analysis,[34,35] total ash, acid-insoluble ash, water-soluble ash, alcohol (90% ethanol) and water soluble extractive values, swelling indices and foreign matter.[34] Calibrated digital pH meter was used to measure the pH of 1 and 10% aqueous extracts and also loss on drying was noted.

Phytochemical screening and high performance

thin layer chromatography (HPTLC) fingerprinting

The preliminary phytochemical investigation of the aqueous and other fractions of Hygrophila auriculata showed the presence of avonoids, terpenoid saponins, and tannins as the major phytoconstituents. In particular, the chemical tests on the n-butanol fraction demonstrated the presence of tepenoids. It has been shown that the plant Hygrophila auriculata is unique with regard to its content of avonoids and terpenoids.[35] A distinct chemopro le of the terpenoid fraction of Hygrophila auriculata (K. Schum) Heine was obtained using HPTLC. Precoated TLC plates of silica-gel 60 F254 (E. Merck, India), 0.2 mm thick, were used. Ten

The leaves occur in whorls, the outer pair of leaves are larger, lanceolate, scalerous, margins are minutely dentate, subsessile, thorns strong straight or curved. Flowers occur in axillary whorls, bract and bracteoles leafy. Calyx four lobed, lobes unequal. Corolla, -5 petals gamopetalous, unequally 2- lipped, middle lobe of the lower lip with yellow palate; corolla purple coloured. Stamens - four, in two pair,

laments unequal; anthers divergent; ovary two celled; four ovules in each cell. Fruit dehiscent capsule.

Collection of Plant materials

The fresh plants of Hygrophila auriculata (K. Schum) Heine were collected from the eld area of Baryahi, Saharsa District Bihar, India, in January 2009. The plant specimen was authenticated by Dr. Anjani kumar Sinha, Principal, M L T Saharsa College Saharsa, Saharsa, Bihar. A voucher specimen no SHC 55/01/2009 has been deposited at the herbarium, Department of Dept. of Botany, M L T Saharsa College Saharsa- 852201. Care was taken to select healthy plants and for normal organs. The required samples of different organs were cut and removed from the plant and xed in formalin acetic acid solution (formalin: acetic acid: 70% ethyl alcohol in the ratio of 0.5:0.5:9). After 24 h of xation, the specimens were dehydrated with graded series of tertiary-Butyl alcohol as per the schedule given by Sass, 1940.[28] In ltration of the specimens was carried by gradual addition of paraf n wax (melting point 58-60 °C) until thiobarbituric acid solution attained super saturation. The specimens were casted into paraf n blocks.

Preparation of sections

The paraf n embedded specimens were sectioned with the help of a rotary microtome. 10-12 m thickness of the sections was made. However, dewaxing of the sections was done using customary procedure.[29] The sections were later stained with toluidine blue as per the method published by O’Brien et al. (1964).[30] Since toluidine blue is a polychromatic stain, the staining was remarkably good and yielded varied cytochemical reactions. The dye rendered pink color to the cellulose walls, blue to ligni ed cells, dark green to suberin, violet to the mucilage, blue to the protein bodies etc. However, where necessary, sections were also stained with safranin, fast-green and iodine-potassium iodide for starch. In studying the stomatal morphology, venation pattern and trichomes distribution, paradermal sections taken parallel to the surface of leaf were cleared with 5% sodium hydroxide and epidermal peeling was prepared through partial maceration by employing Jeffrey’s maceration uid.[28] Cleared sections were then mounted with glycerin for microscopical observation. Moreover, powdered materials of different parts of the plant were also cleared with sodium hydroxide and mounted in glycerin medium after staining.



median vertical plane and 1 m in horizontal plane. In the adaxial part, the epidermis is prominent with squarish cells and prominent cuticle. Beneath the epidermis occur about three layers of small collenchyma cells. Further below the collenchyma occur four or ve layers of wide thin walled parenchyma cells. The abaxial part of the midrib has epidermis similar to adaxial side. These may be one or two layers of

microliters of the n-butanol fraction was spotted in the form of a band using a CAMAG Linomat -V Automatic Spotter (CAMAG, Switzerland) (Figure 14). The TLC pattern of the n-butanol fraction was developed using n- hexane: ethyl acetate (7:3) as a solvent system.[38,39,40] Then, the plates were scanned in the CAMAG TLC Scanner-III and the peaks were recorded at a wavelength of 366 nm. The TLC and HPTLC ngerprinting studies of the n-butanol fraction showed the presence of various terpenoids with their respective Rf values (Table 1). The observed Rf values from TLC agreed with the HPTLC ngerprints.

Determination of total phenolic contents

The total phenolic content was determined according to the method described by Singleton (1999).[41] A suitable aliquot of the TRF was placed in test tubes and made up to 1 ml with distilled water. Then, 0.5 ml Folin-Ciocalteu reagent (1:1 with water) and 2.5 ml sodium carbonate solution (20%) were added sequentially to each tube. Then, the tubes were vortexed for 2 min, kept in the dark for 40 min and the absorbance was recorded at 725 nm. The amount of total phenolics was calculated as gallic acid equivalents/mg of extract (Table 2).

RESULTS AND DISCUSSION

Anatomy of Leaf

The leaf is dorsiventral, smooth and even with fairly prominent midrib (Figure 3). The midrib is Plano convex in sectional view with at adaxial side and broadly semicircular abaxial side (Figure 4). The midrib is 750 m along the

Figure 3: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Anatomy of the leaf - T.S of Leaf through midrib with lamina.

Table 1: TLC profile of n-butanol fraction of Hygrophila auriculata (K. Schum) Heine

S. No. Solvent system Rf value of track-1

Rf value of track-2

1 n- Hexane:Ethyl acetate (7:3)

0.37 0.352 0.45 0.393 0.76 0.544 0.84 0.645 0.94 0.70

Table 2: Determination of total phenolic contents of different extracts/fractions of Hygrophila auriculata (K. Schum) Heine

Total phenolic contents

Extracts/ Fractions Phenolic contents (μg/ml)

Aqueous 70.0Ethanolic 98.0Pet ether 65.54Chloroform 81.0Ethyl acetate 120.0n- Butanol 135.0

Figure 4: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: T.S of Midrib enlarged.



Cystolits

Calcium carbonate crystals of cystoliths are abundant in the adaxial epidermis of the leaf (Figure 5 and 9). The cystoliths are long, spindle shaped, straight or curved with warty surface. They are 200 m long and 20 m thick. They occur is specialized cells which are elongated and candle like; these modi ed cells in which the cystoliths occur are called lithocysts.

Stomata

Occur on both surfaces of the leaf. They are equal in abundance and frequency in the upper and lower sides (Figure 8, 9). The stomata frequency is 45-50/ m2. The stomata are predominantly diacytic type with two subsidiary cells with their common walls at right angles to the stomatal axis. The subsidiary cells may be equal or unequal in size. The epidermal cells are variable in shape; they are narrowly rectangular, slightly lobed or squarish. Their anticlinal walls are fairly thick, straight or slightly undulate. Some of the epidermal cells are elongated, narrow and canal like. These lithocysts contain calcium carbonate cystoliths (Figure 8). Glandular trichomes are occasionally seen in the epidermis. They are circular in surface the cells darkly staining (Figure 10).

Petiole

In cross-sectional view, the petiole is wide at and boat shaped. The adaxial side is at and abaxial side is convex (Figure 11). The petiole has lateral, thick, abaxially hanging wings on either side. The vascular system consists of a at wide main bundle and two wing bundles. The main

collenchyma inner to the abaxial epidermis. The remaining ground tissue consists of wide, compact, thin walled parenchyma cells. The vascular bundle is single and elliptical in cross - sectional view. It is 350 m horizontally and 150 m vertically. It consists of 8-10, parallel rows of xylem elements which are angular, thin walled and narrow. Phloem occurs as a thin sheath along the abaxial side of the xylem. These are two small, less prominent, circular accessory strands on the adaxial part. They are circular with a cluster of xylem elements and small nest of phloem elements.

Lamina

The lamina is uniform in thickness excepting the places where lateral vein’s and veinlets are situated. The lateral view is raised slightly on the adaxial and abaxial sides. It consists of a small top-shaped collateral vascular bundle surrounded by parenchymatous cells which extend both adaxially and abaxially (Figure 5, 6). The lateral vein is 400 m thick. The vein lets also project slightly on the lower side. They have small cluster of xylem and phloem with parenchymatous bundle sheath without extensions.

The mesophyll is differentiated into adaxial zone of palisade cells and abaxial spongy mesophyll tissue. The palisade zone consists of a single row of thin pillar like cells which are 100 m in height. The spongy mesophyll has four or

ve layers of small, lobed cells which are interlinked with each other forming wide aeranchymatous tissue. The adaxial epidermis is 30 m thick. The abaxial epidermis is 20 m thick. Both of them are stomatiferous (Figure 7).

Figure 5, 6: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: T.S of lamina through lateral vein.



phloem (Figure 12). The ground tissue is homogeneous and parenchymatous. The cells are wide, circular, thin walled and compact.

Anatomy of Stem

The stem is roughly four angled in sectional view with wide arenchymatous cortex and four angled stele (Figure 14). The epidermis is thin and less conspicuous. The outer

bundle is placed in the control part of the petiole. It is at measuring 950 m long and 250 m thick (Figure 13). It consists of several short, radial rows of xylem elements, the xylem elements are 2 or 3 in each row; they are angular to circular and thick walled (Figure 11). Phloem occurs in smallest beneath the xylem strands. The wing bundle are circular, nearly 150 m is diameter and have four or

ve short rows of xylem elements and a small patch of

Figure 7: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: T.S of Leaf margin.

Figure 9: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Abaxial epidermis with stomata.

Figure 10: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Glandular trichomes and lythocyst enlarged.

Figure 8: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Epidermal morphology - Adaxial epidermis with stomata and cystolith, under low magnification.



widely separated radial rows xylem vessels and thin arc of phloem. The pith is wide and parenchymatous. The pith cells are circular less compact and thin walled.

cortex consists of four or ve layers of radially aligned, small, compact squarish parenchyma cells. This zone is uniformly 150 m wide. The inner cortex in quite wider comprising of about ve rows wide, circular air chambers formed by reticulate layers of narrow aerenchyma cells. The stele has four semicircular thicker bundles placed at four corners and two smaller bundles positioned opposite to each other. The larger and smaller bundles are interlinked by a thin cylinder of small compact, dense xylem elements. The vascular bundles are collateral with dense xylem bers,

Figure 11: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Anatomy of the petiole - T.S of Petiole ground plane.

Figure 13: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Central median portion vascular bundles enlarged.

Figure 14: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Anatomy of the Stem - T.S of stem – ground plane.

Figure 12: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Wing portion of the petiole enlarged.



vein terminations are long and slender, branched or unbranched.

Abaxial epidermis fragments are seen which are stomatiferous. The stomata are diacytic type. The epidermal cells are small polygonal and thin walled; the walls are straight (Figure 18).

Anatomy of Root

The root has intact, continuous rhizodermis (epidermis) followed by two layers of tangentially oblong compact outer cortex. The inner cortex is wide and aerenchymatous. Wide, radially elongated air– chambers are formed by thin, uniserate partition laments, made up of thin walled parenchyma cells. Some of the partition cells have thick walled, dilated and squarish rectangular (Figure 15, 16). The vascular cylinder has a thin endodermal layer and pericyclic layer.

ylem consists of ve exarch strands and a few wide angular vessels in between the exarch strands. Phloem is in ve small groups alternating with the primary xylem strands. The central Part is narrow and parenchymatous (Figure 16).

Powder microscopy of Hygrophila auriculata

(K. Schum) Heine.

The macerated and powdered materials exhibited the following components:

Organoleptic properties of powder drug

Colour - Brawnish greenOdour - OdourlessTaste - Tasteless

Small fragments of lamina shows the venation patterns and vein lets with vein terminations (Figure 17). The vein lets are prominent and form distinct vein-islets. The Vein-islets are square shaped or rectangular. The

Figure 15: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Anatomy of the root - T.S of root a sector enlarged.

Figure 16: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: T.S of root entire view

Figure 17: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Leaf venation pattern showing vein islet and vein termination.



are multicellular, uniseriate, unbranched and smooth walled. The marginal trichomes are slightly curved (Figure 17) while surface trichomes are straight. The trichomes are up to 1 cm long and 100 m broad at the base. The powdered drug shows vessels element, xylem

bers and xylem parenchyma. Vessel elements are long, cylindrical and thin walled (Figure 21). The vessel elements are 250 m long and xylem bers may be wide

Cystoliths: Large, cylindrical cystoliths are abundant in the mesophyll and epidermis. They are broad at one end and narrow at the other end. The surface of the cystolith is warty or spiny. The cystolith is 200 m long and 30 m thick at the base (Figure 18).

Epidermal trichomes (Figure 19, 20). Covering types of trichomes are frequently seen the powder. The trichomes

Figure 18: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Abaxial epidermis showing stomata and cystolith

Figure 21: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: One vessels element with tail.

Figure 20: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Surface straight trichomes

Figure 19: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Marginal curved trichomes



ash, loss on drying, swelling index, and foreign matter are presented in table 5.

Phytochemical screening and high performance

thin layer chromatography (HPTLC) fingerprinting

The phytochemical and pharmacognostical investigation of the plant Hygrophila auriculata (Schum) was carried out with standard protocol. Earlier literature review of the plant Hygrophila auriculata (K. Schum) Heine revealed that no work has been carried out with regard to pharmacognostical studies except for its taxonomical identi cations. The extraction of plant material was carried (cold maceration) out with water and 95% w/v alcohol. The yield of aqueous extract is 37% w/w and alcoholic yield is 21% w/w. Both aqueous and alcoholic extracts and different fractions revealed that presence of various phytoconstituents like

avonoids, terpenoids saponins, and tannins as major phytoconstituents (Table 6). It has been shown that the plant Hygrophila species is unique for avonoids and terpenoids

and narrow they are thick walled. The bers 450-600 m in length (Figure 22).

Xylem parenchyma: Narrowly rectangular, thin walled parenchyma cells are seen bundles. They are 140 m long. They have no pits (Figure 22).

Quantitative investigation of Hygrophila auriculata

(K. Schum) Heine

The quantitative determination of some pharmacognostic parameters is useful for setting standards for crude drugs. The vein islet, and vein termination numbers and the other parameters determined in the quantitative microscopy, are relatively constant for plants and can be used to differentiate closely related species. Quantitative microscopy was performed using standard procedures (Table 3). The uorescence analysis of the powdered drug of Hygrophila auriculata (K. Schum) Heine in various solvents and chemical reagents was performed under normal and Ultra Violet (UV) light (Table 4). The physico-chemical characters of powdered drug of Hygrophila auriculata (K. Schum) Heine such as total alcohol soluble extractive, water soluble extractive, ash value, acid insoluble ash, water-soluble

Figure 22: Macro- and micrscopical characters of the plant Hygrophila auriculata (K. Schum) Heine: Fibres

Table 3: Quantitative microscopy Hygrophila auriculata (schum) Heine

Parameters Range

Palisade ratio 14.45Stomatal number Upper surface 21.78Stomatal number Lower surface 24.26Stomatal index Upper surface 15.4Stomatal index Lower surface 25.4Vein- islet number 27.4Veinlet termination number 29.2

Table 4: Flourescence analysis of Hygrophila auriculata (K. Schum) Heine

Solvent used Day Light UV light

254 nm 366 nm

1N HCl Light green Green colour Dark green50% HCl Light green Green colour Dark green50% HNO3 Light brown Dark green Blackish

green50% H2SO4 Slightly red Slight green Light green1N NaOH Light yellow Dark green Moderate

greenAlcoholic NaOH Light green Green colour Dark greenMethanol Light green Dark green Slightly pinkBenzene Yellow Slight buff GreenAcetone Brownish

greenFluorescent green

Transparent

Ethyl acetate Light brown Dark green Light yellowChloroform Slight green Green Dark greenFeCl3 Brownish

yellowWhite Green

1% KOH Brown Green Dark greenLead acetate White Florescent

whiteWhite

Distilled water Clear Green Green

Table 5: Physico-chemical analysis of Hygrophila auriculata (K. Schum) Heine

Quantitative parameter Values obtained (%) w/w

Alcohol soluble extractive 5.12Water soluble extractive 24.96Total ash 9.90Acid insoluble ash 1.48Water – soluble ash 8.35Loss on drying 6.30Swelling index 2.0Foreign matter 1.10



n-butanol fraction showed presence of various terpenoids with their respective Rf values. The preliminary HPTLC studies revealed that the solvent system n- hexane: ethyl acetate (7:3) was ideal and gave well resolved sample peaks. The spots of the chromatogram were visualized at 366 nm (Figure 23) with a 500 k lter at Rf values of 0.07, 0.36, 0.50, 0.57, 0.73, 0.84, 0.94 and 0.97. The densitometric

contents. To fractionate the above compounds alcoholic extract was fractionated with various solvents from petroleum ether, chloroform, ethyl acetate and ethyl acetate insoluble fraction was fractioned with n-butanol. Phytochemcial test on n-butanol fraction showed that presence of terpenoids and thus n-butanol fraction was labeled as terpenoid fraction. Further TLC and HPTLC nger printing studies on

Figure 23: HPTLC Finger printing of n-butanol fraction where peaks (1-8) represent Presence of various terpenoids scanned at wavelength 366 nm.Abbreviations: Abs- Abaxial side; GT- Ground Tissue; La- Lamina; LV- Lateral vein; MR- Midrib; Ade-Adaxial epidermis; Adab- Adaxial accessory bundle; Col- Collenchyma; EP-Epidermis; Ph- Phloem; GT-Ground tissue; PM-Palisade mesophyll; X- Xylem; Abe- Abaxial epidermis; Ads-Adaxial side, BS- Bundle sheath extension; LV-Lateral vein; Ph-Phloem; St- Stomata; SM-Spongy mesophyll; SC Subsidiary cells; Cy- Cystolith; GTr- Glandular trichomes; LC- Lithocyst; GT- Ground tissue; VB- Vascular Bundles; W- Wing; WB- Wing bundles; AC- Arenchymatous inner cortex; OC- Outer cortex; Pi-Pith; Co-cortex; SPh- Secondary Phloem; SX- Secondary Xylem; CO- Cortex, Ve- Vessels, XR- Xylem ray; SX- Secondary xylem; LV- Lateral vein; VT- Vein termination; PP- Perforation plate, T- Tail; X P- Xylem Fibres, XF- Xylem fibres

Table 6: Phytochemical analysis of various extracts/fractions of Hygrophila auriculata (K. Schum) Heine

Phytoconstituents Aqueous extract

Alcohlic extract

Pet-Ether fraction

Chloroform fraction

Ethyl acetate fraction

n-butanol fraction

Alkaloids – + – + – –Steroids – + + + – –Flavonoids + + – – + –Terpenoids + + – – – +Tannins + + – – + –Protein – – – – – –Carbohydrate + + – – – –Saponins + + – – – –Glycosides – – – – – –

– Negative = Absent + Positive = Present



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18. Hussain MS, Nazeer Ahamed KFH, Ravichandiran V and Ansari MZH. Evaluation of in vitro free radical scavenging potential of different fractions of Hygrophila auriculata (K.Schum) Heine. Asian J of Trad Med. 2009; 5(2):51-59.

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21. Shanmugasundaram P and Venkatraman S. Hepatoprotective effect of Hygrophila auriculata (K.Schum) Heine root extract. J Ethanopharmacol. 2006; 104(1-2):124-128.

22. Fernando MR, Nalinie Wickramasinghe SMD, Thabrew MI, Ariyananda PL and Karunamayake. Effect of Artrocarpus Heterophyllus and Asteracantha logifolia on glucose tolorence in normal human subject and in maturity onset diabetic patients. J Ethanopharmacol. 1991; 31(3):277-282.

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24. Hussain MS, Nazeer Ahamed KFH, Ansari MZH. Preliminary Studies on Diuretic Effect of Hygrophila auriculata (Schum) Heine in Rats. Inter J of Health Res. 2009; 2(1):59-64.

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27. Patra A, Jha S, Murthy N, Roy D, Vaibhav A, Chattopadhyay P, Panigrahi G. Anti Inflammatory and Antipyretic Activities of Hygrophila spinosa T. Anders Leaves (Acanthaceae). Tro J of Pharm Res. 2009; 8(2):133-137.

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scanning at 366 nm gives ve major spots with an area of 16.87, 20.46, 12.26, 22.33 and 31.40% at Rf value of, 0.57, 0.73, 0.84, 0.94 and 0.97 respectively.

Determination of total phenolic contents

These extracts and fractions were found to possess various phenolic levels in table 2 ranging from 70 to 135 ( g/ml). The highest concentration of total phenolics was present in ethyl acetate and n-butanol fraction of Hygrophila auriculata (K. Schum) Heine. The content of total phenolic compounds in ethyl acetate fraction was 120 g/ml and n-butanol fraction was 135 g/ml.

CONCLUSION

In conclusion, the pharmacognostic investigations on physicochemical characteristics and uorescence analysis shows that authentic botanical of this crude drug prevents adulteration, substitution and has a crucial role in standardization of crude drugs. The preliminary phytochemical screening of the whole Hygrophila auriculata (K. Schum) Heine indicates the presence of secondary metabolities, having an essential role in medicine. The current report will also help researchers and scientists design strategies for resolving cases of misidenti cation of plant material. However advance experimentation is under way in our laboratory to elucidate the speci c mechanism of action of Hygrophila auriculata (K. Schum) Heine.

ACKNOWLEDGEMENT

The authors are thankful to the Dr. K.F.H. Nazeer Ahmaed, Assistant Professor, department of pharmacology, Vel’s college of pharmacy, Chennai, for his assistance and encouragement. We extend our sincere thanks to Dr. Anjani kumar Sinha, Principal, M. L. T. Saharsa College Saharsa, Saharsa, Bihar, for providing authenticated sample of Hygrophila auriculata (K. Schum) Heine. We also extend our sincere thanks to Mrs. Saba Ansari, research scholar, faculty of pharmacy, Integral University, Lucknow, India, for critically reading the manuscript and providing the valuable suggestions.

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preliminary phytochemical and pharmacognostical screening of the ayurvedic drug hygrophila...

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