INTRODUCTION:

Centella asiatica widely known as Brahmi and Withania somnifera known as Ashwagandha are considered as ‘Medhya rasayanas’ in Ayurveda meaning brain tonic. They are being widely used in various ayurvedic formulations to promote intellect and memory as well as in various nervine disorders by virtue of their active constituents.¹ Brahmi is known as longevity herb and used widely in India and Nepal as part of the traditional Ayurvedic medicine. It consists of dried whole plant of Centella asiatica (Linn.) Urban, belonging to the family Apiaceae (Umbelliferae).² The plant is indigenous to warmer regions of both hemispheres. In India it is abundant in tropical and subtropical regions particularly in damp, shady places along marshy banks of rivers, streams, ponds, irrigated fields.

In Himalayas, it grows wild in natural habitat all round the year. It is particularly found in Gujarat and West Bengal. Its roots and leaves are used for medicinal purposes and provide important health benefits related to healthy veins and blood vessels, to treat skin disorders, help with better memory and improve brain function.⁴ Eating a few leaves daily was thought to strengthen and revitalize worn out bodies and brains.⁵ Brahmi was used similarly as Bacopa monniera, it might be interesting to mention that many early literature interchanges both plant names, although they are botanically unrelated.

Ashwagandha, is a valued herb in Ayurveda and cultivated for centuries in India. It consists of dried mature roots of Withania somnifera (Linn.) Dunal belonging to family Solanaceae. This plant grows wildly in all dry parts and subtropical India. It occurs in Madhya Pradesh, Uttar Pradesh, Punjab plains and North Western parts like Gujarat and Rajasthan.⁶ It is an ingredient in many formulations prescribed for a variety of musculoskeletal conditions (e.g., arthritis, rheumatism), and as a general tonic to increase energy, improve overall health and longevity, and prevent disease in athletes, the elderly.⁷ The scientific classification of the plants is mentioned in Table 1.^8-9The present work was undertaken to generate information regarding relevant pharmacognostical, phytochemical and microbiological data needed for proper identification and authentification of two crude drugs belonging to two different families, mainly focusing and exploring the aspects leading to common effect of improving memory.

Table 1: Plant Profile

Scientific Classification	*Centella asiatica*	*Withania somnifera*
Kingdom	Plantae	Plantae
Division	Spermatophyta	Spermatophyta
Sub-Division	Angiospermae	Angiospermae
Class	Dicotyledonae	Dicotyledonae
Sub Class	Polypetalae	Gamopetalae
Series	Calyciflorae	Bicarpellatae
Family	Umbelliferae	Solanaceae
Genus	Centella	Withania
Species	asiatica	somnifera
Synonym	Hydrocotyle asiatica	Physalis somnifera, P. flexuosa, P. arborescence

MATERIALS AND METHODS:

The whole plants of Centella asiatica and Withania somnifera were procured from different places of Valsad District (Gujarat) in the month of October and November and authenticated by Botany Department, The Zandu Pharmaceutical Works Ltd. (Now Emami Limited). Vapi, Gujarat, India. A voucher specimen (14474-Brahmi, 14441-Ashwagandha) was retained for further references.

Fresh entire plant of brahmi and dried roots of ashwagandha were used for pharmacognostic evaluation and some preliminary phytochemical tests. For the anatomical study, leaf, petiole, stem, root of fresh brahmi plant were collected, sectioned and stained with safranin.¹⁰ Leaf constants and quantitative microscopic measurements were carried out using ocular and stage micrometer using fresh leaves of plant after removal of pigments. Powder of the whole plant of brahmi and ashwagandha dried root were used for the observation of powder microscopic characters.¹¹

Photomicrographs were obtained by observing free hand sections of drug under Digital Binocular Motic microscope (DMB series PAL System) with built in analogue camera. Computer Images were captured using Motic Image Software (VQCI038). Physicochemical constants, phytochemical analysis, and chromatographic studies were carried out from shade-dried powder following prescribed methods.^12-15TLC studies were carried out on silica gel plates (Silica gel 60 F₂₅₄). Extract for TLC was prepared by extracting 2 g drug powder in 30 ml methanol by heating on water bath for 1 hour. It was filtered and concentrated to 1-2 ml which was further used for TLC study. Samples were spotted on TLC plate using Toluene-Ethyl acetate-Acetic acid (6:4:0.5) as mobile phase. TLC plates were sprayed Anisaldehyde- sulphuric acid reagent, dried over at 105°C for 10 minutes. Microbiological tests were carried out to determine total viable bacterial count(TVC), Total yeast and mould count(YMC), presence of coliform E.coli, Salmonella, Staphylococcus and pseudomonas using standard procedures.^16,17

RESULTS:

MACROSCOPY:

C.asiatica is a prostrate, perennial herb with slender, glabrous, pink stem, rooting at nodes giving rise to thin, brownish-grey, roots of about 2.5 to 6.0 cm in length; leaves 1 to 3 from each node, orbicular-reniform, crenate, base cordate, petioles channelled with adnate stipules; flowers fascicled umbels each carrying 3 or 4 flowers, short stalked; fruits are oblong, dull brown, with laterally compressed seeds, the pericarp hard, thickened, woody and white. [Figure 1-A, B, C]

Figure 1: A- Habitat of Brahmi; B- Entire Brahmi Plant ; C- Brahmi Leaf

Ashwagandha is a medium height bushy plant that can easily be seen growing in moderate to warm climatic condition. Branches are circular and are widely spread in all the directions. It is a perennial herb that attains a height up to 1.5 meters. Stem and branches are covered with small star shaped hairy structures. The roots can be one long tuberous root or may be many. Leaves are simple and attain a height of 10 centimeters. It is ovate, petiolate and alternate. The plant flowers throughout the year. The flower is small and greenish to yellow in color. Fruit is small in size having about 0.5 cm diameter, globose, smooth, red enclosed in an inflated and membranous calyx. [Figure 2-D,E,F]

Figure 2: D –Ashwagandha Plant; E- A twig with flowers and fruit; F- Roots

MICROSCOPY:

C.asiatica shows greyish green leaves with stomata on both surfaces, mostly rubiaceous type. Palisade cells differentiated into two layers of cells, spongy parenchyma of about three layers of cells with many intercellular spaces, some with crystals of calcium oxalate; Small-sized leaves usually hug the ground and have a short petiole stem; however, large leaves can have a petiole up to 20cm long. When plants are grown in the shade, they tend to have large leaves and very long petioles. This petiole stem can have a pink/purple tinge. Petiole shows epidermis with thickened inner walls, collenchyma of two or three layers of cells, a broad zone of parenchyma; seven vascular bundles within parenchymatous zone, two in projecting arms and five forming the central strand; vessels 15–23μm in diameter. Some parenchymatous cells contain crystals of calcium oxalate. Midrib region shows two or three layers of parenchymatous cells without chloroplastids. Pink flowers 5mm across, usually set two to four, side by side, as an umbel, developing from the stem nodes. Flowers are so small and often hidden underneath leaves. Fruits shows several ridges in outline, epidermis of polygonal cells, each ridge having a vittae and patch of sclerenchyma, trichomes similar to the leaves, sheets of elongated parquetry layer cells, bundles of narrow annular vessels, and parenchymatous cells contain single large prisms of calcium oxalate. Macerates show presence of vessels with spiral, annular and reticulate thickenings. [Figure 3- G- O]

Figure 3: G-T.S. of Brahmi leaf, H-Brahmi leaf vascular bundles, I- Brahmi leaf(collenchyma cells, J- Brahmi leaf stomata, K- T.S of leaf Petiole, L- T.S of Root, M- T.S of root showing Secondary growth, N- T.S. of root showing reticulate and spiral vessels, O- T.S. of brahmi stem.

Transverse section of ashwagandha root shows cork exfoliated or crushed, when present isodiamatric and non-lignified, cork cambium of two to four diffused rows of cells, secondary cortex about twenty layers of compact parenchymatous cells, phloem consists of sieve tubes, companion cells, phloem parenchyma, cambium 4-5 rows of tangentially elongated cells, secondary xylem hard forming a closed vascular ring separated by multiseriate medullary rays, a few xylem parenchyma which contains plenty of starch grains.[Figure 4-P,Q,R,S]

Figure 4: P-T.S.of Ashwagandha root; Q- Multiseriate medullary rays; R-Xylem parenchyma; S- Phloem parenchyma

POWDER MICROSCOPY:

The powder of Centella asiatica is greyish green with characteristic odour and slightly bittersweet taste. When powder was mounted with chloralhydrate, phloroglucinol –HCl , stained with safranin it shows vessels with spiral, reticulate and annular thickening; microsphenoidal and rosette crystals of calcium oxalate; simple, oval to round starch.[Figure 5-T,U]

Figure 5: T- Spiral vessels; U- Rosette crystals of calcium oxalate

The powder of ashwagandha roots is pale yellow in colour with characteristic odour and bitter acrid taste. It shows presence of large number of starch grains-simple, spherical, oval with distinct central hilum. Cork cells cortical and xylem parenchyma, trachieds, vessels with pitted thickening, wood fibres. [Figure 6- V - Z]

Figure 6: V- Starch grains; W- Starch grains packed in cells; X-Xylem Parenchyma; Y- Tracheids; Z-Xylem vessel with pitted thickenings

DISCUSSION:

The physicochemical studies showed the average value of moisture content of brahmi and ashwagandha 9.34 % w/w and 7.03 % w/w indicating presence of high moisture content in brahmi. [Table 2]. Total ash and acid insoluble were found in higher amount in brahmi (4.99 % w/w and 1.32 % w/w) indicating presence of inorganic radicals like carbonates, phosphates, silicates, silica etc. The higher acid insoluble ash value indicates the contamination with siliceous materials like earth and sand. The water soluble extractive value of both the crude drugs were found almost similar but the alcohol soluble extractive value of ashwagangha (11.35% w/w) was found higher than brahmi (9.86 % w/w). The pH of aqueous solutions of both the crude drugs were found to be slightly acidic. Phytochemical analysis showed presence of alkaloids, saponins, glycosides, carbohydrates, proteins, amino acids, fatty acids and starch in both crude drugs. Brahmi also showed presence of phenolic compounds, tannins and volatile oil [Table 3]. The various leaf constant values of brahmi were measured [Table 4]. These values are especially useful for identifying two different crude drugs and also helpful in the determination of the authenticity of the plant. The results of microbiological tests showed presence of coliform and cocci bacteria in both the crude drugs whereas presence of Staphylococcus and pseudomonas in ashwagandha. The TVC and YMC were found to be 2,70,000/g and 3000/g for brahmi and 23,00,000/g and 10,00/g for ashwagandha which were found to be higher than standard values[Table 5]. Methanolic extracts of both crude drugs showed five spots each after spraying Anisaldehyde-sulphuric acid reagent indicating presence of glycosides [Figure 7]. The R_f values are tabulated [Table 6].

Table 2: Results of Physicochemical Analysis

Parameters	Results
Parameters	*Centella asiatica*	*Withania somnifera*
Total Ash	4.99 % w/w	4.87 % w/w
Acid Insoluble Ash	1.32 % w/w	0.46 % w/w
Water Soluble Extractive	20.04 % w/w	20.88 % w/w
Alcohol Soluble Extractive	9.86 % w/w	11.35% w/w
LOD	9.34 % w/w	7.03 % w/w
pH of 1% solution	5.49	5.68

Table 3: Results of Phytochemical Analysis of Ethanolic extract of Centella asiatica and Withania somnifera

Phytochemicals	*Centella asiatica*	*Withania somnifera*
Alkaloids	+	+
Carbohydrates	+	+
Protiens	+	+
Amino acids	+	+
Glycosides	+	+
Saponins	+	+
Tannins	+	−
Steroids	−	+
Phenols	+	−
Fatty acids	+	+
Volatile oil	+	−

+ = Present, − = Absent

Table 4: Results of quantitative analysis of Leaf constants of Centella asiatica

Parameters		Results
Palisade Ratio		1:5
Stomatal Size	Upper Epidermis	Length of Stomata : 88.04 µ
		Breath of stomata : 21.42 µ
	Lower Epidermis	Length of Stomata : 64.26 µ
		Breath of stomata : 21.42 µ
Stomatal number		Upper Epidermis : 7/0.10 mm²
		Lower Epidermis:16/0.10 mm²
Stomatal Index		Upper Epidermis : 15
		Lower Epidermis:28
Vein islet number		15/2.38 mm²

Table 5: Results of Microbiological Evaluation

Parameters	Results
Parameters	*Centella asiatica*	*Withania somnifera*
Total Viable Bacterial count (TVC)	2,70,000/g	23,00,000/g
Total Yeast and Mould Count (YMC)	3000/g	10,000/g
Coliform Bacteria	+	+
E.Coli	−	−
Salmonella	−	−
Staphylococcus	−	+
Pseudomonas	−	+

+ = present, − = Absent

Figure 7: a- TLC of Ashwagandha ; b- TLC of Brahmi

CONCLUSION:

Pharmacognostic, phytochemical, physicochemical and microbiological evaluation of Centella aiatica (whole plant) and Withania somnifera (roots) provided specific parameters that will be useful in scientific evaluation, identification and authentification of both the crude drugs. Leaf constants, microscopic characteristics, physicochemical values and phytochemicals are important demarcating characters.

ACKNOWLEDGEMENT:

The authors are thankful to the authorities of The Zandu Pharmaceutical Works Ltd. (Now Emami Limited), Vapi, Gujarat for evincing interest in this work.

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Received on 05.06.2011 Modified on 15.06.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1707-1713

Conditions	*Centella asiatica*		*Withania somnifera*
Conditions	No. of Spots	R_fValue	No. of Spots	R_fValue
UV 254 nm	-	-	3	0.12,0.25,0.41
UV 366 nm	1	0.68	-	-
After-spraying Anisaldehyde H₂SO₄ Reagent	5	0.28,0.48,0.56, 0.68, 0.85	5	0.02,0.21,0.36,0.57,0.71