INTRODUCTION:

Premna serratifolia Linn., ( Verbenaceae ) is known as ‘Headache tree’ or Agnimantha in Ayurveda. It is a coastal big aromatic shrub cum small tree grows upto 9 meter often spinous, branchelets unarmed¹. It is common along Indian and Andaman coasts, in the plains of Bombay, Gujerat and Kashi hill where it grows under mesophytic conditions². It is a main ingredient in Dasamularistam, Dhanvantaram, Kasayam, Agastyarasayanam, Sukumaraghrtam etc. Traditionally it is highly valued for anti-inflammatory property. It overcomes kapha and vata disorders, piles and abdominal diseases³. Infusion of leaves is used in eruptive fever, colic, flatulence⁴. Decoction of root is good for liver complaints, cardio tonic, gonorrhoea, during convulsions from fever, rheumatism and neuralgia. ⁵

The earlier phytochemical studies of this plant revealed the presence of β – sitosterol, Betulin in stem bark, luteolin in leaves, Aphelandrine, ganiarine, ganikarine, Premnenol, Premnaspirodiene, sandaracopimar – 8 (14), 15-diene in roots⁶.

Eventhough the plant is rich in bio-active richness active constituents and potential therapeutic activities there is a lacuna in the Pharmacognostical standardization on the root of Premna serratifolia Linn. So the present study is undertaken to produce some Pharmacognostical standards for the roots.

MATERIAL AND METHODS:

The fresh plant material of Premna serratifolia Linn., was collected from Plant medicinal centre at Pondycherry and authentified by Prof. Jayaraman, Botanist, Director, Plant Anatomy Research Centre, Thambaram. The herbarium specimen bearing the voucher number MMC/ 2008/11 is kept in the Department for the future reference.

Macroscopic and Microscopic analysis:

The macroscopic characters such as colour, odour, taste, nature, texture etc were studied. For anatomical investigation, customary technique of microtomy was followed⁷. Paraffin sections of 10µm thick were stained with safranin – fast green. Photomicrograph were taken with NIKON lab photo – Microscopic unit⁸

Physicochemical studies:

The ash values, extractive values, crude fibre content, loss on drying and foaming index were performed according to the official methods prescribed in Indian Pharmacopoeia and the WHO Guidelines on quality control methods for medicinal plants materials ^9,10. Fluorescence analysis was carried out according to the method of Chase and Pratt¹¹ and Kokoski et al¹².

Preliminary Phytochemical screening:

The behavior of powder with various chemical reagents and preliminary chemical tests for extracts were also carried out according to the standard procedures described by Kokate¹³ and Horborne¹⁴.

Table1-Quantitative microscopy of root of Premna serratifolia Lin., (Verbenaceae).

Parameters	Values in µm (Average Of 40 Measurements)
Parameters	Minimum	Average	Maximum
FIBRE LENGTH	501	757	1256
FIBRE WIDTH	2.5	4.0	5.5

RESULTS:

Macroscopy:

The plant is a small tree growing free 6 – 10 m in height. It is common in costal shrub, jungles and also grown in gardens. The leaves emit a foetid smell. Leaves are elliptic ovate measuring 5 -10 cm long and 3 – 6 cm broad. The lamina is thin and coriaceous. Leaf margins are crenate. Inflorescence is terminal corymbs. Calyx is samospalous, minute by 5 to bed. Corolla five petals, gamopetalous, two lipped. Stamens four, epipetalous. Ovary four locular, one ovule in each locule. Fruit drupe, hard, two seeded.

Fig. 1.1. Anatomy of young root entire view

Microscopic features of the root:

The microscopies of young and old lateral roots were studied. The root has a superficial periderm, fairly wide cortex, a continuous cylinder of secondary phloem and dense, solid secondary xylem. (Fig. 1.1,2)

In a young root, the periderm is 150 µm wide. It consists of seven or eight layers of thin walled tabular, suberised phellem cells. (Fig. 2.1, 3.1). the cortex is fairly wide and has narrow, tangentially elangated, ellipitical cells. Secondary phloem is wide and continueous. The phloem rays are not prominent.

In the old root, the periderm is wider, deeply fissured and is 300 micrometer wide and homogeneous( Fig.2.2). the periderm consists of radially oblong or transversely tabular, thin walled suberised phellem cells ( Fig. 3.2 ) The secondary phloem zone is wide and has compact radial files of phloem elements, no ray dilation is seen.

Secondary xylem ( Fig. 2 and 3 ):

Secondary xylem consists of vessels, fibres, prominent rays and seanty parenchyma. The distribution of vessels is characteristic. The vessels are wide, fairly thick walled, circular to ovate. Solitary or aggregates of two vessels. The vessels are arranged in more or less regular circles, each circle having almost uniform diameters the circular arrangement of the vessels is more evident in the thick roots the narrow vessels are 40 µm wide, the longer vessels are 70 µm wide.

Fig. 1.2. Anatomy of old root half portion enlarged

Table: 2Physicochemical Constants of roots Premna serratifolia Lin., (Verbenaceae).

S. No.	Parameters	Percentage %w/w
I.	ASH VALUES
1.	Total ash	9.56
2.	Water soluble ash	1.22
3.	Water insoluble ash	1.08
4.	Acid soluble ash	1.39
2.	Acid insoluble ash	0.886
4.	Sulphated ash	6.7
II.	SOLUBILITY
1.	Ethanol soluble extractive	24.00
2.	Water soluble extractive	18.40
3.	Ether insoluble volatile	13.01
4.	Ether insoluble non-volatile	32.00
III.	LOSS ON DRYING	2.6
IV.	ESSENTIAL OIL CONTENT	1.4
V.	CRUDE FIBRE CONTENT	50
VI.	FOAMING INDEX	33.3

Fig. 2.1. T.S of young root a sector enlarged

Xylem fibres are thick walled and lignified. Fibre distribution is random. At the beginning of each ring of vessels occurs a thin line of parenchyma cells, which also have fairly thick walls and but wider lumen. Xylem rays

Table - 3Fluorescence Analysis of Various Extracts of roots of Premna serratifolia Linn.,

S. No	Extracts	Day Light	UV Light
S. No	Extracts	Day Light	Short (254 nm)	Long (365 nm)
1.	n-Hexane	Yellow	Green Fluorescence	Brown
2.	Chloroform	Dark Yellow	Reddish Brown	Brown
3.	Ethyl Acetate	Brownish Yellow	Brown Fluorescence	Dark Brown
4.	Ethanol	Reddish Brown	Yellow Fluorescence	Brown
5.	Aqueous	Brown	Dark Brown	Brown Fluorescence
6.	Powder + 1N NaOH	Greenish Yellow	Dark greenish yellow	Greenish yellow
8.	Powder + ferric chloride	Pale yellowish green	Black	Yellowish green
9.	Powder + Acetic acid	Creamish yellow	Dark black green	Dark greenish yellow
10.	Powder + Ammonia	Pale greenish yellow	Dark brown	Dark greenish yellow

are one or two cells wider. They are straight and prominently visible in transactions.

Fig. 2.2. T.S of old root a sector enlarged

Powder microscopic observations:

In the macerated / powdered preparation of the root, the following elements were observed.

Fig. 3.1. Periderm in the young root and Fig. 3.2. Periderm in the old root

1. Vessel elements: ( Fig. 4 and 5 ):

The vessel elements are common in the powder. They are mostly cylindrical, wide and elongated. Short, cylindrical vessel elements are also occasionally seen. The vessel elements have tails at one end ( Fig.5.1,2) or at the both ends ( Fig. 5.3 ). They have simple circular perforation plate, which is horizontal or slightly oblique ( Fig.5.1.2 ). The lateral walls are densely pitted, the pits are circular, alternate and multiseriate ( Fig. 5.3 ). The vessel elements are 260 µm long.

2. Xylem fibres: ( Fig. 4.1,2 ):

Libriform fibres are dominant component of the powder. They are thick walled and the walls are lignified. The lumen of the fibres is wider in the mid portion and becomes narrow at the both ends. Lateral wall pits are not evident. The fibres range in length from 350 – 700 µm.

3. Xylem parenchyma:

Parenchyma cells are less frequent in the powder. They are rectangular to square shape. They have comparatively thin walls and dense, simple pits.

4. Starch grains: ( Fig: 5.1,2):

The powder was stained with IKI and unstained powder was observed under the polarised light microscope. When stained with IKI, these were starch grains which were sporadic. They are mostly spherical and have concentric hilum. A dark + mark was observed in the starch grains ( Fig: 2.1 arrow ) which indicates polarimark with starch grains which possess birefringent property. Starch grains vary in size from 30 – 70 µm in diameter.

These are also large pieces of xylem ( vessel ) elements with dense, multiseriate, circular lateral wall pits.

Fig. 3.3 Xylem and Xylem fiber enlarges.

5. Crystals:

Calcium oxalate crystals of spindle shaped bodies are abundant in the xylem fibres. They are densely packed inside the cells. The crystals have pointed ends and dilated central part. The crystals are 10 µm long and 5 µm thick (Fig: 6.1 )

Quantitative Microscopic analysis:

Quantitative analysis was also performed by staining the fibre with phloroglucinal and the length, width of the fibres are tabulated in Table – 1

Fig. 4.1. Vessel element and fibres. Fig: 4.2. Fibres

Physiochemical constants:

The physicochemical parameters are mainly used in judging the purity and quality of the drug. Ash values of a drug give an idea of the earthy matter or inorganic composition or other impurities present along with the drug. The ash values of the powdered leaves revealed a high percentage of sulphated ash. Extractive values give an idea about the chemical constituents present in the drug as well as useful in the determination of exhausted or adulterated drugs. The results suggest that the powdered leaves have high water soluble extractive value. The loss on drying reveals the percentage of moisture present in the drug, the crude fibre content and foaming index were also studied and presented in the table (Table No.2).

Fluorescence analysis of drug powder and extracts:

The fluorescence analysis of powdered leaves was studied in both UV and day light. The powder showed green fluorescence with methanol in UV light at 254nm, which indicates the presence of chormophore in the drug (Table No.3,3.1).

Preliminary phytochemical test for extracts and powder:

All the extracts namely hexane, chloroform, ethanol and aqueous were tested with various reagents and results are presented in the Table 4

Fig. 5.1. .Tailed vessel element and fibre.

DISCUSSION:

Pharmacognostical studies of root of Premna serratifolia Linn., has brought to light microscopic features as well as preliminary phytochemical data of diagnostic values. Anatomy of the plant sometimes proves helpful for identification of fragmentary samples. The plant family descriptions of the collective microscopical data of young and old roots have proved to be simple technique of identification. Generally it has a superficial periderm, fairly wide cortex, a continuous cylinder of secondary phloem and dense, solid secondary xylem. Transverse section revealed it showed slight variance between young and old root. In a young root, the periderm is 150 µm wide. It consists of seven or eight layers of thin walled tabular, suberised phellem cells. The cortex is fairly wide and has narrow, tangentially elongated, ellipitical cells. Secondary phloem is wide and continuous. The phloem rays are not prominent where as in old root the periderm is wider, deeply fissured, 300 µm wide and homogeneous. The periderm consists of radially oblong or transversely tabular, thin walled suberised phellem cells. The secondary phloem zone is wide and has compact radial files of phloem elements, no ray dilation is seen.

Secondary xylem consists of vessels, fibres, prominent rays and seanty parenchyma. The distribution of vessels is characteristic (Fig. 1.1.2 ). Solitary or aggregates of two vessels and arranged in more or less regular circle almost uniform diameter which is more evident in the old roots ( Fig: 1.2 )

Xylem fibres are thick walled and lignified random distribution. At the beginning of each ring of vessels occurs a thin line of parenchyma cells, fairly thick walls and but wider lumen. Xylem rays are one or two cells wider. They are straight and prominently visible in transactions.

Fig. 5.2. Tailed vessel elements and parenchyma cells

Fig. 5.3. Vessel element showing lateral wall

Table - 4 Preliminary Phytochemical Test For Different Extracts Of root Of Premna serratifolia Linn., (Obtained By Successive Solvent Extraction).

S. No.	Test	Powder	n-Hexane	Chloroform	Ethanol	Aqueous
1.	Alkaloids	+	-	+	-	-
2.	Glycosides	+	-	+	+	+
3.	Terpenoids	+	+	+	+	-
4.	Carbohydrates	+	-	-	-	+
5.	Proteins	+	-	-	-	+
6.	Steroids	+	+	-	+	-
7.	Flavonoids	+	-	-	+	-
8.	Phenols	+	-	+	+	+
9.	Tannins	+	-	+	+	+
10.	Quinones	+	-	+	-	-

Note: + ve indicates positive result, whereas – ve indicates negative result.

In the macerated / powdered preparation of the root, the following elements were observed vessel elements are seen as dense, multiseriate and circular lateral wall pits tailed at one or both end. Lignified Libriform fibres wider at mid and becomes narrow at both ends. Xylem parenchyma is thin wall, dense and simple pits. IKI Starch grains are mostly spherical, concentric hilum and dark + polarimark which has peculiar birefringent property. Spindle shaped, pointed end, dilated central part calcium oxalate crystals are abundant in the xylem fibres.

Fig. 6.1. Starch grains under low magnification.

Fig. 6.2. Starch grains and pieces of xylem elements, lateral wall pits.

Quantitative microscopic data such as measurement of fibre content has been highly relied upon by pioneer pharmacognost. It is believed that these features are constant for given species and can be employed for interspecific identity of drugs. Physiochemical constants such as solubility, successive extractive values and other parameters of the drugs are corroborative evidences in drug standardisation. The drug powder exhibits specific colour reaction when mixed with different reagent, thereby indicating the presence and absence of different compounds in the drug. As shown in the table 2. the drug powder and extracts of Premna serratifolia was found to contain alkaloid, flavonoid, terpenoid, steroid, resin and phenols etc. fluorescence analysis of the drug powder as well as drug extracts is an another test for standardising the drug for the presence of chromophores. Thus, anatomical characters coupled with preliminary

phytochemical results are specific for the woody roots of Premna serratifolia Linn.,

Fig. 7.1: Starch grains showing + mark in the grains (←).

Fig. 7.2. Crystals in the xylem fibres.

CONCLUSION:

Establishing standards is an integral part of establishing the correct identity and quality of a crude drug. Before any drug can be included in the Pharmacopoeia these standards must be established. The majority of the information on the identity, purity and quality of the plant material can be obtained from its macrscopy, microscopy and physico-chemical parameters. As there is no record on Pharmacognostical work on roots of Premna serratifolia Linn., the present work is undertaken to produce some pharmacognostical standards The above studies provide information in respect of their identification, chemical constituents and physicochemical characters which may be useful for pharmacognostical study and standardization of herbal drugs of folk medicinal practice of present era and enrichment of Ayurvedic Pharmacopoeia. It will also determine therapeutic diagnostic tools for the scientists who are keen and sincere to evaluate the herbal medicine of indigenous resources.

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10. WHO/PHARM/92.559/rev.1, Quality Control Methods for Medicinal Plant Materials Geneva:Organization Mandiale De La Sante, Geneva, 1992; pp.9, 22-34.

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Received on 31.01.2009 Modified on 28.02.2009

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 180-185

S. No.	Treatment	Day Light	UV Light
S. No.	Treatment	Day Light	Short (254 nm)	Long (365 nm)
1.	Powder	Pale yellowish brown	Dark greenish brown	Pale greenish brown
3.	Powder + 1N HCl	Dark greenish brown	Black colour	Dark green colour
4.	Powder + 1N HNO₃	Pale yellow	Dark greenish brown	Pale greenish brown
5.	Powder + 1N H₂SO₄	Dark Reddish brown	Dark Brownish black	Pale Greenish brown