INTRODUCTION:

In latest decades there has been a wide development in interest for drugs of herbal origin sources in divergence to the synthetics which are viewed as hazardous to population and environment ^[1].Medicinal plants and traditional systems of prescription have been constantly connected as therapeutic plants are spine of these systems.^[2]. India is a rich in natural heritage legacy of traditional systems of medicine.

The advancement of these traditional systems of medicines with the points of view of safety, efficacy, and quality will encourage to safeguard the traditional heritage as well as to justify the utilization of natural products in healthcare^[3]. Duranta repens L. (Syn. Duranta plumieri Jacq.) (Verbenaceae) is local to clean and open forests in the West Indies, the northern region of Pakistan, and Central and South-America. It is utilized as an ornamental plant as a part of numerous tropical nations^[4,5]. There are around 35 Duranta species, evergreen bushes conveyed in tropical and sub-tropical areas. Duranta repens was presented in Egypt as an ornamental plant^[6]. Duranta repens (Golden dewdrop) otherwise called sky blossom, angels-whisper, additionally called Katamehedi. Duranta is an upright to hanging bush that occasionally takes the type of a scrambling bush or once in a while a little tree^[7,8]. Literature proposes that Duranta repens L. has been accounted for Antishigellosis Cytotoxic Potency^[9], antimalarial action^[10]and Antiviral Activity^[11]. The pharmacological significance was noted due to the presence of various bioactive compounds. Plant is reported to produce C-alkylated flavonoids, steroids, flavonoids, terpenoidsand triterpenes^[12-14]. As of late reported six known Phytoconstituents alongside beta-sitoserol, naringenin^[15], sucrose, raffinose, acetosides, triterpenes saponins ^[16], and steroidal glycosides^[17]. From the class Duranta a few iridoid glycosides as durantosides I, II, III, IV and lamiide were isolated^[18]. New constituents has been isolated for first time from the species, for example, coumarinolignoids, Repenins A–D, cleomiscosin A, and durantin A^[19]. In view of the various medicinal constituents and utilizations credited to Duranta repens L, there is no pharmacognostic report on the leaves of the plant to decide the anatomical and other physicochemical guidelines required for quality control of the crude drug material. Thus this work was undertaken to assess different pharmacognostic parameters like macroscopic and microscopic, powder characteristics and physicochemical properties of Duranta repens L.

MATERIALS AND METHODS:

Procurement of Chemicals:

All the chemicals utilized were of analytical grade and were obtained from Loba chemicals Limited India Mumbai, India.

Procurement of plant materials:

Fresh leaves of Duranta repens were collected from nearby cantonment territory of St John Institute of pharmacy and research Palghar. Identification of the plant was done by Dr. Vinayak Naik, Senior Botanist Primal health care. A specimen sample (DEL-08) is deposited in the Dept. of Pharmacognosy, at St. John Institute of Pharmacy and Research, Palghar, Maharashtra.

PHARMACOGNOSTIC STUDIES:

Organoleptic evaluation:

Diverse sensory parameters of the plant material, (Colour, Odor, Size, Shape, and Taste) were examined by organoleptic evaluation.

Macroscopic evaluation:

Diverse macroscopic characters of fresh leaves of Duranta repens L.were documented, type of leaf base, presence or absence of petiole and characters of lamina. Lamina comprises of characteristic elements, for composition, incision, shape, venation, margin, apex, base, surface and texture.

Qualitative microscopy:

In this study, transverse sections of leaf were examined under microscope (10X and 40X). Staining reagents (Phloroglucinol- Hydrochloric) were employed according to standard techniques ^[20-23]. The different distinguishing characters were observed with or without staining and images were recorded.

Leaf microscopy:

In this study, leaf was plunged in chloral hydrate solution for several hours until it lost its pigments. The cube of leaf was selected and vertically cut, leaf was embedded and fine sections were obtained. Fine sections mounted on glass slide with help of glycerin without any staining reagent utilized was set under microscope. Various dermal characters, such as type of trichomes, epidermis cells, and stomata were identified. ^[24] To observe vascular tissue transverse sections was stained by reagent 0.1% w/v phloroglucinol took after by concentrated hydrochloric acid. The stained and unstained sections were observed under microscope^{[25, 26]}. Diverse layers of cells and recognizing characters were observed.

Powder microscopy:

Dried leaves of Duranta repens L. was powdered and studied under microscope. Diverse staining reagents, were used for location of starch grains, calcium oxylate and identification of lignified tissue components. A little amount of dried leaf powder was taken onto a slide; 1-2 drops of 0.1% w/v phloroglucinol and a drop of concentrated hydrochloric acid were included and secured with a cover slip. The slide arrangement was mounted in glycerol and inspected under microscope. The vicinity of starch grain and calcium oxalate crystal was distinguished by the solution of blue shading on expansion of 2-3 drops of 0.01 M iodine solution^[27]. The characteristic structures and cell parts were watched and their photos were taken utilizing photomicrography.

Quantitative microscopy:

Determination of stomatal number and stomatal index:

Stomatal number is the average number of stomata per square millimeter of epidermis. The percentage proportion of the definitive divisions of the epidermis of a leaf which can be converted into stomata is termed as stomatal index. Stomatal index can be calculated by using following equation: I = S / E + S X 100, where, I = Stomatal index, S = number of stomata /mm² and E = number of ordinary epidermal cells / mm². A piece of leaf was cleaned and the upper and lower epidermis was peeled out was peeled out independently by method for forceps and mounted in glycerin Prism type Camera Lucida was attached and black cardboard sheet was set for drawing the cells. A square of 155 mm²by means of stage micrometer was drawn on it. The slide mounted cleared leaf was placed on the stage and the epidermal cells and stomata were traced. The number of stomata and the number of epidermal cells in each field were counted. The numbers of stomata were counted as stomatal number and the stomatal index utilizing the above formula was calculated independently for upper and lower surface.

Determination of vein-islet and vein termination Number:

Vein islet is the minute area of photosynthetic tissue enclosed by the ultimate division of the conducting patterns. Vein termination number is the number of veinlet terminations per mm of leaf surface. A piece of the leaf was cleared by boiling in chloral hydrate solution and camera Lucida and drawings board were arranged and 1 mm line was drawn with help of stage micrometer. A square was constructed was developed on this line in the focal point of the field. The slide was set on the stage. The veins included inside of the square were traced off, finishing the diagram of those islets which cover two conformity side of the square. The average number of vein islet from the four adjoining square, to get the value for one square mm was calculated ^[28]. The number of veinlet termination present inside of the square was counted and the average number of veinlet termination number from the four adjoining square to get the value for 1 square mm was discovered known as vein termination number.

Determination of palisade ratio:

A piece of the leaf was boiled in chloral hydrate and was placed under microscope. Camera Lucida and drawing board were arranged and the outline of were orchestrated and the layout of four cells of the epidermis was traced utilizing 4 mm objective. At that point, palisade layer was focused down and adequate cells for covering the epidermal cells were traced off. The layout of those palisade cells which were converged by the epidermal walls was completed. The palisade cells under the four epidermal cells (counting cells which are more than half and barring cells which are not as much as half inside of the territory of epidermal cells) were counted. The determination for five groups of four epidermal cells from various part of the leaf was repeated. The average number of cells underneath epidermal cells was calculated known as palisade ratio^[29].

Physiochemical analysis:

The physicochemical constant for example percentage of Total Ash value, Acid-Insoluble Ash value, Water soluble Ash value. Water soluble extractive value, Alcohol soluble extractive value and Petroleum ether soluble extractives were determined. Moisture content were figured according to the WHO guidelines^[30].

Preliminary Phytochemical analysis:

Fresh shade dried leaves of Duranta repenes Linn. Was powdered and extracted by Ethanol using Soxhlet apparatus. The ethanolic extract was concentrated under reduced pressure utilizing a rotating evaporator and dried in vacuum. A portion of the crude ethanolic extract was suspended in water and fractionated with petroleum ether, chloroform, and ethyl acetate. All fractions, including the aqueous fraction, were concentrated under reduced pressure using a rotary evaporator and dried in vacuum and subjected to phytochemical screening^[^31-33].

Fluorescence analysis:

Fluorescence analysis of powder of dried leaves of Duranta repenes Linn. was done by standard methodology^[34]. In this investigation dried leaf powder were treated with various acidic and basic solvents and were then observed in UV / visible chamber under visible, short wave and long wave regions simultaneously^[35,36] Fluorescence is an imperative wonder displayed by different phytoconstituents show fluorescence in the visible range in day light. The UV light delivers fluorescence in many natural products which don't obviously show fluorescence in day light. In the event that the substances themselves are not fluorescent, they might regularly be changed over into fluorescent derivatives by applying distinctive reagents thus some unrefined crude drugs are often assessed qualitatively along these lines and it is an essential parameter of pharmacognostic assessment ^[37]. The changes in appearance and colour were observed and recorded.

RESULTS:

The macroscopic evaluation involved study of morphological characters. The organoleptic features of the leaves indicates dark green colour on both surfaces. The morphological characters were seen as deciduous, leaf base is decurrent and symmetric petiole. The composition of lamina was observed to be simple, pinnate reticulate venation, entire margin, acuminate apex, subribs alternate, surface glabrous and texture subcoriaceous. The size of leaves varied from 1.5 to 4.6cm in length and 2.4cm in breath (Figure 1).

Figure. 1: Macroscopical characteristics of Duranta Repenes Linn. Leaf

The microscopical characters shows lamina of the leaf was dorsiventral sort. The upper epidermis comprised of little polygonal parenchymatous cells which seemed wavy in diagram and after epidermis, chlolenchyma cells were available. The chlolenchyma cells were further seen as upper palisade and lower spongy tissue. The palisade with a solitary layer of customary, long, columnar cells, underneath which, 3 to 4 layered mass of firmly stuffed cells loaded with chloroplast was available. The cortex comprised of 4 to 5 layers of paranchymatous cells in the midrib region. The vascular bundle isovoid in shape, collateral and beneath them a zone of sclerenchymatous tissues was present. In the middle of the upper epidermis and the vascular bundle, 5 to 6 layers of sporadic formed collenchyma cells were available. Xylem cells (lignified) were seen on the upper side though phloem was seen towards the lower side of the epidermis. There were 2 to 3 layers of pericycle cells in the middle of the xylem and phloem. The phloem comprised of strainer tubes, partner cells and phloem parenchyma while xylem comprised of scalariform xylem vessels and parenchyma. The uniseriate covering and unicellular trichomes were available on both surfaces of leaf, more along the midrib district and less along the lamina. The covering trichomes were simple, uniseriate, unbranched and unicellular with blunt apex and smooth walls (Figure 2).

Figure. 2: Transverse section of Duranta Repenes Linn. Leaf through midrib

The powder of leaf appears dark green in colour with coarse texture, unpleasant odour with bitter taste. Powder microscopy demonstrates presence of trichomes, lignified fibers, xylem vessels and calcium oxalate crystals of tetragonal type were observed which have been presented in (Figure 3).

Repenes Linn. Leaf

A- Tracheid B - Xylem

C -Epidermis cells D- Lignified fibers

E - Xylem vessels F- Calcium oxalate crystals

G -Phloem Fibers H- Covering trichomes
I - Anisocytic Stomata

Figure. 3: Powder microscopy of Duranta

Figure 4a: Stomatal Figure 4b: Vein Islet Figure 4c: Palisade Number Vein Terminals Ratio

Uantitative microscopy showed Anisocytic sorts of stomata were available on the lower surface of leaves. The stomatal number of upper surface and lower surface was found as 122.5 and 245, separately. The stomatal indexes of upper surface and lower surface were found 10.17 and 5.56, respectively from (Figure 4a). The vein islet and vein termination were calculated as 18 and 39 from (Figure 4b). The palisade ratio was observed to be 4.4 from (Figure 4c)

Physicochemical parameters essential in quality control of crude drug were also determined and were inside the official limit results are tabulated (Table 1 and 3). The consequences of fluorescence investigation are displayed in Table 4. Standardization is an essential device for natural medications keeping in mind the end goal establish their identity and purity.

Table 1: Physicochemical parameters of Duranta repenes Linn. Leaves

Sno.	Parameters	Leaf
1.	Loss on drying	3.9 ± 0.25
2.	Total ash	9.6 ±0.58
3.	Water soluble ash	6.0 ± 0.54
4.	Acid insoluble ash	7.3 ± 0.23
5.	Petroleum ether soluble extractive value	1.49 ± 0.60
6.	Acetone soluble extractive value	5.17±0.93
7.	Alcohol soluble extractive value	14.76 ± 0.87
8.	Aqueous soluble extractive value	8.74 ± 0.42

Table 2: Quantitative microscopy of Duranta repenes Linn. Leaves

Characteristics	Values (Average value of 3 replicates ± SD)
Stomatal Number	122.5 UE
Stomatal Number	245 LE
Stomatal index	10.17 ± 0.22 UE
Stomatal index	05.56 ± 0.35 LE
Vein Islet no	18.00 ± 0.41
Vein Terminal no.	39.31 ± 0.63
Palisade Ratio.	4.4 ± 4.6

Table 3: Qualitative phytochemical analysis of ethanolic extract of Duranta repenes Linn. Leaves

Sr.No.	Phytochemicals Test Crude powder	Ethanolic Extract
1.	Alkaloids Dragendroff’s test	+++
2.	Mayer’s test	++
3.	Wagner’s test	++
4.	Hagers’s Test	_
5.	Flavonoids Alkaline reagent	_
6.	Flavonoids Shinoda Test	_
7.	Tannins FeCl3 test	_
8.	Phlobatanins Hcl test	_
9.	Triterpenes H2SO4 test	+
10.	Steroids Liebermann and Burchard test	++
11.	Saponins Frothing test	+
12.	Cardiac glycosides Keller-kilianni test	_

-: Absent; +: Less present; ++: Moderate present; +++: High present

Table 4: Florescence analysis of powdered leaves of Duranata repenes Linn

Sr. No	Reagents	Visible	Short ultra violet	Long ultra violet
1.	Chloroform	Dark green	Fluorescent green	Red
2.	Methanol	Dark green	Fluorescent green	Red
3.	Pet ether	Pale green	Green	Red
4.	Ethyl acetate	Green	Fluorescent green	Orange
5.	50% Sulphuric acid	Green	Dark green	Black
6.	50% Nitric acid	Green	Black	Green
7.	50% Hydrochloric acid	Green	Black	Green
8.	10% Sodium hydroxide	Green	Black	Green
9.	Ethyl acetate:	Dark green	Dark green	Black
10.	Hydrochloric acid (1:1)	Green	Green	Orange
11.	Acetone	Pale green	Green	Orange

DISCUSSIONS:

With a specific end goal to standardize a natural medication, various macroscopic, microscopic, fluorescence examination are completed. Microscopic method is one of the least expensive and most straightforward strategies to begin with building up the right distinguishing proof of the source material. Morphological and microscopical investigations of the leaf will empower to distinguish and identify the crude drug. The quantitative determination of some pharmacognostic parameters is helpful for setting measures for crude medications Stomatal number, stomatal index value and palisade ratio, vein islet and vein termination value determination are just as imperative in the assessment of crude drugs. These qualities help in the evaluation of purity of drugs. The information obtained from preparatory phytochemical screening will be valuable in discovering the nature and quality of the drug. Morphological and microscopic studies of leaves go about as a solid guide for detecting adulteration. These studies can likewise help the manufacturers for identification and selection of the raw material. Taking everything into account, the parameters which are accounted for here can be considered as enough to identify and decide the authenticity of this drug in herbal industry.

ACKNOWLEDGEMENTS:

The authors would like to thank Principal, St John Institute of Pharmacy and Research for generous support and providing necessary facilities to carry out the present research work. We sincerely express gratitude to Mr. Albert W. D’Souza, Chairman Adel Education Trust for his motivation and encouragement.

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Received on 30.08.2017 Modified on 10.09.2017

Res. J. Pharmacognosy and Phytochem. 2017; 9(3): 141-146.

DOI: 10.5958/0975-4385.2017.00026.7