4. PHARMACOGNOSTIC STUDY 4.1...

CHAPTER 4 PHARMACOGNOSTIC STUDY

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Pharmacognostic, Phytochemical and Pharmacological studies on

Clerodendrum splendens. Family- Verbenaceae. 22

4. PHARMACOGNOSTIC STUDY

4.1 Introduction

Quality control standards of various medicinal plants used in indigenous systems of

medicines are becoming more relevant today in view of commercialization of

formulation. The quality control parameters for the crude drugs as raw materials were

established with the help of several official determinations based on morphology,

microscopy, and physico-chemical studies. These studies were aimed at ensuring

standardization of herbal drug under investigation.

Botanical authentication of a drug depends entirely on macroscopical and microscopical

characters. Each crude drug derived from the vegetable kingdom consists of a definite

part of the plant.

Ash values are helpful in determining the quality and purity of crude drugs in powdered

form. The total ash usually consists of inorganic substances like carbonates, phosphates,

silicates and silica of sodium, potassium, magnesium, and calcium. Sometimes inorganic

variables like calcium oxalate, silica, carbonate content of crude drug affects ‘total ash’

values, such variables are then removed by treating with acid (as they are soluble in

hydrochloric acid) and then acid insoluble ash value is determined (Mukherjee, 2002).

Ash value found to be high water soluble ash value was found to be greater than the acid

insoluble ash value, it indicates that the crude drugs contains low amount of inorganic

variables, like calcium oxalate, silica and carbonate content. The value of moisture

content was found within limits which indicate good stability of the carious crude drugs

used.

Ash value found to be high water soluble ash value was found to be greater than the acid

insoluble ash value, it indicates that the crude drugs contains low amount of inorganic

variables, like calcium oxalate, silica and carbonate content. The value of moisture

content was found within limits which indicate good stability of the carious crude drugs

used. Hence, process of standardization can be achieved by stepwise pharmacognostic

and phytochemical studies helping proper identification of crude plant material.


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4.1.1. Collection and authentification of the plant

Plant parts of Clerodendrum splendens G. Don was collected from Nashik district of

Maharashtra in February 2011 and later on authenticated by Botanical Survey of India,

Pune, where herbarium voucher specimen No. SBP-1 has been deposited.

Figure 6: Authentication certificate of plant Clerodendrum splendens.


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4.1.2. Pharmacognostic study of Leaves of Clerodendrum splendens

4.1.2.1. Macroscopic study

Procedure:

Macroscopy of the leaves of Clerodendrum splendens was studied according to the

methods of Brain and Turner, 1975.

a. Size – Length : 6 -12 cm.

Width: 2- 6.5 cm.

b. Shape – The leaves are opposite, simple with an entire margin. Leaf is petiolate.

c. Color – Dark green colored from inner side and pale green colored from outside.

d. Surface characters – Texture is smooth.

e. Odor – Characteristic.

f. Taste – Slightly bitter.

(Figure 7)


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Figure 7: Macroscopic characteristic of Clerodendrum splendens Leaf.


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4.1.2.2. Microscopic study

Procedure:

Microscopy of leaves of Clerodendrum splendens stem was studied according to the

methods of Brain and Turner, 1975. The powder microscopy was performed according to

the methods of Kokate (1994) and Khandelwal (2007).

For microscopic study paraffin embedded specimens were sectioned with the help of

Rotary Microtome. The thickness of the sections was 10-12 μm. Dewaxing of the

sections was by customary procedure (Johansen, 1940). The sections were stained with

Toluidine blue as per the method published by O’Brien et al. (1964). Since Toluidine

blue is a polychromatic stain. The staining results were remarkably good; and some

cytochemical reactions were also obtained. The dye rendered pink colour to the cellulose

walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the

protein bodies etc. wherever necessary sections were also stained with safranin and Fast-

green and IKI(for starch).

4.1.3. Result and Discussion

Midrib of the leaf: The midrib is more or less circular in the sectional profile with flat

adaxial side and thick circular abaxial part. It is 1.8 mm in horizontal plane and 1.6 mm

in vertical plane. The epidermal layer of the midrib is thin, comprising small squarish or

rectangular cells. Inner and outer epidermis is a narrow regions of thick walled cells

(Figure 8). The ground tissue includes thin walled, angular, compact parenchyma cells.

The vascular tissues constitute a complex system of hollow wide cylinder of unequal

thickness. Two vascular strands at abaxial-lateral part are larger than those that are

located along the lateral and adaxial portions of the cylinder (Figure 9).

The two abaxial bundles are horizontally stretched collateral bundles, they have several

diffusely distributed, narrow thick walled angular xylem elements and thick band of

pholem situated on the outer part of the xylem strands. The vascular bundles in the other

regions of the cylinder are wedge shaped and collateral having a group of diffusely

distributed xylem elements mixed with narrow fibres. The pholem units occur on the

outer end of each xylem strand. The entire cylinder of vascular bundles is ensheathedly a

thin layer of fibres (Figure 9) short, elongated prismatic calcium oxalate crystals are

sparsely distributed in the central ground tissue (Figure 11).


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Lamina: The lamina (leaf-blade) is distinctly dorsiventral; the adaxial and abaxial sides

are heteromorphic. The upper epidermal cells are larger and squirish in shape; the lower

epidermal cells are narrow and cylindrical, it is stomatiferous and short stalked peltate

types of glandular trichomes are located in shallow epidermal pits (Figure 10).

Powder microscopical results:

Powder preparation of the leaves shows the following inclusion when viewed under the

microscope-

Fragment of epidermal peeling of the lamina are frequently seen in the powder.

The abaxial epidermal peelings have highly wavy anticlinical walls and the epidermal

cells appear amoeboid on outline (Figure 12, 13, 14). The stomata are abundant in the

epidermis. The stomata are anomocytic type; no definite subsidiary cells are evident

around the stomata (Figure 13). The stomata have circular guard cells and the elliptical

stomatal pore (Figure 14). The guard cells are 55 µm in diameter.

Ventation: (Figure 15, 16 and 17) Fragments of leaf-blade are common in the powder.

These fragments exhibits vein-islets and vein terminations to branched once or twice type

(Figure 16, 17). The vein-islets are polygonal in outline with thick vein boundary (Figure

15).

Epidermal Trichomes:

On the surface of broken leaf, two type of epidermal trichomes were seen some of the

trichomes are glandular type (Figure 18). They are peltate type of glands. They have short

two or three celled stalk which is very short and attached on the epidermal pit. The head

part of the gland is circular plate compromising eight wide radiating cells. These cells

have dense cytoplasm and are secretory in function.

Non glandular trichomes are seen mostly located on the veins (Figure 19). These

trichomes are short and thickened, they are unicellular or two walled and unbranched.

They are conical in shape. These trichomes are 60-100 µm long and 20 µm thick.


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Figure 8: T.S of midrib of the leaf.

Figure 9: T.S of enlarged midrib to show the vascular system.

Where, AbB-Abaxial Bundle, AdB-Adaxial Bundle, Ep- Epidermis, LB-Lateral Bundles,

GT-Ground Tissue, Ph-Phloem, SC-Sclerenchyma, X-Xylem.


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Figure 10: Calcium oxalate crystals in the central pith cells.

Figure 11: T.S of lamina.

Where, AbB- Abaxial Bundle, AbE- Abaxial Epidermis, AdB- Adaxial Bundle, AdE-

Adaxial Epidermis, AbS- Abaxial Side, AC- Air Chamber, Ep- Epidermis, GT- Ground

Tissue, GTr- Glandular Trichomes, La- Lamina, MR- Midrib, PM- Palisade Mesophyll,

SC- Sclerenchyma, SM- Spongy Mesophyll.


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Figure 12: Adaxial epidermal layer in surface view in powder microscopy.

Figure 13: Enlarged stomata observed in powder microscopy.

Figure 14: Single enlarged stoma observed in powder microscopy.

Where, AW-Anticlinical wall, EC- Epidermal Cells, GTr: Glandular Trichomes, St: Stomata)


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Figure 15: Vein-islet of the lamina in powder microscopy.

Figure 16: Branched Vein-termination in powder microscopy.

Figure 17: Unbranched Vein-termination in powder microscopy.

Where, VI- Vein Islet, Vt- Vein Termination.


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Figure 18: Glandular trichomes- in surface view in powder microscopy.

Figure 19: Non-glandular trichomes- in surface view in powder microscopy.

Where, Gtr-Glandular trichomes, NGTr- Non-glandular Trichomes.


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4.1.4. Pharmacognostic study of Stem of Clerodendrum splendens


Procedure:

Macroscopy of the stem of Clerodendrum splendens was studied according to the


a. Size – Width : 2 mm.

b. Shape – The stem of Clerodendrum splendens is hollow cylindrical.

c. Color – Dark green colored surface.

d. Surface characters – Even outline.



(Figure 20)

Figure 20: Macroscopic characteristic of Clerodendrum splendens Stem.


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Procedure:

Microscopy of stem of Clerodendrum splendens stem was studied according to the


For microscopic study paraffin embedded specimens were sectioned with the help of

Rotary Microtome. The thickness of the sections was 10-12 μm. Dewaxing of the

sections was by customary procedure (Johansen, 1940). The sections were stained with

Toluidine blue as per the method published by O’Brien et al. (1964). Since Toluidine

blue is a polychromatic stain. The staining results were remarkably good; and some

cytochemical reactions were also obtained.


The stem is a hollow cylinder with even outline. The central part is occupied by a wide

hollow canal which is formed by lysis of the pith cells (Figure 21). The stem is about 2

mm thick. It consists of a thin intact epidermal layer of darkly stained spindle shaped

cells. There is a narrow cylinder of superficial periderm compromising two to four layers

of periderm derivatives. Inner to the periderm, there is a narrow region cortex; the cortex

includes about five layers of compact parenchymal cells. The inner boundary of the

cortex is a thin, broken cylinder of sclerenchyma cells.

The vascular cylinder consists of wide, continuous cylinder of secondary pholem,

compromising radial files of sieve elements and parenchyma cells (Figure 22 & 23). The

hollow secondary xylem cylinder includes diffusely distributed, solitary elliptic or

circular, thin walled vessels and thick walled, lignified xylem fibres (Figure 23) Calcium

oxalate crystals of prismatic type are located in cortical sclerenchyma elements and

pholem rays (Figure 25).


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Figure 21: T.S of stem- ground plan.

Figure 22: T.S of stem- sector enlarged.

Figure 23: Secondary Xylem element enlarged.

Where, Co- Cortex, CFi- Cortical fibres, Ep- Epidermis, Pe- Periderm, Pi- Pith, Pc- Pith Canal,

SPh- Secondary Pholem, Sx- Secondary xylem.


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Figure 24: T.S of Stem showing secondary Pholem.

Figure 25: Occurrence of calcium oxalate pholem, crystal in the cortical fibres and

phloem rays.

Where, Cr- Crystals, Fi- fibers, Ph- Pholem, X- Xylem,Ve- Vessel.


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4.1.6. Pharmacognostic study of Flowers of Clerodendrum splendens


Procedure:

Macroscopy of the flowers of Clerodendrum splendens was studied according to the


a. Size – Length– 2 - 3 cm

b. Shape – The flower is pentamerous with 5 free sepals, 5 gamopetalous corolla and five

free petal lobes. The stamens are five, incurved within the corolla tube and

become exerted when the corolla tube open

c. Color – Scarlet red.

d. Surface characters – Even outline.



(Figure 26)


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Figure 26: Macroscopic characteristic of Clerodendrum splendens Flowers.


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Procedure:

Microscopy of flower of Clerodendrum splendens flower was studied according to the


For the microscopical studies transverse sections were prepared and stained as mentioned

previously.


Sepals: The sepals are thick with blunt margins and are concave on the abaxial side

(Figure 27). It is 150 µm thick. The sepal has their epidermal layers of narrow rectangular

cells. The ground mesophyll tissue includes undifferentiated polygonal, thin walled

compact parenchyma cells. Small vascular strands are located in the median part of the

leaf blade. Grandular trichomes are frequently seen on the inner epidermis (Figure 28)

Petals: There are five petal lobes which are imbricate and aestivation. The petals are 120

µm thick. There is no distinct midrib; it is uniform in thickness. The outer epidermal cells

of the petal are semicircular or slightly papillate. The inner epidermal cells are also

papillate with beak-like outer tangential walls. The mesophyll tissue consists of

homogenous, parenchymatous circular compact cells (Fig. 31). Small collateral vascular

bundles are located in the mesophyll tissue. The petals are thicker in the middle and

gradually tapering towards margins (Fig. 30).

Anther: The anther is dithecous and four chambered (Figure 32). The wall of the mature

dehisced anther is 80 µm thick. It consists of the outer prominently papillate epidermal

layer and wide, thick radially oblong enthelial cells (Figure 33). The inner layer of

endothelial cells has several annular thickenings. The endodermal layer is disintegrated.

The anther dehisces longitudinally through the stomium.

Pollen grains: The pollen grains are circular and they are 40 µm in diameter. They have

slightly echinate exine and thin smooth infine. (Figure 34, 35).

Corolla tube: The basal part of the petal forms a tubular structure. The corolla is 200 µm

thick (Figure 36). It consists of the outer epidermis compromising large thick walled

circular cells. The inner epidermal layer also consists of large papillate thick outer

tangential walls. The ground tissue consists of 7-9 layers of large, thin walled compact


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parenchyma cells. Fairly prominent circular vascular strands are located along the median

part of the corolla tube. The vascular strands have large groups of thick walled xylem

elements and a thin layer of darkly stained pholem elements (Figure 37).

Figure 27: T.S of flower through sepals and corolla tube.

Figure 28: T.S of sepals.


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Figure 29: T.S of sepals enlarged.

Where, Co- Corolla tube, GTr - Glandular Trichomes, IEp - Inner Epidermis, MT-

Mesophyll Tissue, OEp-Outer Epidermis, Se - Sepal, St- Style).

Figure 30: T.S of flower through petal lobes and anthers.

Figure 31: T.S of petals- enlarged.

Where, An- Anther, IEP- Inner Epidermis, OEp- Outer Epidermis, MT- Mesophyll

Tissue, Pe - Petal, Po- Pollen, VB - Vascular Bundle).


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Figure 32: T.S of mature anther with pollen.

Figure 33: T.S of anther wall enlarged.

Where, An- Anther wall, Ep- Epidermis, Et- Endothecium; Pc- Pollen chamber, Po-

Pollen, ST- Spiral thickenings.

Figure 34: T.S of anther liberating the pollen.


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Figure 35: T.S of pollen grains enlarged.

Where, An- Anther, Ep- Epidermis, Et- Endothecium, Po- Pollen; St-Stomium.

Figure 36: T.S of corolla tube and style.

Figure 37: T.S of a sector of the corolla tube enlarged.

(CT: Corolla tube; GT: Ground Tissue; IEp: Inner Epidermis; OEp: outer Epidermis; VB:

Vascular Bundle; SC: Stylar canal; St: style.


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4.1.8. Physicochemical analysis

Physiochemical values such as the foreign organic matter, moisture content, ash values

and extractive values were determined according to the official methods (Anonymous,

1996; Anonymous, 1996) and as per WHO guidelines on quality control methods for

medicinal plant materials (WHO, 1998; WHO, 1992).

4.1.8.1. Determination of foreign organic matter

Five gm of air dried coarsely powdered drug (leaves, stem and flowers) was spread in a

thin layer. The sample was inspected with the unaided eye or with the use of 6X lens. The

foreign organic matter was separated manually as completely as possible. Sample was

weighed and percentage of foreign organic matter was determined from the weight of the

drug taken (Anonymous, 1996).

4.1.8.2. Determination of moisture content

Accurately weigh dried, stoppered glass shallow weighing bottle. Two gm of each sample

(leaves, stem and flowers) was transferred to the bottle and covered. Weight was taken

and sample was distributed evenly and poured to a depth not exceeding 10 mm. Then

loaded bottle was kept in oven and stopper was removed. The sample was dried to

constant weight. After drying it was collected to room temperature in a desiccator.

Weighed and calculated moisture content in terms of percent w/w (Anonymous, 1996).

4.1.8.3. Determination of ash values

Ash value is used to determine quality and purity of crude drug. Ash value contains

inorganic radicals like phosphates, carbonates and silicates of sodium, potassium,

magnesium, calcium etc. sometimes inorganic variables like calcium oxalate, silica,

calcium carbonate content of the crude drug affects ‘total ash value’. Such variables are

then removed by treating with acid and then acid insoluble ash value is determined

(Anonymous, 1996; Khandelwal, 2007).

4.1.8.3.1. Total ash

Accurately weighed 2 g of the air-dried crude drug (leaves, stem and flower) was taken in

a tare silica dish and incinerated at a temperature not exceeding 450 C until free from

carbon, cooled in a desiccator and weight was taken. The process was repeated till

constant weight was obtained. The percentage of ash was calculated with reference to air-

dried drug (leaves, stem and flowers) (Anonymous, 1996).


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4.1.8.3.2. Water-soluble ash

The ash, obtained as per the method described above was boiled for 5 minutes with 25

ml of water, filtered and collected the insoluble matter in a Gooch crucible, washed with

hot water and the filtrate was ignited for 15 minutes at a temperature not exceeding 450°

C and weight was taken. Subtracted the weight of the insoluble matter from the weight of

the ash; the difference in weight represents the water-soluble ash. The percentage of

water-soluble ash was calculated with reference to air-dried drug (leaves, stem and

flower) (Anonymous, 1996).

4.1.8.3.3. Acid-insoluble ash

The ash obtained as per method described above and boiled with 25 ml of 2 M

hydrochloric acid for 5 minutes, filtered, and collected the insoluble matter in a Gooch

crucible or on an ash less filter paper, washed with hot water, ignited and cooled in a

desiccator and weighed. The percentage of acid-insoluble ash was calculated with

reference to the air-dried drug (leaves, stem and flowers) (Anonymous, 1996).

4.1.8.4. Determination of extractive values

Different extractive values like water soluble and alcohol soluble extractive values were

performed by standard method (Anonymous, 1996).

4.1.8.4.1. Determination of water-soluble extractive value

Five gm of air dried coarsely powdered drug (leaves, stem and flowers) was macerated

with 100 ml of chloroform water in a closed flask for 24 hours, and it was shaken

frequently during first 6 hours and allowed to stand for 18 hours. Then it was filtered, 25

ml of the filtrate was evaporated in a flat shallow dish, and dried at 105° C and weighed.

Percentage of water-soluble extractive value was calculated with reference to air-dried

drugs (leaves, stem and flower) (Anonymous, 1996).

4.1.8.4.2. Determination of alcohol-soluble extractive value

Five gm of air-dried coarsely powdered drug (leaves, stem and flowers) was macerated

with 100 ml of ethanol of specified strength in a closed flask for 24 hours, and it was

shaken frequently during first 6 hours and allowed to stand for 18 hours. Then it was

filtered, during filtration precaution was taken against loss of ethanol, 25 ml of the filtrate

was evaporated in a flat shallow dish, and dried at 105° C and weighed. Percentage of


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ethanol soluble extractive value was calculated with reference to air dried drugs (leaves,

stem and flower) (Anonymous, 1996).

Table 1: Various physiochemical values of leaves stem and flowers of C. splendens.

Sr. No. Physicochemical Parameter (%w/w)

Leaves Stem Flowers

Foreign organic matter 0.25 0.56 0.31

2 Moisture content 5.63 6.81 4.90

3 Total ash 6.79 7.28 5.83

4 Water soluble ash 0.1 0.40 0.94

5 Acid insoluble ash 0.21 0.47 0.20

6 Water soluble extractive value 7.8 13.7 7.5

7 Alcohol soluble extractive value 8.61 12.1 9.0

4.1.9. Results and Discussion

Foreign organic matter of leaves, stem and flower of C. splendens is found to 0.25, 0.56

and 0.314% w/w, respectively. Moisture content of leaves, stem and flowers of C.

splendens is found to 5.63, 6.81 and 4.90% w/w, respectively. Total ash value of leaves,

stem and flowers of C. splendens is found to 6.79, 7.28 and 5.83% w/w, respectively.

Water soluble ash value of leaves, stem and flowers was found to be 0.1, 0.40 and 0.94%

w/w, respectively. Acid insoluble ash value of leaves, stem and flowers was found to be

0.21, 0.47 and 0.20% w/w, respectively. Water soluble extractive value of stem, leaves

and flowers was found to be 7.8, 13.7 and 7.5 % w/w, respectively. Alcohol soluble

extractive value of leaves, stem and flowers was found to be 8.61, 12.1 and 9.0 % w/w,

respectively.


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4.2. Extraction Methodology

The leaves, stem and flower of Clerodendrum splendens were collected from Nashik

district and was air dried in shade avoiding exposure to direct sunlight, then was

pulverized in grinder. Dried and powdered leaves, stem and flower of C. splendens were

extracted successively with various solvents as mentioned below.

Technique: Soxhlet extraction.

Different solvents in their sequence of increasing polarity were used for extraction.

1. Leaves: Petroleum ether (60°-80C), Chloroform, Ethyl acetate, Methanol, Water.

2. Stem : Petroleum ether (60°-80C), Chloroform, Methanol,

3. Flowers: Petroleum ether (60°-80C), Methanol

The extraction was carried out in several batches.

Petroleum ether extraction:

The extraction with non-polar solvent like petroleum ether was called as defattation.

Extraction carried out by continuous hot extraction method using soxhelt extractor till all

constituents are removed. The end of completion of extraction was indicated by no colour

with iodine in iodine chamber. After completion of extraction, solvents were distilled out

and dried extract obtained. These extracts were kept in desiccators till used. Petroleum

ether extract mainly contain coloring matter like chlorophyll, carotenoids, lipids, free

sterol and triterpenes.

Chloroform extraction: (successive)

The marc after petroleum ether extraction was air-dried and subjected to (exhaustive)

soxhlet extraction with chloroform. The chloroform extract usually separates heterocyclic

types of acids, long chain compounds, naphthoquinone type of substances, benzoquinone

type of substances, xanthophylls, chlorophyll, triterpenoid glycosides, free alkaloid and

free flavones.

The chloroform extract was subjected to solvent recovery by vacuum distillation.

Ethyl acetate extraction: (successive)

The marc after exhaustive chloroform extraction was air-dried and subjected to

exhaustive soxhlet extraction with ethyl acetate. The end point of extraction was

determined by reaction with iodine vapors.

The ethyl acetate extract usually contain flavonoid and alkaloid.


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Methanol extraction: (successive)

The marc after exhaustive ethyl acetate extraction was air-dried and subjected to

exhaustive soxhlet extraction with Methanol (high polarity). The end point of extraction

was determined by reaction with iodine vapors.

The methanol extract usually contain flavonoid, glycosides, tannins, resinous substances,

and alkaloids.

Distilled water extraction: (successive)

The marc after exhaustive methanol extraction was air-dried and subjected to exhaustive

soxhlet extraction with Methanol. The end point of extraction was determined by reaction

with iodine vapors.

The aqueous extract usually contain flavonoids and glycosides.


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Figure 38: Flow chart for extraction scheme of C. splendens leaves, stem & flowers.

,

Dried Leaves powder of C. splendens

Dried Flower powder of C. splendens

Dried Stem powder of C. splendens

Soxhlet extraction with Petroleum ether (60-800C)

Petroleum ether extract of leaves

Marc

Chloroform extract of leaves

Marc

Soxhlet extraction with chloroform

Ethyl acetate extract of leaves

Marc

Soxhlet extraction with Ethyl acetate

Methanol extract of leaves

Marc

Soxhlet extraction with Methanol

Aqueous extract of leaves

Marc

Soxhlet extraction with distilled water

Petroleum ether extract of stem

Marc

Chloroform extract of stem

Marc

Soxhlet extraction with chloroform

Methanol extract of stem

Marc


Petroleum ether extract of Flower

Marc

Methanol extract of Flower

Marc


Solvent ether insoluble fraction

Solvent ether soluble fraction

Solvent ether treatment

Ethyl acetate treatment

Ethyl acetate soluble fraction Ethyl acetate insoluble fraction


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4.2.1. Preliminary phytochemical screening

Preliminary phytochemical screening of various extracts was carried out using the

standard procedures (Khandelwal, 2007; Kokate, 1994).

4.2.1.1. Test for Carbohydrates

4.2.1.1.1. Molish test: Two ml of extracts solution was treated with few drops of 15

percent ethanolic α- napthol solution in a test tube and 2 ml of concentrated sulphuric

acid was added carefully along the side of tubes. The formation of reddish violet ring at

the junction of two layers indicates the presence of carbohydrates.

4.2.1.1.2. Fehling’s test: Five ml of extract solution was mixed with 5 ml Fehling’s

solution (equal mixture of Fehling’s solution A and B) and boiled. Development of brick

red precipitate indicates the presence of reducing sugars.

4.2.1.2. Test for Proteins

4.2.1.2.1. Biuret test: The extract was treated with 1 ml of 10% sodium hydroxide

solution and heated. A drop of 0.7% copper sulphate solution was added to the above

mixture. The formation of purple violet color indicates the presence of proteins.

4.2.1.2.2. Millon’s test: The extract was treated with 2 ml of Millon’s reagent. Formation

of white precipitate indicates the presence of proteins and amino acids.

4.2.1.3. Test for Amino acids

Ninhydrin test: The extract was treated with Ninhydrin reagent at pH range of 4-8 and

boiled. Formation of purple color indicates the presence of amino acid.

4.2.1.4. Test for Steroids

4.2.1.4.1. Salkowski test: One ml of concentrated sulphuric acid was added to 10 mg of

extract dissolved in 1 ml of chloroform. A reddish brown color exhibited by chloroform

layer and green fluorescence by the acid layer suggests the presence of steroids.

4.2.1.4.2. Liebermann-Buchard test: 10 mg extract was dissolved in 1 ml of chloroform

and 1 ml of acetic anhydride was added following the addition of 2 ml of

concentrated sulphuric acid from the side of the test tube. Formation of reddish violet

color at the junction indicates the presence of steroids.


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4.2.1.5. Test for triterpenes

Vanillin-Sulphuric acid test: 10 mg extract was dissolved in 1 ml of ethanol and added

20 mg vanillin and 0.5 ml concentrated sulphuric acid and heated for 10 min. Formation

of bluish violet color indicates the presence of triterpenes.

4.2.1.6. Test for Glycosides

Keller-killani test: To 2 ml of extract, glacial acetic acid, one drop 5 % Ferric chloride

and conc. Sulphuric acid was added. Presence of cardiac glycosides is indicated by

formation of reddish brown color at the junction of the two liquid layers and upper layer

appeared bluish green.

4.2.1.7. Test for Saponins

Foam formation test: One ml solution of the extract was diluted with distilled water to

20 ml and shaken in a graduated cylinder for 15 minutes. The development of stable foam

indicates the presence of saponins.

4.2.1.8. Test for Alkaloids

4.2.1.8.1. Dragendroff’s test: 0.1 ml dilute hydrochloric acid and 0.1 ml Dragendroff’s

reagent was added in 2 ml of extracts in a test tube. Formation of orange brown

precipitate indicates the presence of alkaloids.

4.2.1.8.2. Mayer’s test: To 2 ml of extracts, 0.2 ml of dilute hydrochloric acid and 0.1 ml

of Mayer’s reagent was added. Formation of yellowish buff precipitate indicates the

presence of alkaloids.

4.2.1.9. Test for Tannins and Phenolic compounds

4.2.1.9.1. Ferric chloride test: Five ml of extract solution was allowed to react with 1 ml

of 5% ferric chloride solution. Greenish black coloration indicates the presence of

tannins.

4.2.1.9.2. Dilute nitric acid test: Two ml of extract solution was allowed to react with

few drops of dilute HNO3 solution. Formation of reddish to yellow color indicates the

presence of tannins.

4.2.1.10. Test for Flavonoids

4.2.1.10.1. Shinoda test: To the extract, 5 ml (95%) ethanol, few drops of con. HCl and

0.5 g of magnesium turnings was added to give pink color.


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4.2.1.10.2. Lead acetate test: Few drops of 10% lead acetate were added to the extract.

Development of yellow colored precipitate confirms the presence of flavonoids.

Table 2: Preliminary phytochemical screening of Clerodendrum splendens leaves

extracts.

Chemical

constituent Chemical test PEE CLE EAE MTE AQE

Alkaloid Dragendorff’s test - - + + -

Mayer’s test - - + + -

Steroids Salkowaski test + + - - -

Liebermann-burchard test + + - - -

Triterpene Vanillin-sulphuric acid test + + - - -

Tannin Ferric chloride test - - - + +

Dilute nitric acid test - - - + +

Glycoside Keller-killani test - - - - +

Carbohydrate Molish test - - - - +

Fehling’s test - - - - +

Flavonoid Shinoda test - - + + +

Lead acetate test - - + + +

Saponins Foam formation test - - - - -

Proteins Biuret test - - - - -

Millon’s test - - - - -

Amino acids Ninhydrin test - - - - -

PEE - Petroleum ether extract , CLE - Chloroform extract, EAE - Ethyl acetate extract,

MTE - Methanol extract, AQE - Aqueous extract , ‘+’ indicates present and ‘-‘ indicates

absent.

Table 3: Preliminary phytochemical screening of Clerodendrum splendens stem and

flower extracts.

Chemical

constituent

Chemical test Clerodendrum splendens

Flower

Clerodendrum splendens

Stem

PEE MTE PEE CLE MTE

Alkaloid Dragendorff’s test - + - - +

Mayer’s test - + - - +

Steroids Salkowaski test + - + + -

Liebermann-burchard test + - + + -


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Triterpene Vanillin-sulphuric acid test + - + + -

Tannin Ferric chloride test - + - - +

Dilute nitric acid test - + - - +

Glycoside Keller-killani test - - - - -

Carbohydrate Molish test - - - - -

Fehling’s test - - - - -

Flavonoid Shinoda test - + - - +

Lead acetate test - + - - +

Saponins Foam formation test - - - - -

Proteins Biuret test - - - - -

Millon’s test - - - - -

Amino acids Ninhydrin test - - - - -

PEE - Petroleum ether extracts CLE - Chloroform extract, MTE - Methanol extract ‘+’

indicates present and ‘-‘indicates absent.


Preliminary phytochemical screening of leaves extracts mainly revealed the presence of

steroids and triterpenes in petroleum ether and chloroform extract, alkaloid and

flavonoids in ethyl acetate extract, alkaloids, flavonoids and tannins in methanol extract

and tannins, glycosides, carbohydrates and flavonoids in aqueous extract (Table 2 ).

Preliminary phytochemical screening of Clerodendrum splendens stem extract mainly

revealed the presence of steroids and triterpenes in petroleum ether and chloroform

extract and alkaloid, tannins and flavonoids methanol extract (Table 3).

Preliminary phytochemical screening of Clerodendrum splendens flower extract mainly

revealed the presence of steroids and triterpenes in petroleum ether extract and alkaloid,

tannins and flavonoids in methanol extract. (Table 3).

CHAPTER 5 PHYTOCHEMICAL STUDY

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5. PHYTOCHEMICAL STUDY

5.1. Introduction

Thin Layer Chromatography (TLC):

TLC enables the qualitative, semi-quantitative and quantitative evaluation of

phytoconstituents of herbal drugs. The quality control and quality assurance of herbal

drug still remains a challenge because of the high variability of chemical components

involved. Due to natural variability, chemical analysis of plant material is a great

challenge and requires special approaches. Planner chromatography is most versatile

option for the required identification tests for the quality control of herbal products. In its

traditional form, thin layer chromatography (TLC) is frequently used for the analysis of

botanical raw materials. Thin layer chromatography has a long record in almost all

Pharmacopeias for its use in the identification of herbal medicines.

TLC identity test given in the individual monographs includes the identification of the

drug based on co-chromatography with reference standard marker compound and

recording the fingerpoint profiles. TLC technique involve application of sample

(dissolved in suitable solvent) at one end of the precoated plate, development of the plate

in solvent system in a closed chamber to the specified height and visualization of the

plate for the compound in natural light, under U.V 254 and 366 nm and/or by

derivatization of the plate with suitable reagent. The Rf values and the colours of the

resolved bands are recorded and fingerpoint profile are established. Identification of the

chemical marker is by comparision of the Rf value, absorption spectra, response to

derivatizing reagent etc.

Rf =

Distance travelled by the Solute

Distance travelled by the Solvent

Rf is a retardation factor which depends on the physiochemical properties of the

substance. It is a measure of relative affinity of substances for stationary phase. It is a

ratio of the distance travelled by substance to the distance travelled by solvent front.


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5.2. Material and methods

Precoated silica plates (E. Merk), Chromatogram development chamber, Glass capillaries

(applicator) , U.V chamber & spraying bottles for reagents

Petroleum ether extract of leaves, stem and flowers, Chloroform extracts of leaves and

stem, Ethyl acetate extract of leaves, methanol extract of leaves, stem and flowers and

aqueous extract of leaves of plant C. splendens.

5.2.1. Experimental

Preparation of sample:

Petroleum ether extract: Petroleum ether extract of leaves, stem and flower (10 mg)

was transferred to a 10 ml of volumetric flask. It was dissolved in petroleum ether and

volume was made up to the mark with petroleum ether.

Chloroform extract: Chloroform extract of leaves and stem (1mg/ml) was prepared as

by dissolving (10 mg) of each extract to 10 ml volume of solvent.

Methanol extract: Methanol extract of leaves, stem and flower (1mg/ml) was prepared

as by dissolving (10 mg) of each extract to 10 ml volume of solvent in volumetric flask.

Ethyl acetate extract of leaves and aqueous extract of leaves was prepared (1 mg/ml) by

dissolving in respective solvent in volumetric flask.

Thin Layer chromatography of extracts of leaves, stem and flowers were performed to

develop methods for chromatographic separation of phytoconstituents and also to

preliminary characterize each separable phytoconstituents from respective extracts.


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5.2.1.1. Thin layer chromatography of petroleum ether extracts of leaves, stem and

flowers of Clerodendrum splendens.

Stationary phase : Precoated silica plates (E. Merk)

Mobile phase : Benzene : Ethyl acetate (9:1)

Visualization : Anisaldehyde-sulphuric acid reagent and heat at 100°C

Figure 39: TLC of petroleum ether extract of leaves, stem and flowers of C. splendens

visualized by spraying anisaldehyde-sulphuric acid reagent.

Table 4: Rf values of phytoconstituents from TLC of petroleum ether extracts of leaves,

stem and flowers of C. splendens after spraying anisaldehyde-sulphuric acid reagent.

Sr. No. Flower extract Leaves extract Stem extract

Rf Remark Rf Remark Rf Remark

1 0.1 Violet 0.06 Violet 0.12 Violet

2 0.24 Light blue 0.2 Light blue 0.3 Light blue

3 0.28 Purple 0.28 Purple 0.43 Dark purple

4 0.4 Purple 0.52 Yellowish 0.52 Light purple

5 0.72 Violet 0.8 Violet 0.58 Light purple

6 0.66 Purple

7 0.76 Violet


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Visualization : Vanillin-sulphuric acid reagent and heat at 110°C

Figure 40: TLC of petroleum ether extract of leaves, stem and flowers of C. splendens

visualized by spraying vanillin-sulphuric acid reagent.

Table 5: Rf values of phytoconstituents from TLC of petroleum ether extracts of leaves,

stem and flowers of C. splendens after spraying vanillin-sulphuric acid reagent.



1 0.28 Violet 0.28 Violet 0.28 Violet

2 0.70 Violet 0.40 Purple

3 0.63 Light blue

4 0.78 Indigo


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5.2.1.2. Thin layer chromatography of chloroform extracts of leaves and stem of

Clerodendrum splendens.




Figure 41: TLC of chloroform extract of leaves and stem of C. splendens visualized by

spraying anisaldehyde-sulphuric acid reagent.

Table 6: Rf values of phytoconstituents from TLC of chloroform extracts of leaves and

stem of C. splendens.

Sr. No. Leaves extract Stem extract

Rf Remark Rf Remark

1 0.20 Light blue 0.11 Purple

2 0.33 Light blue 0.20 Light blue

3 0.4 Violet 0.44 Violet

4 0.50 Light blue 0.53 Light purple

5 0.80 Purple 0.95 Violet

6 0.96 Violet


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5.2.1.3. Thin layer chromatography of Methanol extracts of leaves, stem and

flowers of Clerodendrum splendens.


Mobile phase : Toluene : Ethyl acetate : formic acid (8: 2 : 0.5)


Figure 42: TLC of methanol extract of leaves, stem and flowers of C. splendens

visualized by spraying anisaldehyde-sulphuric acid reagent.

Table 7: Rf values of phytoconstituents from TLC of methanol extracts of leaves, stem

and flowers of C. splendens.



1 0.25 Purple 0.16 Reddish green 0.24 Violet

2 0.49 Light Purple 0.13 Yellowish green 4.9 Light blue

3 0.65 Purple 0.23 Purple 0.75 Violet

4 0.73 Purple 0.43


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5.2.1.4. Thin layer chromatography of ethyl acetate extracts of leaves of C.

splendens.


Mobile phase : Toluene : Ethyl acetate : formic acid (8: 2 : 0.5)


Figure 43: TLC of ethyl acetate extract of leaves of C. splendens visualized by spraying

anisaldehyde-sulphuric acid reagent.

Table 8: Rf values of phytoconstituents from TLC of ethyl acetate extracts of leaves of

C. splendens.

Ethyl acetate leaves extract

Sr. No. Rf Remark Sr. No. Rf Remark

1 0.08 Reddish green 5 0.52 Violet

2 0.15 Yellowish green 6 0.56 Yellowish blue

3 0.31 Yellow 7 0.67 Light blue

4 0.45 Light blue 8 0.76 Violet


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5.2.1.5. Thin layer chromatography of aqueous extracts of leaves of C. splendens.


Mobile phase : Ethyl acetate: Formic acid: Glacial acetic acid: Water

(100:11:11:26)

Visualization : Spray ferric chloride acid reagent at 100°C

Figure 44: TLC of aqueous extract of leaves of C. splendens visualized by spraying

ferric chloride acid reagent.

Table 9: Rf values of phytoconstituents from TLC of aqueous extract of leaves of C.

splendens.

Sr. No. Aqueous leaves extract

Rf Remark

1 0.21 Greyish blue

2 0.62 Greyish blue

4. PHARMACOGNOSTIC STUDY 4.1...

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