Pharmacognostic, Phytochemical and Pharmacological studies on 22 Clerodendrum splendens. Family- Verbenaceae.

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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. Pharmacognostic, Phytochemical and Pharmacological studies on 22

2 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. Pharmacognostic, Phytochemical and Pharmacological studies on 23

3 Pharmacognostic study of Leaves of Clerodendrum splendens Macroscopic study Procedure: Macroscopy of the leaves of Clerodendrum splendens was studied according to the methods of Brain and Turner, a. Size Length : 6-12 cm. Width: 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) Pharmacognostic, Phytochemical and Pharmacological studies on 24

4 Figure 7: Macroscopic characteristic of Clerodendrum splendens Leaf. Pharmacognostic, Phytochemical and Pharmacological studies on 25

5 Microscopic study Procedure: Microscopy of leaves of Clerodendrum splendens stem was studied according to the methods of Brain and Turner, 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 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 Fastgreen and IKI(for starch) 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). Pharmacognostic, Phytochemical and Pharmacological studies on 26

6 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 µm long and 20 µm thick. Pharmacognostic, Phytochemical and Pharmacological studies on 27

7 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. Pharmacognostic, Phytochemical and Pharmacological studies on 28

8 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. Pharmacognostic, Phytochemical and Pharmacological studies on 29

9 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) Pharmacognostic, Phytochemical and Pharmacological studies on 30

10 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. Pharmacognostic, Phytochemical and Pharmacological studies on 31

11 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. Pharmacognostic, Phytochemical and Pharmacological studies on 32

12 Pharmacognostic study of Stem of Clerodendrum splendens Macroscopic study Procedure: Macroscopy of the stem of Clerodendrum splendens was studied according to the methods of Brain and Turner, 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. e. Odor Characteristic. f. Taste Slightly bitter. (Figure 20) Figure 20: Macroscopic characteristic of Clerodendrum splendens Stem. Pharmacognostic, Phytochemical and Pharmacological studies on 33

13 Microscopic study Procedure: Microscopy of stem of Clerodendrum splendens stem was studied according to the methods of Brain and Turner, For microscopic study paraffin embedded specimens were sectioned with the help of Rotary Microtome. The thickness of the sections was 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 Result and Discussion 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). Pharmacognostic, Phytochemical and Pharmacological studies on 34

14 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. Pharmacognostic, Phytochemical and Pharmacological studies on 35

15 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. Pharmacognostic, Phytochemical and Pharmacological studies on 36

16 Pharmacognostic study of Flowers of Clerodendrum splendens Macroscopic study Procedure: Macroscopy of the flowers of Clerodendrum splendens was studied according to the methods of Brain and Turner, 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. e. Odor Characteristic. f. Taste Slightly bitter. (Figure 26) Pharmacognostic, Phytochemical and Pharmacological studies on 37

17 Figure 26: Macroscopic characteristic of Clerodendrum splendens Flowers. Pharmacognostic, Phytochemical and Pharmacological studies on 38

18 Microscopic study Procedure: Microscopy of flower of Clerodendrum splendens flower was studied according to the methods of Brain and Turner, For the microscopical studies transverse sections were prepared and stained as mentioned previously Result and Discussion 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 Pharmacognostic, Phytochemical and Pharmacological studies on 39

19 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. Pharmacognostic, Phytochemical and Pharmacological studies on 40

20 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). Pharmacognostic, Phytochemical and Pharmacological studies on 41

21 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. Pharmacognostic, Phytochemical and Pharmacological studies on 42

22 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. Pharmacognostic, Phytochemical and Pharmacological studies on 43

23 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) 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) 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) 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) 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 airdried drug (leaves, stem and flowers) (Anonymous, 1996). Pharmacognostic, Phytochemical and Pharmacological studies on 44

24 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) 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) Determination of extractive values Different extractive values like water soluble and alcohol soluble extractive values were performed by standard method (Anonymous, 1996) 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) 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 Pharmacognostic, Phytochemical and Pharmacological studies on 45

25 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. (%w/w) Sr. No. Physicochemical Parameter Leaves Stem Flowers Foreign organic matter Moisture content Total ash Water soluble ash Acid insoluble ash Water soluble extractive value Alcohol soluble extractive value 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. Pharmacognostic, Phytochemical and Pharmacological studies on 46

26 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 (60-80 C), Chloroform, Ethyl acetate, Methanol, Water. 2. Stem : Petroleum ether (60-80 C), Chloroform, Methanol, 3. Flowers: Petroleum ether (60-80 C), 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. Pharmacognostic, Phytochemical and Pharmacological studies on 47

27 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. Pharmacognostic, Phytochemical and Pharmacological studies on 48

28 Figure 38: Flow chart for extraction scheme of C. splendens leaves, stem & flowers. Dried Leaves powder of C. splendens Dried Stem powder of C. splendens Dried Flower powder of C. splendens Soxhlet extraction with Petroleum ether ( C), Petroleum ether extract of leaves Marc Petroleum ether extract of stem Marc Soxhlet extraction with chloroform Soxhlet extraction with chloroform Chloroform extract of leaves Marc Chloroform extract of stem Marc Soxhlet extraction with Ethyl acetate Soxhlet extraction with Methanol Ethyl acetate extract of leaves Marc Methanol extract of stem Marc Soxhlet extraction with Methanol Methanol extract of leaves Marc Petroleum ether extract of Flower Soxhlet extraction with Methanol Marc Soxhlet extraction with distilled water Methanol extract of Flower Marc Aqueous extract of leaves Marc Solvent ether treatment Solvent ether insoluble Solvent ether soluble fraction Ethyl acetate treatment Ethyl acetate soluble fraction Ethyl acetate insoluble fraction Pharmacognostic, Phytochemical and Pharmacological studies on 49

29 Preliminary phytochemical screening Preliminary phytochemical screening of various extracts was carried out using the standard procedures (Khandelwal, 2007; Kokate, 1994) Test for Carbohydrates 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 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 Test for Proteins 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 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 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 Test for Steroids 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 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. Pharmacognostic, Phytochemical and Pharmacological studies on 50

30 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 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 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 Test for Alkaloids 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 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 Test for Tannins and Phenolic compounds 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 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 Test for Flavonoids 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. Pharmacognostic, Phytochemical and Pharmacological studies on 51

31 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 Alkaloid Steroids Chemical test PEE CLE EAE MTE AQE Dragendorff s test Mayer s test Salkowaski test Liebermann-burchard test Triterpene Vanillin-sulphuric acid test Tannin Ferric chloride test Dilute nitric acid test Glycoside Keller-killani test Carbohydrate Flavonoid Molish test Fehling s test 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 Pharmacognostic, Phytochemical and Pharmacological studies on 52

32 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 Result and Discussion 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). Pharmacognostic, Phytochemical and Pharmacological studies on 53

33 CHAPTER 5 PHYTOCHEMICAL STUDY 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. Pharmacognostic, Phytochemical and Pharmacological studies on 54

34 CHAPTER 5 PHYTOCHEMICAL STUDY 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 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. Pharmacognostic, Phytochemical and Pharmacological studies on 55

35 CHAPTER 5 PHYTOCHEMICAL STUDY 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 Violet 0.06 Violet 0.12 Violet Light blue 0.2 Light blue 0.3 Light blue Purple 0.28 Purple 0.43 Dark purple Purple 0.52 Yellowish 0.52 Light purple Violet 0.8 Violet 0.58 Light purple Purple Violet Pharmacognostic, Phytochemical and Pharmacological studies on 56

36 CHAPTER 5 PHYTOCHEMICAL STUDY Stationary phase : Precoated silica plates (E. Merk) Mobile phase : Benzene : Ethyl acetate (9:1) 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. Sr. No. Flower extract Leaves extract Stem extract Rf Remark Rf Remark Rf Remark Violet 0.28 Violet 0.28 Violet Violet 0.40 Purple Light blue Indigo Pharmacognostic, Phytochemical and Pharmacological studies on 57

37 CHAPTER 5 PHYTOCHEMICAL STUDY Thin layer chromatography of chloroform extracts of leaves and stem of Clerodendrum splendens. Stationary phase : Precoated silica plates (E. Merk) Mobile phase : Benzene : Ethyl acetate (7:3) Visualization : Anisaldehyde-sulphuric acid reagent and heat at 100 C 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 Light blue 0.11 Purple Light blue 0.20 Light blue Violet 0.44 Violet Light blue 0.53 Light purple Purple 0.95 Violet Violet Pharmacognostic, Phytochemical and Pharmacological studies on 58

38 CHAPTER 5 PHYTOCHEMICAL STUDY Thin layer chromatography of Methanol extracts of leaves, stem and flowers of Clerodendrum splendens. Stationary phase : Precoated silica plates (E. Merk) Mobile phase : Toluene : Ethyl acetate : formic acid (8: 2 : 0.5) Visualization : Anisaldehyde-sulphuric acid reagent and heat at 100 C 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. Sr. No. Flower extract Leaves extract Stem extract Rf Remark Rf Remark Rf Remark Purple 0.16 Reddish green 0.24 Violet Light Purple 0.13 Yellowish green 4.9 Light blue Purple 0.23 Purple 0.75 Violet Purple 0.43 Pharmacognostic, Phytochemical and Pharmacological studies on 59

39 CHAPTER 5 PHYTOCHEMICAL STUDY Thin layer chromatography of ethyl acetate extracts of leaves of C. splendens. Stationary phase : Precoated silica plates (E. Merk) Mobile phase : Toluene : Ethyl acetate : formic acid (8: 2 : 0.5) Visualization : Anisaldehyde-sulphuric acid reagent and heat at 100 C 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 Reddish green Violet Yellowish green Yellowish blue Yellow Light blue Light blue Violet Pharmacognostic, Phytochemical and Pharmacological studies on 60

40 CHAPTER 5 PHYTOCHEMICAL STUDY Thin layer chromatography of aqueous extracts of leaves of C. splendens. Stationary phase : Precoated silica plates (E. Merk) 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. Rf Aqueous leaves extract Remark Greyish blue Greyish blue Pharmacognostic, Phytochemical and Pharmacological studies on 61