Weekly Science Research Journal Primary Article Histochemical Studies Of Cochlospermum Religiosum (l) Alston Sasikala A, Linga Rao M And Savithramma N Sasikala A, Linga Rao M And Savithramma N From 1,2,3 Research Scholar Department of Botany, Sri Venkateswara University, Tirupati – 517 502. Andhra Pradesh, INDIA. Te Article is published on September 2013 issue & available at www.weeklyscience.org DOI : 10.9780/ 2321-7871/1102013/29 ABSTRACT Traditional medicines are prepared from a single plant or combination of plants. Histochemical studies are helpful in drug adulteration and biosystematics arrangement. The present study deals with the location or identification of phytochemicals like tannins, polyphenols, crystals and starch grains in various regions of leaf, stem and stembark of Cochlospermum religiosum by using different chemicals or reagents (FeCl3, Iodine solution, Toluidine blue reagent and HCl). The results showed that the bluish black, purple or blue, bluish green and dark black colour indicates the presence of tannins, starch grains, polyphenols and crystals in various regions like epidermis, endodermis, midrib, cortex and vascular bundle of leaf, stem and stembark of Cochlospermum religiosum. These observations could be of immense value in the botanical identification and standardization of crude drug. KEY WORDS: INTRODUCTION Cochlospermum Religiosum, Stembark, Phenols, Tannins. Plants are the great sources of medicines, especially in traditional system of medicine, which are useful in the treatment of various diseases. Indian contribution to herbal market and emphasis on novel research is continuously increasing. Cochlospermum religiosum (L) Alston is a sparsely branched small tree, belonging to the family Cochlospermaceae. It is commonly called as Yellow Silk Cotton, Buttercup Tree and Torchwood Tree because of flowers are large, bright golden yellow and seeds covered with silky hairs. C. religiosum stem bark and root powder is traditionally used for fertility and ash of fruit mixed with coconut is used for the treatment of scabies (Goud et al., 2005). The gum of C. religiosum is also found to be an ingredient of unani medicine Qurs-e-Sartaan Kafoori which is used for Styptic, Antipyretic, Phthisis, Tuberculosis, Hectic fever and Qurs-e-Suzak Cicatrizant, Diuretic, Gonorrhea. These formulations were found to possess good antibacterial and antifungal activity (Cecilie et al., 2005). Sasikala and Savithramma (2012) studied the antimicrobial activity of biological synthesis of silver nanoparticles from leaves of C. religiosum , preliminary phytochemical screening (Sasikala and Savithramma, 2012), quantification of phytochemical (Sasikala and Savithramma, 2013) and also studied the invitro propagation (Sasikala and Savithramma, 2012). Histochemistry or cytochemistry deals with localization of chemical compounds within the cells by means of specific colors of the compounds. Staining the cells with different stains or dyes, which render the compounds visible under the microscope, makes the specific color reaction compounds. The importance of histochemistry in solving critical biosystematic problems is as popular as the use of other markers. According to botanical literatures, the use of histochemical characters in taxonomic conclusions is now a common practice. For example, the presences of calcium oxalate crystals in various plant families 2321-7871 th Vol-1, Issue-10, 26 September 2013 Page No-1
Transcript
Weekly Science Research Journal
Primary Article
Histochemical Studies Of Cochlospermum Religiosum (l) Alston
Sasikala A, Linga Rao M And Savithramma N
Sasikala A, Linga Rao M And Savithramma N
From1,2,3Research Scholar
Department of Botany,Sri Venkateswara University,
Tirupati – 517 502.Andhra Pradesh, INDIA.
Te Article is published on September 2013 issue &
available at www.weeklyscience.org
DOI : 10.9780/ 2321-7871/1102013/29
ABSTRACT
Traditional medicines are prepared from a single plant or combination of plants. Histochemical studies are helpful in drug adulteration and biosystematics arrangement. The present study deals with the location or identification of phytochemicals like tannins, polyphenols, crystals and starch grains in various regions of leaf, stem and stembark of Cochlospermum religiosum by using different chemicals or reagents (FeCl3, Iodine solution, Toluidine blue reagent and HCl). The results showed that the bluish black, purple or blue, bluish green and dark black colour indicates the presence of tannins, starch grains, polyphenols and crystals in various regions like epidermis, endodermis, midrib, cortex and vascular bundle of leaf, stem and stembark of Cochlospermum religiosum. These observations could be of immense value in the botanical identification and standardization of crude drug.
Plants are the great sources of medicines, especially in traditional system of medicine, which are useful in the treatment of v a r i o u s d i s e a s e s . I n d i a n contribution to herbal market and emphasis on novel research is c o n t i n u o u s l y i n c r e a s i n g . Cochlospermum religiosum (L) Alston is a sparsely branched small tree, belonging to the family Cochlospermaceae. It is commonly called as Yellow Silk Cotton, Buttercup Tree and Torchwood Tree because of flowers are large, bright golden yellow and seeds covered with silky hairs.C. religiosum stem bark and root powder is traditionally used for fertility and ash of fruit mixed with coconut is used for the treatment of scabies (Goud et al., 2005). The gum of C. religiosum is also found to be an ingredient of unani medicine Qurs-e-Sartaan Kafoori which is used for Styptic, Antipyretic, Phthisis, Tuberculosis, Hectic fever and Qurs-e-Suzak Cicatrizant, Diuretic, Gonorrhea. These
formulations were found to possess good antibacterial and antifungal activity (Cecilie et al., 2005). Sasikala and Savithramma (2012) studied the antimicrobial activity of biological synthesis of silver nanoparticles from leaves of C. r e l i g i o s u m , p r e l i m i n a r y phytochemical screening (Sasikala a n d S a v i t h r a m m a , 2 0 1 2 ) , quantification of phytochemical (Sasikala and Savithramma, 2013) and also studied the invitro propagat ion (Sas ika la and Savithramma, 2012).
H i s t o c h e m i s t r y o r c y t o c h e m i s t r y d e a l s w i t h localization of chemical compounds within the cells by means of specific colors of the compounds. Staining the cells with different stains or dyes, which render the compounds visible under the microscope, makes the specific color reaction compounds. The importance of histochemistry in solving critical biosystematic problems is as popular as the use of other markers. According to botanical l i t e r a t u r e s , t h e u s e o f histochemical characters in taxonomic conclusions is now a common practice. For example, the presences of calcium oxalate crystals in various plant families
have been reported by various scientists (Mbagwu et al., 2009) reported that the size and shape of calcium oxalate crystals though variable in each species showed enough interspecific differences that may be used for taxonomic references in Vigna species. This has been done in other groups of plants such as Dioscoreaceae, (Edeoga and Okoli, 1995). Icacinaceae, (Heintzelman and Howard, 1948) Nyctaginaceae, (Edeoga and Ikem, 2002) and Verbenaceae (Mathew and Shah, 2006). The biosystematic importance and implications of histochemical features of ergastics, calcium oxalate crystals, nature of tannins and saponins have been investigated in various plants families such as Dioscoreaceae (Edeoga and Ogbegbor, 1999), Leguminosae-papilionoideae (Mbagwu and Edeoga, 2006). Identification of localization of secondary metabolites in plant parts which are using in the preparation of drug is an immense importance to prevent adulteration and also helpful in taxonomic hierarchy. Hence in the present study an attempt has been made to identification and localization of secondary metabolites in the medicinal plants.
Fresh and healthy leaf, stem and sembark of Cochlospermum religiosum were collected from Tirumal hills of Chittoor district, Andhra Pradesh, India during the year May, 2012. These specimens were initially fixed in FAA (1:1:18) glacial acetic: 40% formaldehyde: 70% ethanol (v/v) for 48-72 h. after 72 h the specimens were transverse (T.S) section were taken using a rotary microtome (RMT-30). Anatomical staining was done by initially staining with few drops of alcian blue for 5 min and counter stained with safranin solution for 2 min. The specimens were treated with FeCl3, Iodine solution, Toluidine blue reagent and HCl for identification of polyphenols, tannins, c r y s t a l s a n d s t a r c h g r a i n s . Photomicrographs of the anatomical features were then taken from the slides using Nikhon Labhot 2 microscopic unit (Asokan, 2006).
The fluorescence studies were carried out as per the method of Bhattacharya and Zaman (2009). A small quantity of the leaf powder was placed on a grease free clean microscopic
MATERIAL AND METHODS
Fluorescence Studies
slide and added 1-2 drops of the freshly prepared reagent solution, mixed by gentle tilting the slide and waited for 1-2 min. Then the slides were placed inside the UV-viewer chamber and viewed in day light short (254 nm) and long (365 nm) ultraviolet radiations. The colours observed by application of different reagents in different radiations were recorded.
Behavior of the leaf powder with different chemical reagents was studied to detect the presence of major phytoconstituents with color changes under day light by reported method of Pratt and Chase (1949)
Present day's adulteration of the drugs is a common phenomenon. It is inevitable to study the pharmacognostic studies to avoid adulteration.Hence the following experiments were carried out.
Histochemical color reactions were carried out through transverse sections of leaf, stem and bark of Cochlospermum religiosum. The results were showed in Table-1. Fig-1 showed various secondary metabolites present in leaf with treatment of different reagents. The presence of tannins was indicated by the development of bluish black colour, when treated with ferric chloride (FeCl3). The tannins were found mainly in the parenchyma tissue of the midrib region, whereas the starch grains were indicated by the development of blue color or purple, when treated with iodine solution. The starch grains were located in the mesophyll and parenchymatous region of midrib. The presence of polyphenols was indicated by the development of bluish green color, when treated with toluidine blue reagent. The polyphenols were found surrounding the vascular bundle sheath. The presences of crystals were indicated by the development of dark black color, when treated with HCl, the crystals were present in the midrib region and vascular bundles.
Fig-2 showed the histochemical studies of stem, the presence of tannins were indicated by the development of bluish black color, when treated with ferric chloride (FeCl3). The tannins were present in endodermis and cortex region. The starch grains were indicated by the development of blue color or purple, when treated with iodine solution found in epidermis, cortex and vascular bundles.
RESULTS AND DISCUSSIONHistochemical Studies
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The polyphenols were found mainly in the endodermis and cortex region, which were indicated by the development of bluish green colour, when treated with toluidine blue reagent. The presences of crystals were indicated by the development of dark black color, when treated with HCl the crystals were present mainly in the cortex.
The histochemical analysis of bark showed in Fig-3 the presence of tannins were indicated by the development of bluish black color, when treated with ferric chloride (FeCl3) and these were found in cortex and vascular bundle region. The polyphenols were found mainly in the cortex region which was indicated by the development of bluish green color, when treated with toluidine blue reagent. The presence of crystal indicated by the development of dark black color, when treated with HCl the crystals were present mainly in the cortex. The starch grains were indicated by the development of blue or purple colour, when treated with iodine solution the starch grains were present mainly in the cortex and vascular bundle region.
Toluidine blue is a cationic dye that binds to negatively charged groups. An aqueous solution of this dye is blue, but different colors are generated when the dye binds with different anionic groups in the cell for example, a pinkish purple colour will appear when the dye reacts with carboxylated polysaccharides such as pectic acid; green, greenish blue or bright blue with polyphenolic substances such as ligning and tannins; and purplish or greenish blue with nucleic acids (Baker, 1966). Plants store glucose as the polysaccharide starch, it can be separated into two fractions-amylose and amylopectin. Amylose forms a colloidal dispersion in hot water, whereas amylopectin is completely insoluble. The structure of amylose consists of long polymer chains of glucose units connected by an alpha acetyl linkage. Amylose in starch is responsible for the formation of a deep blue colour in the presence of iodine. The iodine molecule slips inside of the amylose coil. When added the Iodine-KI reagent to a solution or other materials blue colour is present. If starch amylose is not present, then the colour will stay orange or yellow.
Behavior of the leaf powder with different chemical reagents was studied
Leaf Powder Behavior
to detect the presence of major phytoconstituents with color changes under day light by reported method (Pratt and Chase, 1949). And the results were showed in Table-2.
The fluorescence characteristics of leaf powder with different chemical reagents were summarized in Table-3. Although a change in color was observed by the addition of various reagents under day light, none of the reagents induced any fluorescence to the leaf powder under both short and long UV radiations. Under UV light dark brown and black colors were prominent. Fluorescence is the phenomenon exhibited by various chemical constituents present in the plant material. Some constituents showed fluorescence in the visible range in the day light. The natural products (Alkaloids) produce fluorescence in UV light but do not produce fluorescence in visible day light. If the substances themselves are not fluorescent, they often be converted fluorescent derivatives or decomposition products by applying different reagents (Mukherjee, 2002).
The pharmacognostical studies and histochemical studies and behavior analysis of fluorescence studies of Cochlospermum religiosum are useful to supplement the information with regard to its botanical identification and drug standardization. Moreover, it also helps in distinction from other allied species and adulteration.
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Cochlospermum religiosum 2013; 4:
Quantification of Primary And secondary metabolites From leaves and stem bark of
(L.) Alsten
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