1.4. DISCUSSION. traditional medicinal system. It is very important to undertake Pharmacognostic

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1 1.4. DISCUSSION Most of the traditional knowledge about medicinal plant was in the form of oral knowledge. There is no uniform or standard procedure for maintaining the inventory of these plants and the knowledge about their medicinal properties. Therefore, it is necessary that such procedures to be documented and studied for systematic regulation and widespread application. The leads for a significant number of modern synthetic drugs have originated either from isolated plant ingredients, since the search for new entities begins from derivatives of the existing drug or from traditional medicinal system. It is very important to undertake Pharmacognostic studies and phytochemical investigations along with biological screening to understand therapeutic dynamics of medicinal plants and to develop quality parameters. In the present day, despite the availability of more advanced analytic techniques, identification and evaluation of plant drugs by pharmacognostic and physicochemical parameter study is still more reliable, accurate, and inexpensive. According to World Health Organization (WHO) the macroscopic and microscopic determination of the plant is the first step towards establishing the correct identity and purity of the source materials (WHO 2002). Standardization of natural products is a complex task due to their heterogeneous composition, which is in the form of whole plant, plant part/extracts obtained thereof. To ensure reproducible quality of herbal products, proper identification of starting material is essential (Reddy et al., 2010). As there is no pharmacognostic work recorded on rhizome of C. pseudomontana J.Graham, the present work has undertaken to lay down the standards, which could be useful for establishing its authenticity. The parameter observed may be useful for 48

2 identification of the plant material. Macroscopic and microscopic analysis of the rhizome acts as a reliable source for detecting adulteration Macro and microscopic analysis of rhizome of Curcuma pseudomontana J. Graham Macro and microscopic studies of C. pseudomontana reveals that the rhizome was aromatic, bitter in taste, buff colored and fleshy white inside, 3 to 6 cm in size with stout rootstock bearing small almond like sub-globose tubers at the ends of the fibrous roots to that of C.aromatica (Shantha et al., 1991). However, it is different in other Curcuma species. C.amada rhizome was pale brown with nodes and internodes, branched obliquely, with scars of undeveloped buds covered with leafy scales (Chitra et al., 2002). C.harita rhizome was laterally flattened, longitudinally wrinkled, branched, non-aromatic, pale yellowish green inside, sweet taste, tubers sessile, fleshy, finger shaped, branch roots branching sympoidal, horizontal, thread like yellowish brown (Srivastava et al., 2006). C.domestica rhizome was irregular in shape, orange yellow; surface rough, pungent, and slightly bitter aromatic (Mazumder et al., 2007). C.caesia rhizome was tuberous with camphoraceous sweet odor, irregularly shape, laterally flattened covered with adventitious roots and scars with longitudinal circular zones, bluish black (Paliwal et al., 2011). Z.zerumbet rhizome are irregularly branched with nodes and internodes, scales present at the nodal region, surface smooth, covered with trichomes and fibers, light grayish yellow, hard and brittle, fragrant, aromatic, spicy and slightly bitter, with abundant starch grains and oxalate crystals (Prakash et al., 2011). The rhizome of C.neilgherrensis was conical in shape, brownish with mild aromatic flavor (Yasodamma et al., 2014). Transverse section (T.S) of rhizome of C. pseudomontana shows an outermost single layered epidermis and wide central stellar region. Single celled 49

3 trichomes. Cortex is multilayered, wider, parenchymatous cells containing starch grains, oleoresin cells, and prism shaped calcium oxalate crystals. Vascular bundles are collateral, conjoint, closed and scattered. Xylem vessels are spiral shaped. Fibres occur in the groups and found associated with vessels. Xylem vessels walls are thin marked with numerous pits, which was similar to that of C.aromatica rhizome (Shantha et al., 1991) Physicochemical analysis of rhizome of Curcuma pseudomontana J. Graham The physicochemical parameters are mainly used in judging the purity and quality of the drug. An ash value of a drug gives an idea of the earthy matter or inorganic composition or other impurities present along with the drug. The ash values are important since ash may be derived from the plant itself (physiological or natural ash) as well as from the extraneous matter, especially sand and soil adhering to the surface of the drug (non-physiological ash). The determination of physiological and non-physiological ash together is called as total ash. The total ash may vary within wide limits for specimen of genuine drug due to variable natural or physiological ash, in such cases the ash obtained is treated with acid in which most of the natural ash is soluble leaving the silica as acid- insoluble ash which represents most of the ash from the contaminating soil. Higher values of Total ash content and less acid insoluble ash value in the rhizome clearly indicated the purity of the test drug and the presence of higher inorganic contents. The watersoluble ash used to estimate the amount of inorganic compound present in the drug. The ash values of the powdered rhizome revealed a high percentage of sulphated ash. 50

4 In current study C.pseudomontana rhizome contains 8% total ash, 8 % water insoluble ash, 3% acid insoluble ash, 13% sulphated ash. Present observation reveled that higher values of total ash content and less acid insoluble ash values in both the species clearly indicated the purity of the test drugs and the presence of higher inorganic contents. Similarly, the earlier studies of physicochemical results showed that the total ash content was 6, 7.4, 8.5, and 20 %. Watersoluble ash content was 13.6, 19.5, 19.5 and 24 % in rhizomes of C.caesia, C.harita, C.domestica and C.amada respectively (Chitra et al., 2002; Srivastava et al., 2006; Mazumder et al., 2007; Paliwal et al., 2011). Whereas alcohol insoluble ash content was 6, 7.4, 8.5 and 9.5% in rhizome of C.caesia, C.domestica, C.harita and Z. zerumbet respectively (Sharad Srivastava et al.,2006, Mazumder et al., 2007; Paliwal et al., 2011; Prakash et al., 2011). In rhizome of C.caesia, acid insoluble ash content was 4 % and sulphated ash content was not determined in any of above plant species. Extractive values give an idea about the chemical constituents present in crude drug and help to estimate the specific constituents, which are soluble in a particular solvent. Results of the present study suggest that the powdered rhizome of C. pseudomontana has high alcohol soluble extractive value then water-soluble extractive value. Similarly, in C.harita rhizome water-soluble extractive was higher than alcohol soluble (Srivastava et al., 2006). The extractive value of rhizome of C.caesia highest in methanol, followed by hexane (Paliwal et al., 2011). The loss on drying reveals the percentage of moisture present in the drug, since moisture facilitates the enzyme hydrolysis or growth of microbes that leads to deterioration (Ansari 2006; Thomas et al., 2008). In the present study, C.pseudomontana has 6.45 % loss on drying or moisture content. Similarly, the 51

5 moisture contents in rhizomes of C.caesia, Z. zerumbet and C.domestica has 9 %, 11 %, and 12.5 % respectively (Mazumder et al., 2007; Paliwal et al., 2011; Prakash et al., 2011). In the present investigation, the moisture content of rhizome of C. pseudomontana is less, whereas in above reported plants moisture content was more. This shows that C. pseudomontana less proven to microbe infection Fluorescence analysis of rhizome of Curcuma pseudomontana J. Graham Fluorescence is an important observable fact exhibited by various chemical constituents show fluorescence in the visible range in daylight. The UV light produces fluorescence in many natural products (Alkaloids like berberine) which do not visibly fluorescence in day light. If the substances themselves are not fluorescent, they may often be converted into fluorescent derivatives by applying different chemical reagents hence some crude drugs are often assessed qualitatively in this way and it is an important parameter of pharmacognostical evaluation (Ansari 2006; Thomas et al., 2008). Fluorescence analysis of powder rhizome of C.pseudomontana powder alone shows Brown color in visible, short (254 nm) and long (365 nm) wavelength light. The rhizome powder with NaOH showed pale yellow, yellowish green and dark blue color. In 1NHCL, rhizome powder showed pale orange, dark brown, brown color show in C.pseudomontana rhizome. Powder with nitric acid showed pale yellow, pale green and dark green color. Brownish red, brown and blackish brown were observed in powder with sulphuric acid. under visible, short, and long wavelength lights respectively in C.pseudomontana rhizome. Kadam et al., (2013) have reported that reddish, brown, and yellowish brown in 10% NaoH with rhizome powder of Clonga. Yellow, pale green and yellow color in concentrated HCL. Orange, green, and brown 52

6 color in HNo 3. Brownish black, dark brown and greenish black observed in sulphuric acid under visible, short, and long wavelength lights. Yasodamma et al., (2014) have reported that, reddish yellow, reddish brown, and brown in NaoH with rhizome powder of C.neilgherrensis. Black, green and thick brown in 50% HCL. C.neilgherrensis rhizome showed ash, black, and black in HNO 3 and reddish brown, black, black observed in sulphuric acid Analysis of mineral elements and nutritive value of rhizome of Curcuma pseudomontana J. Graham The nutritive value shows that the rhizome of C. pseudomontana is a good source of carbohydrate and protein; these may serve as a source of energy and nutrients for the body metabolic activities in addition to its medicinal properties. The carbohydrates and proteins present in the plant may be a conglomerate of bioactive sugars and proteins, which give the plant its medicinal potency against certain diseases. Some plants were known to contain certain sugars that are biologically active against some diseases (Hokputsa et al., 2004). The dietary fibre is important for lowering blood cholesterol and blood sugar, which reduce the risk of diseases such as obesity, diabetes, breast cancer, hypertension, and gastrointestinal disorder (Saldanha et al., 1995). The mineral nutrition is an important aspect and it play pivoted role in human life for healthy growth. Minerals such as sodium and potassium, which are present in the intracellular and extracellular fluid, help to maintain electrolyte balance and membrane fluidity. Iron is a component of some metalloenzymes, myoglobin, and hemoglobin. Magnesium and zinc are considered necessary as cofactor in enzyme catalysis in the body (Ahmed et al., 2009). Calcium was known to play a significant role in muscle contraction, bone and teeth formation and blood clotting (Heaney et al., 53

7 2009; Peters et al., 2010). Mn is essential for hemoglobin formation, but excess is harmful (Indrayan et al., 2005). Cu is also a component of many enzyme systems such as cytochrome oxidase, lysyl oxidase and ceruloplasmin, an iron-oxidizing enzyme in blood (Mills et al., 1981). Based on analysis the primary metabolite such as protein, carbohydrate, starch and secondary metabolites has found to be higher in rhizome due to the presence of larger chemical storage parts like vascular bundles and cortex in rhizome Behaviors of rhizome powder of Curcuma pseudomontana J. Graham Prior to the phytochemical screening rough estimation of phytoconstituents was done by the behavior of powder drug with different chemical reagents in which powdered drug showed different colors when it got mixed with the particular reagents and found that different drugs exhibit different colors. Those colors were characteristic for the particular drug, which reflects the presence of phytochemicals in accordance with the colors obtained (Pratt et al., 1949; Kokashi et al., 1958; Pharmacopeia of Indian. 1996). Behaviors of rhizome powder with different chemical regents showed the presence of various phytochemical compounds in the rhizome which are known to have various therapeutic importance in medical sciences Phytochemicals Analysis of rhizome extracts of Curcuma pseudomontana J. Graham Phytochemicals are compounds found in plants that are not required for normal functioning, but have a beneficial effect on health or play an active role in amelioration of diseases. In fact, some people claim that many of the diseases afflicting human beings are the result of lack of phytonutrients in their diet (Ayoola et al., 2008). Natural products such as secondary metabolites of plants are of restricted occurrence. They are non-essential for the normal physiology and hence, in the 54

8 survival of plants, in contrast to the other organic compounds in nature: sugars, amino acids, nucleotides, etc. which are essential to the plant for survival. They fall into major groups such as alkaloids, terpenoids, steroids, phenylpropanoids, etc. Phytonutrients have various health benefits, for example, they may have antimicrobial, anti-inflammatory, cancer preventive, antidiabetic, and antihypertensive effects. The phytochemical constituents of a plant will often determine the physiological action on the human body (Ayoola et al., 2008). Phytochemicals are produced by shoot part of the plants, their functions are unknown, but certain phytochemicals have structural, functional and defence against plant pathogens hence preliminary phytochemical analysis is important, because crude drugs posses varying compositions of secondary metabolites (Naidu and Shridar, 2011). In the study, phytochemicals analysis of rhizome extracts of C.pseudomontana mainly revealed the presence of alkaloids, steroids, saponins, glycoside in hexane extract. Alkaloid, flavonoid, saponins, carbohydrate, and proteins, amino acids present in chloroform and methanol extract. Alkaloid, flavonoid, tannin, steroids, saponins, glycoside carbohydrate, and proteins, amino acids present in the crude rhizome extract of ethyl acetate. All other species of curcuma contains all of the above secondary metabolites, except saponins (Chitra et al., 2002, Srivastava et al., 2006, Mazumder et al., 2007, Paliwal et al., 2011). Present finding lends support the important fact that this plant possesses a serum cholesterol lowering effects (Yasni et al., 1993). However, rhizomes of C.neilgherrensis and C. xanthorrhiza contain saponins (Yasodamma et al., 2014). The results of phytochemical analysis showed the presence of various phytochemical compounds in the rhizome which are known to have various therapeutic importance in medicinal sciences. 55

9 Alkaloids and flavonoids are the source of antimicrobial activities. Tannins may have the potential values as cytotoxic agent (Aguinaldo et al., 2005). Tannins are present in the ethyl acetate extract. Hence, the ethyl acetate extract may be used as anti-cancer agent. Phenolic compounds are the important source for antimicrobial and insecticidal activities. Saponins are naturally occurring glycosides, that were typically characterized by their foam forming ability in water, hemolytic activity, and cholesterol binding properties (Sodipo et al., 2000). Saponins have been implicated, as bioactive antibacterial agents (Mandal et al., 2005). The interesting feature of saponins was the ability of this compound to inhibit or kill cancer cells without destroying normal cells in the process. Cancer cells possess more of cholesterol type compounds on their membrane compared to that of normal cells. Saponins therefore bind to the cholesterol and thus interfere with cell division and cells growth (Ryan et al., 1994). TLC profile of the present study will help in further isolation of bioactive compounds from C. pseudomontana rhizome. 56