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Research Article Pharmacognosy

International Journal of Pharma and Bio Sciences ISSN

0975-6299

PHARMACOGNOSTIC EVALUATION OF RHIZOME OF

CURCUMA PSEUDOMONTANA J. GRAHAM

GURUSIDDESH B. HIREMATH AND BASAPPA B. KALIWAL *

Department of Studies and Research in Biotechnology and Microbiology,

Karnatak University, Dharwad – 580 003, INDIA

ABSTRACT

The present work carried out to establish the necessary pharmacognostic standards, according to the WHO guidelines for evaluating the plant material of Curcuma pseudomontana J. Graham rhizome. Pharmacognostic parameters such as macroscopic, microscopic, physicochemical constituents, fluorescence analysis, nutritive value, behavior analysis of rhizome powder and TLC studies were performed. Macroscopic and microscopic evaluation of the rhizome show aromatic, fleshy white inside, starch grains, oleoresin cells, and prismatic calcium oxalate crystals. Physicochemical analysis of the rhizome powder showed 8% total ash, 3% acid insoluble ash, 8 % water insoluble ash, 13% sulphated ash, 11.5% water-soluble extractive, 13.5% alcohol soluble extractive and 6.45% loss on drying. Analysis of nutritive value of rhizome showed, rich source of protein, carbohydrate, dietary fibre and minerals. Behavior analysis of rhizome powder showed presence of steroid, tannins, starch, alkaloid, flavonoid and protein. This is the first report on the pharmacognostic studies of Curcuma pseudomontana J. Graham rhizome. The results of pharmacognostic analysis were helpful in developing standards for quality, purity, and sample identification.

KEYWORDS: Curcuma pseudomontana J. Graham, Physicochemical properties, Fluorescence analysis and Nutritive value.

BASAPPA B. KALIWAL

Department of Studies and Research in Biotechnology and Microbiology,

Karnatak University, Dharwad – 580 003, INDIA

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INTRODUCTION

Curcuma pseudomontana J. Graham belongs to the family Zingiberaceae, commonly known as Hill Turmeric. C. pseudomontana has, small root stock, bearing small almond like or subglobose tubers at the ends of the fibres (but no sessile tubers); tubers pure white inside and it is edible. Leaves are uniformly green, reaching 2ft or more long (including the petiole), 4-6’ broad, lanceolate oblong acuminate, tapering to the base, petioles 8-15 in long. Flowers are bright yellow appearing with the bracts, 2 or 3 in each bract, in autumnal central narrowly oblong spikes 2-5 by 1-1 ¾ inch; peduncles 3-4in long embraced by leaf- sheaths; flowering bract 1 ¼- 1 ¾ by 5/8 – 7/8 inch., obovate- lanceolate, the lowest with purple edges only1.Flowering starts from the month of June and ends in the month September. Curcuma pseudomontana is endemic to the Western and Eastern Ghats, of peninsular India. The plant found in Karnataka, Maharashtra and Andhra Pradesh, it is potentially ornamental species and so far the Curcuma pseudomontana J. Graham known only from India 2 Curcuma pseudomontana J. Graham known as Tavaksheera (Ayurveda) Kachura (Hindi), Raan halada, shindalavana or shindalavani (Marathi), Kattu manjal (Tamil), Kattu manjal (Malayalam). The Savara, Bagata, Valmiki tribes of Andhra Pradesh use tuber extracts to cure jaundice and Bagata tribes use this plant for Diabetes3 . Jatapu and Kaya tribes apply warm tuber paste to treat body swellings. Women of Jatapu and Savara tribes eat boiled tubers to increase lactation4 . Khand tribes apply the tuber paste on the head for cooling effect, crushed and boiled rhizome is edible5 . The Kukus-Mukus eat fresh tubers as a blood purifier6 . The tubers are edible7 . Rhizome past used to apply to wounds and cuts8 -9 . C. pseudomontana rhizome is also beneficial against leprosy, dysentery, cardiac diseases10. In the recent decades, peoples are more attracted towards plant origin drugs as they are highly biocompatible with lower side effects than the synthetic drugs. Such drugs are very prone to adulteration if the supply of crude drug

is poor. This adulteration could be prevented by various pharmacognostic parameters such as morphological, anatomical and biochemical characteristics of plant material11. Nutrient analysis of wild edible plants plays an essential role in assessing their nutritional importance12

.The considerable use of wild edible rhizome species by the local people in their diet impelled us to carry out the present nutrient analysis. In spite of the various medicinal uses attributed to this plant, there is no pharmacognostical report on the rhizome of the plant till date. There are also no data available on the anatomical and other physicochemical standards required for the quality control of its crude drug. Hence, the present investigation has been undertaken to determine the pharmacognostical standards for authenticating the plant material of rhizome of Curcuma pseudomontana J. Graham.

MATERIALS AND METHODS

Procurement of Plant Materials Fresh and healthy rhizomes of Curcuma pseudomontana J. Graham (Zingiberaceae) was collected from the Western Ghats, Karnataka, India, during September 2012. Taxonomist Prof, G.R. Hegde, Department of Botany, Karnataka University Dharwad, India, authenticated the plant. The rhizomes were dried in hot air oven at 450C for 72 hours and powdered for further analysis. Macroscopic and microscopic analysis The macroscopic and microscopic analyses were carried out according to the procedure of WHO guidelines11 and Khandelwal13 . Physiochemical analysis Physiochemical parameters such as total ash, water-soluble ash, acid-insoluble ash and sulphated ash, loss on drying and extractive value were performed according to the pharmacopoeia of India14 .

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Fluorescence analysis Fluorescence analysis was performed by the method described in the Kokashi (1958)15 and Patt (1949)16 . The rhizome powder was treated with various acidic and basic solvents and were then observed in visible and UV short wave and long wave regions at the same time. Behaviors of rhizome powder Behaviors of rhizome powder of C. pseudomontana with various chemical regent has performed to detect the occurrence of phytoconstituents along with color change under ordinary daylight by standard method of Sing and Govil17 . Analysis of Nutritive value and minerals For determination of nutritive value and various minerals in the rhizome of C. pseudomontana the following parameters were studied by using the powder of rhizome. Estimation of ash, moisture, crude fat, crude fibre, crude protein, and carbohydrate. Estimation of minerals, such as Potassium, calcium, magnesium, manganese, iron, copper, sulphur and zinc in plant material by Indrayan18 . Thin Layer Chromatography The sample was applied with the help of micropipette on precoated silica gel G60 F254 TLC plates (Merck) Different combinations of solvent system were tried for best separation of constituents by Stahl19 .

RESULTS

Macroscopic and microscopic analysis Based on the macroscopic evaluation the plant rhizome is aromatic, bitter in taste, buff colored and fleshy white inside, 3 to 6 cm in size with stout root stock bearing small almond like sub-globose tubers at the ends of the fibrous roots (Fig. 1A, B and C). Microscopic observation illustrates that T.S of rhizome shows an outermost single layered epidermis and wide central stellar region occupying 2/3rd the area of the section. Single celled trichomes. The 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 vessel walls were thin marked with numerous pits (Fig.1D-H). Powder microscopy show that the presence of spherical shaped xylem vessels, fibers and varying size of starch grains (Fig. 1I-K) in the rhizome. Physiochemical analysis In this study, various physiochemical parameters such as total ash, water-soluble ash, acid-insoluble ash and sulphated ash, loss on drying and extractive value were determined and results are given in Table 1.

Table 1

Physicochemical analysis of Curcuma pseudomontana J.Graham rhizome.

Sr.No Parameters Value(%) w/w

1 Ash 8

2 Acid insoluble ash 3

3 Water insoluble ash 8

4 Sulphated ash 11.5 5 Loss on drying 6.45

6 Water soluble extractives 11.5

7 Alcohol soluble extractives 13.53 * w/w: weight/weight

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Fluorescence analysis

The fluorescence analyses of C.pseudomontana J.Graham rhizome were done with different acidic and basic solvents and were then observed in visible light, short and long wavelength. The result is was given in Table 2.

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Table 2 Fluorescence Analysis of Curcuma pseudomontana J.Graham rhizome.

Sr.No Treatment

Visible light

UV short (254 nm)

UV long (365 nm)

1 Powder Brown Brown Brown

2 Powder + Noah Pale Yellow Yellowish

Green Dark Blue

3 Powder + 1N HCL Pale orange Dark Brown Brown

4 Powder + Nitric acid Pale Yellow Pale Green Dark Green

5 Powder +Sulphuric acid

Brownish red Brown Blackish Brown

Analysis of Nutritive value and minerals The analysis of Nutritive value and minerals showed the presence of N 1.63%, P 0.134%, K 2.194%, Na 0.197 %, S 0.905 %, Ca 0.368 %, Mg 0.253 %, Cu 15.4 ppm, Zn 212.1 ppm, Fe 314.89 ppm and Mn 255.35 ppm in powder of rhizome and is given in Table 3 and 4.

Table 3 Mineral elements of Curcuma pseudomontana J. Graham rhizome

Sr.No Elements Elemental content

1 2 3 4 5 6 7 8 9 10 11

N P K Na S Ca Mg Cu Zn Fe Mn

1.63% 0.134% 2.194% 0.197 % 0.253 % 0.905 % 0.368 % 15.4 ppm 121.1ppm 314.89 ppm 255.35 ppm

Table 4 Analysis of Nutritive value of Curcuma pseudomontana J. Graham rhizome

Behaviors of rhizome powder The behavior of rhizome powder with different chemical reagents was studied to detect the presence of phytoconstituents with color changes under day light and the results showed the presence of steroid, tannins, starch, alkaloid, flavonoid and protein given in Table 5.

Sr.No Ash Fat Moisture Protein Carbohydrate Crude fiber Nutritive value

1 8 % 8.36% 11.02% 10.18% 62.44% 14.0 % 365.72%

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Table 5 Behavior of powdered Curcuma pseudomontana J.Graham rhizome

Sr.No Reagents Colour/ppt Constituents

1 Powder as such Brown -

2 Concentrated H2SO4 Brownish red Steroid (+)

3 Aqueous ferric chloride black color Tannins (+)

4 Aqueous iodine solution Blue color Starch (+)

5 Aqueous mercuric chloride Brown Alkaloid (+)

6 Picric acid Yellow ppt Alkaloid (+)

7 Magnesium HCl Pink Flavonoids (+)

8 Aqueous silver nitrate solution No change Protein (+)

+: present, -: absent

Thin Layer Chromatography By optimization of the solvent system, Hexane (petroleum ether 9.5: Ethyl acetate0. 5), Chloroform (toluene 9: ethyl acetate1), Ethyl acetate (ethyl acetate 9: actone1) and Methanol (chloroform 9.5: ethyl acetate 0.5) were found to be the best solvent system for separation of bands in TLC and Rf value is given in Table 7 (Fig. 2) It plays pivotal in the purification of bioactive compounds.

Table 7 The TLC profile of the powdered Curcuma pseudomontana J.Graham rhizome

Sr.No Extract Wavelength Number of Spots

Rf values

1 Hexane 254nm 8 0.87, 0.76, 0.58, 0.52, 0.30, 0.22, 0.15, 0.04

366nm 4 0.93, 0.60, 0.42, 0.38

2 Chloroform 254nm 9 0.91, 0.81, 0.72, 0.62, 0.49, 0.40, 0.32, 0.25, 0.16

366nm 3 0.96, 0.74, 0.59,

3 Ethyl acetate 254nm 7 0.87,0.43,0.36,0.32,0.23,0.16,0.12

366nm 3 0.6, 0.41, 0.34

4 Methanol 254nm 9 0.96, 0.72, 0.55, 0.96, 0.32, 0.29, 0.20, 0.17, 0.13

366nm 4 0.74, 0.46, 0.31, 0.22

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DISCUSSION

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 essential20 . 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 materials21 . 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 identification of the plant material. Macroscopic and microscopic analysis of the rhizome acts as a reliable source for detecting adulteration. Ash values are used to find out the quality and purity of crude drugs. It indicates the presence of various impurities like carbonate, oxalate and silicate. The water-soluble ash used to estimate the amount of inorganic compound present in the drug. The acid insoluble ash consists mainly silica and indicates contamination with earthy material. 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. Moisture content of drugs could be at minimal levels to depress the growth of bacteria, yeast or fungi during storage. The extractive values are useful to evaluate the chemical constituents present in crude drug and also help with estimation of specific constituents soluble in a particular solvent22 .

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 (e.g. 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 evaluation22. 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 obtained15 -17. 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. TLC profile of the present study will help in further isolation of bioactive compounds from C. pseudomontana rhizome. The nutritive value shows that the rhizome of Curcuma pseudomontana J. Graham 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 are known to contain certain sugars that are biologically active against some diseases24. 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 disorder25 .

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

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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 body25. Calcium is known to play a significant role in muscle contraction, bone and teeth formation and blood clotting27-28. Mn is essential for hemoglobin formation, but excess is harmful18 . Cu is also a component of many enzyme systems such as cytochrome oxidase, lysyl oxidase and ceruloplasmin, an iron-oxidizing enzyme in blood29 . Based on the 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.

CONCLUSION

The rhizome used under this study can be utilized as potential source of useful therapeutics and the outcome data will be beneficial for herbal preparation containing rhizome of C. pseudomontana. Analysis of nutritive value and minerals of rhizome

showed, rich source of protein, available carbohydrate, dietary fibre and minerals. This rhizome can be used for nutritional requirement of human being attributable to the good qualities of nutrient and it may give adequate protection against diseases arising malnutrition. Further studies are in progress on this rhizome in order to isolate, identify, characterize and elucidate the structure of bioactive compounds along with investigation of their pharmacological activity. CONFLICT OF INTEREST Conflict of interest declared none.

ACKNOWLEDGEMENT

The authors are grateful to the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, New Delhi, for funding the Bioinformatics Infrastructure Facility Project (BT/BI/25/001/2006 VOL II dt 05-03-2012). Interdisciplinary Program for Life Science Project (BT/PR/4555/INF/22/126/2010 dated 30-09-2010) and P. G Departments of Microbiology and Biotechnology Karnatak University, Dharwad for providing the facilities.

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