INTRODUCTION:

Quirivelia frutescens Botanical name: Ichnocarpus Frutescens (Linn) comes from the family Apocynaceae.It has many synonyms such as Kalidudhi, Shyamalata Ananta, Bhadra, Chandanagopy, Dudhhd, Krishna-Sarwa , Kante-bhouri etc.^[1,2,3]

According to the literature survey done, the plant is reported to be effective for mouth ulcer, sore throat etc. Fresh roots have been found to be very effective in jaundice. The methanolic extract of the aerial parts of plant have shown strong anti-oxidant activity.The literature survey for this plant also suggests that the plant is not much explored pharmocognostically and no extensive work is seen with respect to its phytochemical account. Therefore the present work is planned to explore the plant for its pharmocognostic, phytochemical, spectroscopic and chromatographic behavior.

MATERIALS AND METHODS:

Authentification of Plant was done by Dr. S.C. Mujumdar, Botanical Survey of India, Pune.(BSI/WC/Tech/2008/510-ANAP1)

Pharmacognostic study:

The fresh plant of the Quirivelia frutescens was collected from Kolkatta West Bengal. External visible characters such as size,shape, diameter and organoleptic properties of fruits were studied and recorded. The characters were matched with the morphological characters reported in the literature.

Microscopical Study:

A thin transverse section and powder characteristics of the stem were studied.

Microscopic descriptions of tissues were supplemented with micrographs. Photographs of different magnifications were taken with Nikon Labphot 2 Microscopic Unit .For normal observations bright field was used. For the studies of crystals, starch grains and lignified cells, polarized light was employed. Since these structures have birefringent property, under polarized light they appear bright against background. Magnifications of the figures are indicated by the scale-bars.

Proximate analysis:

Physicochemical constants such as Extractive values, Ash values (Total, Acid insoluble and water soluble ash values),L.O.D. were studied using standard methods.

Qualitative Phytochemical Screening:

Powder was extracted by continuous hot extraction (soxhlet) method using methanol. This extract was concentrated and subjected to phytochemical screening. for presence of alkaloids, glycosides, flavonoids, tannins, terpenes, steroids etc^.[4,5]

Thin layer Chromatoraphy:

TLC of methanol extract of stem of Quirivelia frutescens was carried out. The resolution of TLC plates was tried in different solvent systems and best resolving system was chosen for running the plates. The plates were then exposed to i) UV-light at 365 nm, ii) Iodine-vapour .

Extraction:

Extraction Procedure was carried out using the following procedure:

Fresh plant material was taken and homogenized with the methanol and water (4:1) for 30 minutes, filtered .Filtrate was concentrated, acidified and extracted with chloroform. The chloroform layer and the acid layer were separated. The acid layer was basified to pH 10 with ammonium hydroxide and again extracted with the chloroform. The extract was subjected to various forms of analysis mentioned below.^[6,7,8]

Spectroscopic Evaluation:

UV Analysis:

UV Visible analysis was carried out using Jasco spectrophotometer V-7000 SERIES. For determination of λ_max 100mg of the above extract was dissolved in 100ml of methanol which gave 1mg / ml solution. The above stock solution was used for making dilutions of 100 μg/ml, 50 μg/ml, 20 μg/ml, 10 μg/ml.

Infrared Analysis:

Jasco FTIR was used for infrared analysis.

Nuclear Magnetic Resonance:

Varian 700 MHz NMR Magnet was used to perform NMR .

Gas Chromatography Mass Spectroscopy:

GCMS was carried out from Department of Chemistry, University of Pune.

RESULTS

The results are computed in tables and figures.

Table I: showing the T.S. stained with different reagent.

Sr. No.	Reagent	Observation	Inference
1	Phlorglucinol + HCl	RED	Lignified xylem
2	Dil. Iodine solution	BLUE	Starch grains present
3	Sudan red	RED	Cuticle
4	Sulphuric acid	SOLUBLE	Ca.Oxalate crystals

Pharmacognostic study, of the stem of Quirivalia fructescens showed presence of well defined tissues like xylem vessels,cortical parenchyma etc., leaves showed anomocytic stomata, bicollateral vascular bundle. Powder showed presence of the calcium oxalate crystals in the form of thick bundle called raphides. xylem elements with circular bordered pits were also observed .

Figure I: T.S. of Stem (stained with Phlorglucinol)

Figure II: T.S. of Stem (Stained with iodine)

Figure III: Palisade layer

Physicochemical Constants

Table II: Table representing the physicochemical constants

Sr No	Parameters	Result (%w/w)
1	Total ash	9.75
2	Acid Insoluble Ash	1
3	Water soluble Ash	4
4	Alcohol Soluble Extractive	8
5	Water Soluble Extractive	10

Table III: Phytochemical Investigation of Quirivelia frutescens Extract

Sr No	Test	Ethanol extract
1	Alkaloids	+
2	Carbohydrates	-
3	Glycosides	-
4	Flavonoids	-
5	Saponins	-
6	Proteins and Amino acids	-
7	Phytosterols	-
8	Tannins	+
9	Fixed Oil and Fats	-
10	Mucilage	-

Table IV: TLC profile of Ethanol extract of Quirivelia frutescens

Compound	Tannins	Alkaloids
Solvent for preparation of Sample	90% Ethanol	90% Ethanol
Stationary phase	Silica Gel	Silica Gel
Mobile phase	Ethylacetate: glacialacetic acid: water (90:10:10)	Chloroform: Methanol: Diethylamine (80:20:10)
Visualizing agent	5% FeCl₃ solution	Iodine vapors
Rf value	0.77	0.79

Figure IV TLC of alkaloid Figure V TLC of Tannins

ULTRAVIOLET AND VISIBLE SPECTROSCOPY PROFILE

Sr No	Concentration (µg/ml)	Absorbance
1	100	331
2	50	332
3	20	336
4	10	337

Table V: showing absorbance at given concentration

Figure VI: Infrared Spectroscopy graph, peaks indicating presence of alkaloids and tannins.

Interpretation of Infrared spectroscopy:

The Alkaloid extract subjected to Infrared spectroscopy show peaks between 1419.35 to 1646.91 cm ^-1 indicating presence of aromatic amides.Peaks between 2299.7 to 28.48 cm^-1 show stretching of C-H bond .Peak at 3598.52 cm^-1 shows presence of hydroxyl group and peak at 744.388 cm^-1 shows presence of substitution of aromatic ring in structure.

Nuclear Magnetic Resonance Spectroscopy Graph

Figure V11: Graph of H- NMR

Figure V111: Graph depictingC₁₃ NMR

Interpretation of nuclear magnetic resonance spectroscopy

In the above graph, three parallel peaks between 78 to 82 ppm are because of solvent CDCl_3.The peak between 117 to 126 ppm shows presence of unsaturation.A high peak is obtained in the graph which shows the presence of the CH₃chain. Many small peaks show presence of alkaloids and steroids. From general observation, we can conclude that the compound possesses saturated ring substituted with aliphatic chain.

Gas Chromatography and Mass spectroscopy

Figure IX :Chromatogram obtained from Dept of chemistry, Pune University showing probability of presence of alkaloids and tannins,

Interpretation of GCMS

The methanolic extract of the powder was subjected to GCMS. The above graph shows presence of 8 chemical moieties present in the extract .For the elucidation of structure first priority is given to the peak which is having the highest retention time. The peak which is obtained at the retention time of 12.726 occupied maximum area of the total ion chromatogram.

DISCUSSION:

Pharmacognostic study, of the stem of Quirivalia fructescens showed presence of well defined tissues like xylem vessels, cortical parenchyma. Leaves showed anomocytic stomata, bicollateral vascular bundle. Powder showed presence of the calcium oxalate crystal in the form of thick bundle called raphides^[9,10,11]. Xylem bundle is seen in the powder as short, thick xylem vessels with circular bordered pits. The total ash, acid insoluble and water soluble ash were found to be within the limit as compared to other plant belonging to this family. The preliminary phytochemical tests of extract indicated the presence of alkaloid and tannins. In phytochemical analysis, TLC, UV, IR, NMR, GCMS were carried out to study the chemical moiety and functional groups present in the plant. In TLC, the R_f value was found to be 0.77 which is near to many alkaloids. In NMR, the peak showed presence of unsaturation between conjugated carbon, alkaloid and steroid group and saturated ring substituted with aliphatic chain. GCMS spectrum of the extract showed the presence of eight chemical constituents. These structures were very similar to those seen in alkaloids and tannins . In UV, the λmax is found between 331 to 337 nm .Thus the main aim of this project to investigate the important functional groups present in plant is achieved. ^{[12, 13]}

ACKNOWLEDGEMENTS:

We are grateful the Principal, Dr. Kiran Bhise, and the management of the college for providing us the necessary infrastructure for the research work.

REFERENCES:

1. Patwardhan, B., Joshi, K., Chavan, P., Warude, D.Bioprospecting Laboratory, Interdisciplinary School of Health Sciences, University of Pune, current science, 2004;87, 2: 159-165.

2. Cox, P, Balick M. The ethnobotanical approach to drug discovery. Scientific American, 1994; 82-87.

3. Ramakrishnappa, K. Impact of cultivation and gathering of medicinal plants on biodiversity a case study from India, biodiversity and the ecosystem approach in agriculture, Forestry and Fisheries, 2004;A: 021.

4. Butler, M., Buss, A. D. A new model for utilizing chemical diversity from natural sources, Drug development research 2004; 62: 362–370.

5. Mathur, A.Who owns Traditional Knowledge, Working Paper No. 96, Indian Council for Research on International Economic Relations, 2003; 1-33.

6. Pal, S., Shukla, Y.Herbal medicine current status and future, Asian pacific journal of cancer prevention, 2004; 4:281-288.

7. Raskin, I., Ribnicky, D.M., Komarnytsky, S., Ilic, N., Poulev, A., Borisjuk, N., Brinker, A. Plants and human health in the twenty-first century, Trends in biotechnology,2004; 20:2,522-531.

8. Endress, M.E. and P.V. Bruyns. A revised classification of the Apocynaceae.2002; 66: 1-56.

9. K.R Khandelwal; Practical Pharmacognosy; 17^th edition; Nirali Publication, pp30-37.

10. The Wealth of India; Raw materials; vol-7^th; pp200-201.

11. Indian Medicinal Plant; Text Vol-3rd by Kirtikar and Basu; pp1602.

12. Trease and Evans; Pharmacognosy by William Charles Evans; 15^thedition.

13. Henry, A.N., Nair, N.C., Flora of Indian medicinal plants, Botanical survey of India, pp 184.

Received on 09.08.2009 Modified on 29.09.2009

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 509-512