Phytochemical Investigations of the Medicinal Plant Swertia Chirata Ham

Natural product chemistry is an ancient science. Different medicinal compounds such as antitumor, anticancer, anti AIDS etc were isolated from natural plants. Isolation of any compound from any plants is uncertain. None can assure it before investigation. But generally secondary metabolites are obtained by proper investigation. Plants are unique in their ability to synthesize fats, carbohydrates, proteins that constitute three major food classes for human race [1]. The array of medicines derived from them is impressive and includes hypotensive drugs. analgesics, anaesthetics, anticancer and antiparasitic compounds, antiinflammatory drugs, steroids, laxatives, diuretics and many others [2]. Theophrastus [3] a student of plato, in his historia plantarum described the use of nearly 500 medicinal plants. A vast compilation of ancient knowledge of medicinal plant, can be seen in the natural history of pliny [4], the famous Roman physician. The Chinese Emperor Shen Nung (3000 BC) [5] compiled a treatise on medicinal plants. The medicinal plants that have been used since ancient time, many have yielded most useful drugs that are very much in use in current medicine. Such asThe most important natural analgesic, morphine [6] was isolated from opium poppy (latex of Papaver somniferum fruit). Quinine [6], an antimalarial alkaloid, was isolated from cinchona bark (Cinchona succirubra) which was used by the South Americans and the Indians. Emetine [7], which is considered as an important medicine for amoebiasis, is the main alkaloid obtained from the root of ipecacuanha (Cephaelis ipecacuanha) was used in Brazil and Far east for dysentery and diarrhoea. Reserpine [8], used as a hypotensive drug and tranquilizer was isolated from the plant Rauwolfia serpentia, considered to be a common remedy for mental illness and snake bite in the Indian sub continent. Bronchodilating effect of ephedrine from Ephedra vulgaris [6].


Introduction
Natural product chemistry is an ancient science. Different medicinal compounds such as antitumor, anticancer, anti AIDS etc were isolated from natural plants. Isolation of any compound from any plants is uncertain. None can assure it before investigation. But generally secondary metabolites are obtained by proper investigation. Plants are unique in their ability to synthesize fats, carbohydrates, proteins that constitute three major food classes for human race [1]. The array of medicines derived from them is impressive and includes hypotensive drugs. analgesics, anaesthetics, anticancer and antiparasitic compounds, antiinflammatory drugs, steroids, laxatives, diuretics and many others [2]. Theophrastus [3] a student of plato, in his historia plantarum described the use of nearly 500 medicinal plants. A vast compilation of ancient knowledge of medicinal plant, can be seen in the natural history of pliny [4], the famous Roman physician. The Chinese Emperor Shen Nung (3000 BC) [5] compiled a treatise on medicinal plants. The medicinal plants that have been used since ancient time, many have yielded most useful drugs that are very much in use in current medicine. Such as-The most important natural analgesic, morphine [6] was isolated from opium poppy (latex of Papaver somniferum fruit). Quinine [6], an antimalarial alkaloid, was isolated from cinchona bark (Cinchona succirubra) which was used by the South Americans and the Indians. Emetine [7], which is considered as an important medicine for amoebiasis, is the main alkaloid obtained from the root of ipecacuanha (Cephaelis ipecacuanha) was used in Brazil and Far east for dysentery and diarrhoea. Reserpine [8], used as a hypotensive drug and tranquilizer was isolated from the plant Rauwolfia serpentia, considered to be a common remedy for mental illness and snake bite in the Indian sub continent. Bronchodilating effect of ephedrine from Ephedra vulgaris [6].
Curare used as arrow poison by the South American natives has now given as tubocurarine [9], a quaternary alkaloid now being used as an adjunct to anesthesia for surgery. Anti-spasmodic effect of atropine [6], from Atropa belladona. Analeptic effect of strychnine [6] from Nux vomica and pelletierine, from pomegranate. Antileprotic effect of chaulmoogra fruit [6] was well known to the ancient Indians. At present thousands of plant metabolites are also now being successfully used in the treatment of a variety of diseases. A few striking examples of plant metabolites are as follow: Taxol [10], from Taxus brevifolia, Vincristine [11] and Vinblastine [11] from Vinca rosea Linn. (Periwink Plant), all of which are important anticancer drugs. Arrow poison of foxglove [12] consisting of digitalis glycosides is cardiotonic for man. The root bark of Mexican yam [13] is used in making cortisone and other steroidal drugs. Salicylic acid [14] isolated from willow bark has a variety of pharmacological effects on platelet aggregation, pain and immune system. Artemisinin, a new antimalarial from Chinese herbal medicine [15].
The plant under investigation is Swertia chirata Ham which belongs to the family Gentianaceae. The Gentianaceae is a tropical family of small trees, herb and bitter tonic. It consists of 180 species. About 8-10 species exist in India [16]. This plant is indigenous to temperature Himalayas at altitudes above 4000 feet from Kashmir, Nepal and Bhutan [17]. In this family all plants are use as medicine. In the present investigation the plant Swertia chirata Ham was selected for phytochemical and biological studies.
The plant Swertia chirata (Family: Gentianaceae) Ham was chosen for investigation since it has a folkloric reputation. So far literature surveyed, Swertia chirata Ham has tremendous uses in traditional medicines. It has anti-microbial activity against Gram positive and Gram negative bacteria. All the plant are used as astringent, unani-tonic to heart, liver, eyes, cough, scanty-urine, melancholia, dropsy, sciatia, skin diseases, the plant is used as a bitter tonic in gastrointestinal disorders, like dyspepsia/anorexia, it is used as digestive, febrifuge and laxative. It is used to prevent malaria, particularly useful in fever. The plant is also effective against intestinal worms burning of the body, bronchial asthma, regulating the bowels [18] (Figure 1).

Materials and Methods
Dried stem of Swertia chirata Ham (Locally known as chirata) were collected from Bhangura Kabiraji Shop of Rajshahi Shaheb Bazar. The stems of the plant were cut into small pieces by a sharp knife. About 650 Page 2 of 7 gm of the plant pieces were weighed by the electric balance and made paste by pestle-moter. The paste materials (stem) were taken in a clean flat bottomed glass container (2.5 litre) and macerated with sufficient amount of rectified spirit and with occasional shaking. After 15 days the solvents was decanted and filtered by Tincture filter press (Karl Kolb, Scientific-Technical Supplies, Frankfurt/M Germany) and then filtered through fresh cotton. The filtrate thus obtained was taken in a 1 litre beaker. The solvent (R.S) of the extract was evaporated under normal pressure and normal temperature to obtain a gummy mass. This mass was preserved in a refrigerator for chemical investigation. Rectified spirit extract of the plant of Swertia chirata Ham a crude product.
It contained a mixture of compounds. In this chapter, an attempt was made to isolate the individual compounds. TLC examination of rectified spirit extract, petroleum ether: ethyl acetate (1:2) showed 3 spots of R f values 0.1, 0.54 and 0.73, respectively.
The crude rectified spirit extract was subjected to an alumina column for fractionation. The column containing 10 gm of alumina was prepared as described before. Crude extract (2.5 gm) for the purpose was mixed with a little rectified spirit in a morter to get a free following mass. The sample was then placed carefully on the top of the prepared column and was successively eluted with n-hexane, n-hexane with increasing portions of petroleum ether; ethyl acetate and finally with methanol (Table 1). A number of colour bands were observed during the development of the column. The subsequent eluants were collected in 100 ml beakers ( Table 2).

Examination of the combined fractions
Fraction A: Fraction A gave no spot on TLC examination and was discarded.

Fraction B:
The residue (35 mg) from fraction B did not give any discrete spot with different solvent system and tailed badly and was not worked out further.

Fraction C:
The fraction showed two spots on the TLC, placed with solvent system n-Hexane: Ethyl acetate (3:1). The spots were pink in color under UV light. The band were contains R f value=0.67 and 0.583, respectively. The residue from fraction C was subjected to a small chromatographic column fitted with a cotton-plug, using Ethyl acetate: Toluene (1:4). The two eluants were collected and evaporated to get small amount of compounds C 1 and C 2 . The compounds C 1 and C 2 were insufficient quantities and were not worked out further.

Fraction D:
The fraction showed two spots (R f =0.53 and 0.73) on TLC plate using the solvent system Petroleum ether: Ethyl acetate (1:2). The spot having R f =0.73 showed a violet fluorescence under UV light and yellowish spot in iodine vapour. While the other spot having R f =0.53 was pink under UV light and yellow in iodine vapor. The components on chromatoplate did not react with potassium permanganate reagent and reacted with vanillin sulfuric acid reagent giving bluish violet spots.
The residue (110 mg) from fraction D was subjected on alumina to a small chromatographic column using petroleum ether: ethyl acetate (1:1). The two eluants were colleted and evaporated to get two compounds designated as AJ-1 (45 mg) and AJ-2 (8 mg).
The compound AJ-1 was crystalline but the compound AJ-2 was not crystalline. The compound AJ-2 was insufficient quantities and was not worked out further.
Fraction E and F: These two fractions appeared to be a mixture of components, which had R f values very close to each other and could not be separated. The fractions were therefore preserved for further studies in future.  The fractions were combined on the basis of their preliminary TLC examination to give combined fractions A, B, C, D, E, F (Table.2). Each combined fraction was evaporated to dryness under reduced pressure.

Isolation of the compound AJ-1 from small chromatographic column
From the TLC analysis of the fraction obtained from small column chromatography, it was observed that the compound AJ-1 contained only one compound. This was recystallized from methanol-ethyl acetate mixture.
Purification of the compound AJ-1: The compound AJ-1 was further recrystallized dissolving in petroleum ether: ethyl acetate (1:1) and the crystals were washed with different solvents of varying polarity when needle-shaped crystals were obtained.
Purity Test: This isolated compound AJ-1 was tested in different solvent systems for its purity. The compound showed a single spot on TLC examination. So this compound is pure. Finally its R f values were determined using the various solvent systems (Table 3).

Spectral characteristics of the compound AJ-1
Infrared (IR) Spectrum of the compound: Infrared (IR) spectrum ( Figure 2) of the compound in KBr (spectroscopic grade) pellet was recorded with a pye-unicam SP 3 -300 spectrophotometer. The samples were put in an agate mortar and thoroughly powdered with KBr and then transferred in a disc holder and a disc was made by hydraulic press. The KBr pellet was mounted in the sample cavity of the machine.

Preliminary extraction
The paste materials were taken in a clean flat bottomed glass container (2.5 litre) and macerated with sufficient amount of rectified spirit and with occasional shaking. After 15 days the solvents was decanted and filtered by tincture press. Then the extract was evaporated under normal pressure and normal temperature to obtain a gummy mass.

Examination of the rectified spirit extract
The rectified spirit extract showed at least three components (R f =0.4, 0.54 and 0.73) on thin layer chromatographic plates with Petroleum ether: Ethyl acetate (1:2). The rectified spirit extract was then subjected to column chromatography on alumina. The column was eluted successively with n-hexane, n-hexane-petroleum ether mixtures and finally with methanol ( Table 1). The fractions were combined on the basis of their preliminary TLC examination to give combined fractions A, B, C, D, E and F ( Table 2). The combined fractions were evaporated to dryness under reduced pressure. Fraction A did not give any residue.  TLC plates using solvent system Petroleum ether: Ethyl acetate (1:2). The fraction D was further subjected on mini column chromatography using Petroleum ether: Ethyl acetate (1:1). The two eluants were collected and evaporated to get two components designated as AJ-1 (40 gm) and AJ-2 (5 mg). The component AJ-1 was crystalline but the component AJ-2 was not crystalline and was insufficient quantities.

Isolation and purification of pure compound
The compound AJ-1 showed single spot on TLC analysis with some impurities. The compound AJ-2 was recrystallized dissolving in Petroleum ether: Ethyl acetate (1:1) and the crystals were washed with different solvents of varying polarity. The isolated compound AJ-1 was tested in different solvent systems (Table 1) for its purity. The compound showed a single spot on TLC examination. So, this compound was pure.
Finally its R f values (Table 3) were determined using the various solvent systems.

Characteristics of the compound AJ-1 (A) Physical characteristics
Physical form: Fine crystals (needles shaped) having single R f value was obtained.

Color:
The compound was all most colorless.

Solubility:
The solubility data of the compound have been summarized below.
The compound is highly soluble in ethyl acetate and chloroform.
The compound is insoluble in n hexane, petroleum ether.
The compound is sparingly soluble in ethanol, diethyl ether.

(B) Chemical characteristics:
The compound gave positive test for ketone group, tertiary amine group, alcohol, ester, unsaturation, alkaloid and negative test for carbohydrate, phenol and hydrocarbon.

H and 13 C-NMR (Neuclear Magnetic Resonance) Spectra:
1 H and 13 C-NMR spectra were recorded on a JEOL-AX-500 (500 MHz) and JEOL-JNM AX 400 (400MHz), FT NMR spectrometers, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabedori, Mizuho-ku, Nagoya 467, Japan. CDC13 was used solvent with Tetramethylsilane (TMS) as an internal standard and the chemical shifts are given in δ-values. All organic extracts were dried over anhydrous Na 2 SO 4 and solvent was evaporated off under reduced pressure in a rotary evaporator.  In J resolved DEPT 45° spectrum four down field signals appeared at 173.08, 174.36, 174.41 and 176.02 ppm and are assumed to be carbonyl carbon. Chemical examination showed the positive test for carbonyl group and absence of aldehyde which is also supported by the appearance of three peaks in its IR spectrum at 1762, 1743 and 1705 cm -1 for carboxyl functions. Signal for aldehyde group proton did not appear in 1 H-NMR spectrum. From the above findings, four ketone functions are present in the molecule. Six aromatic carbons appeared at 126.49, 126.5, 130.55, 138.03, 140.85 and 144.63 among which two are quaternary and the other four are methane carbon atoms, identified by DEPT 135° spectrum, which indicates the presence of a benzene ring in the molecule.

Study of spectral data
Four benzene ring protons are present in the 1 H-NMR spectrum and their splitting pattern is 1:3 i.e. a singlet equivalent to one proton and a multiplet equivalent to three protons. From the above 13 C-NMR and 1 H-NMR spectra, the benzene ring, substituted in two portions which are presumed to be ortho and para and are evident form the assymmetrical splitting pattern of the benzene ring protons (1:3) (Figure 4).
From the 1 H-NMR spectrum of the compound four benzene ring protons appeared in an unsymmetrical pattern, one as singlet at δ: 7.73 and other three as multiplet between δ: 7.65~7.67. Five doubly bouded methane protons appeared at δ: 6.51 (1H, dd, J=2.  (Figure 6). One doublet, equivalent to two protons, appeared at δ: 6.70 with J values of 1.3 and 12.2 Hz and other singlet of two protons appeared at δ: 5.26. These two sets of protons seems to be a doubly bonded terminal methylene group present in the molecule. The above discussion field proton signals satisfy all the carbon signals appeared in the down field region in 13 C-NMR spectrum.
Rest of the saturated CH 2 protons appeared between δ: 1.75 and 2.94 ppm (plesse, vide experimental). A peak of four protons appeared as singlet at δ: 1.52 which seems to be NH or OH protons or mixture of these two. Positive test with alkaloidal reagents indicated the presence of nitrogen and the presence of OH group can also be predicted from the IR spectrum since a broad peak for OH stretching centered at 3509 cm -1 appeared in the IR spectrum. Four methyl protons signals also appeared which have been discussed above. Form the above spectral evidences; the compound is an alkaloid containing 37 carbon atoms with 52 hydrogen along with secondary or tertiary nitrogen and several OH groups in the molecule.

Conclusion
From the research work only one compound is isolated and the above spectral evidence, the compound is an alkaloid containing 37 carbon atoms with 52 hydrogen along with secondary or tertiary nitrogen and several OH groups in the molecule. For complete structure elucidation, Mass spectrum, HSCOSY, HSQC and HMBC spectra will be required. From the above discussion, it is clear that this plant may contain other medicinal compounds. Further research may be extended to isolate active compounds specially from fractions B, C, E and F.