Isolation and Structural Characterization of Phytoconstituents from Strobilanthes kunthianus

Natural products offer huge structural varieties and up-to-date techniques for isolation, structural characterization, screening and combinatorial preparations have led to renaissance of plant products as sources of new drugs.1-4 The importance of plant based drugs, the introduction of medicinal plants in the form of nutraceuticals and dietary supplements are also altering the current trend dominated by synthetic drugs to plant-based drug market.5


INTRODUCTION
Natural products offer huge structural varieties and up-to-date techniques for isolation, structural characterization, screening and combinatorial preparations have led to renaissance of plant products as sources of new drugs. [1][2][3][4] The importance of plant based drugs, the introduction of medicinal plants in the form of nutraceuticals and dietary supplements are also altering the current trend dominated by synthetic drugs to plant-based drug market. 5 Strobilanthes kunthianus (S. kunthianus) is a shrub in the grasslands of Western Ghats in India. The Nilgiris, which literally means the blue mountains got its name from the purplish blue flowers of Neela kurinji that blossoms gregariously once in twelve years. 6,7 S. kunthianus is well known for its medicinal properties and reported to possess many biological activities. 8 Everlyne et al. 9 studied FT-IR and GC-MS analysis of ethanolic leaves extract of S. kunthianus and reported that it may contains aromatic hydrocarbons, straight chain primary alcohols, fatty acids, alkenes, phenolic compounds, titerpene alcohols and triterpenes, primary aliphatic alcohols and trienoic fatty acids. Prabakaran and Kirutheka 10 identified ten phytoconstituents based on GC-MS analysis in callus methanolic extract of S. kunthianus and studied for antioxidant properties. However, so far there is no phytoconstituents are isolated individually in various plant parts of S. kunthianus. Therefore, the present study was aimed to isolate and characterize the phytoconstituents present in various parts of S. kunthianus.

MATERIALS AND METHODS
An electrothermal IA-9200 was used to determine melting points of the isolated compounds. IR spectrum was obtained from Perkin Elmer FTIR 1275X. The EIMS spectrum was obtained from a 5989 B MS spectrometer from Hewlett Packard. A Bruker AM500 FT-NMR spectrometer was used to record 1 petroleum ether, chloroform, ethyl acetate and methanol in a Soxhlet apparatus separately for 18-20 h. The extracts were concentrated in a rotary evaporator under reduced pressure at 35-40 o C and stored at 4 o C in a refrigerator till further use.

Cold maceration
The powdered leaves and flowers (250 g) were extracted with 1.5 l of methanol by cold maceration separately by agitation for 7 days and filtered. The mass was squeezed out and again subjected for remaceration for 7 days and filtered off. The combined filtrate was concentrated similarly as stated in successive extraction method.

Crude extraction
The powdered root, stem, leaves and flowers of S. kunthianus (500 g) were extracted separately with 2.5 l of methanol in a Soxhlet apparatus for 18-20 h. The extracts were concentrated similarly as stated in successive extraction method.

Isolation and characterization of phytoconstituents
Isolation is a part of natural product research, through which it is possible to separate components. The biologically active ones can be incorporated as ingredients in the modern system of medicine. The column chromatographic technique (adsorption chromatography) is widely used for the separation, isolation and purification of the natural products. The principle involved in this is the adsorption towards the adsorbent packed in the column. By changing the polarity of the mobile phase, the separation was achieved by column chromatography. Characterization of the isolated compounds were carried out by different analytical techniques like IR, NMR and Mass spectroscopy (MS).

Column chromatography of petroleum ether root extract of S. kunthianus
The petroleum ether root extract (6 g) was chromatographed over silica gel 60-120 mesh of column length 60 cm and diameter 3 cm. Elution was carried out with solvents and solvent mixtures of increasing polarities. The fractions were collected in 25 ml portions and monitored on TLC and the fractions showing similar spots were combined. The fractions 143-172 eluted with chloroform:ethyl acetate (80:20) gave a white precipitate which showed two major spots on TLC. Hence, it was subjected for re-column chromatography to isolate the two compounds. The remaining fractions were not worked out because of lower yields. The white precipitate (3.2 g) obtained from fractions 143-172 was further chromatographed over silica gel 100-200 mesh of column length 50 cm and diameter 3 cm. Elution was carried out with solvents and solvent mixtures of increasing polarities. The fractions were collected in 10 ml portions and monitored on TLC. The fractions 25-48 eluted with petroleum ether: acetone (90:10) gave a white residue and showed single spot with tailing. Repeated recrystallisation in methanol yielded colorless needle shaped crystals which showed a single spot on TLC with petroleum ether: acetone (85:15). TLC studies were carried out in different solvent systems to prove the homogeneity. It was designated as compound 1 (Yield 1.4 g, 23.33%). The next fraction (49-56) which gave colourless white residue was washed several times with petroleum ether and on recrystallisation in chloroform yielded a colorless compound and its homogeneity was confirmed by TLC studies, designated as compound 3 (Yield 0.31 g, 5.17%). Compounds 1 and 3 were subjected to physical and spectral studies for confirming their purity and characterization. The remaining fractions were not worked out because of lower yields.

Column chromatography of petroleum ether stem extract of S. kunthianus
The petroleum ether stem extract (5.6 g) was chromatographed over silica gel 100-200 mesh of column length 100 cm and diameter 1.2 cm. Elution was carried out with solvents and solvent mixtures of increasing polarities. The fractions were collected in 50 ml portions and monitored on TLC and the fractions showing similar spots were combined. The fractions 46-64, on elution with chloroform: ethyl acetate (60:40) yielded a yellow residue (2.1 g) which showed on TLC single spot with tailing. On repeated recrystallisation with methanol it gave a colorless needle shaped crystalline compound and its homogeneity was proved by TLC studies and designated as compound 2 (Yield 0.9 g, 16.07%). This compound was subjected to physical and spectral studies for confirming the purity and characterization. The remaining fractions were not worked out because of lesser yields.

Column chromatography of chloroform stem extract of S. kunthianus
The chloroform stem extract (2.5 g) was chromatographed over silica gel 100-200 mesh of column length 100 cm and diameter 1.2 cm. Elution was carried out with solvents and solvent mixtures of increasing polarities. The fractions were collected in 25 ml portions and monitored on TLC and the fractions showing similar spots were combined. The fractions, 25-55 eluted with petroleum ether: chloroform (80:20) gave a light green residue. On repeated washing with acetone, it yielded a colourless residue. The colourless residue was dissolved in minimum quantity of chloroform and filtered off. The filtrate on evaporation yielded a white semisolid compound, which on repeated washing with acetone and recrystallisation with chloroform yielded a colorless residue and its homogeneity was confirmed with various solvent systems by TLC and designated as compound 4 (White semisolid; Melting point 118 ºC; Yield 0.016 g, 0.64%). This compound was subjected to physical and spectral studies for confirming the purity and characterization. The remaining fractions were not worked out because of lesser yields.

Column chromatography of macerated methanol flower extract of S. kunthianus
The macerated methanol flower extract (6 g) was chromatographed over silica gel 60-120 mesh of column length 50 cm and diameter 3 cm. Elution was carried out with solvents and solvent mixtures of increasing polarities. The fractions were collected in 50 ml portions and monitored on TLC and the fractions showing similar spots were combined. The fractions 59-76 eluted with chloroform: ethyl acetate (60:40) gave a yellow residue and showed one major spot along with other minor impurities on TLC. Repeated recrystallisation with chloroform: methanol (90:10) gave a colorless compound. The colorless compound showed a single spot in petroleum ether: ethyl acetate (88:12) and was further proved for its homogeneity with different solvent systems by TLC and designated as compound 5 (Yield 0.015 g, 0.25%).
The fractions 89-95 eluted with ethyl acetate gave a brown residue. On washing with acetone, followed by methanol washing it gave a white crystalline compound. The white crystalline compound which showed a single spot with TLC by using different solvent systems was designated as compound 6 (White crystalline; Melting point 132 ºC; Yield 0.012 g, 0.20%). These compounds were subjected to physical and spectral studies for confirming their purity and characterization. The remaining fractions were not worked out because of lesser yields.

Column chromatography of crude methanol flower extract of S. kunthianus
The crude methanol flower extract (50 g) was chromatographed over silica gel 60-120 mesh of column length 60 cm and diameter 10 cm. Elution was carried out with solvents and solvent mixtures of increasing polarities. The fractions were collected in 100 ml portions and monitored on TLC and the fractions showing similar spots were combined. The fractions 11-30 eluted with petroleum ether: chloroform (50:50) gave a colorless residue which on purification with acetone gave a fluffy white precipitate and its homogeneity was checked by TLC in n-hexane: ethyl acetate (65:35) and was further confirmed with different solvent systems by TLC. It was designated as compound 7 (Yield 0.012 g, 0.02%). The fractions (31-50) eluted in chloroform were mixed together, evaporated to a minimal volume and then added acetone. White amorphous sticky residue was obtained. Repeated washing with acetone and recrystallisation with chloroform yielded a colorless precipitate, showed a single spot on TLC in n-hexane: ethyl acetate (50:20) and it was designated as compound 8 (Colorless powder; Melting point 87 ºC; Yield 0.01 g, 0.02%). The fractions 148-189 eluted with ethyl acetate: methanol (60:40) gave a sticky brown residue. This was washed several times with methanol and repeatedly recrystallized with water. A reddish brown compound was obtained. Its homogeneity was checked with chloroform: methanol: water (8:2:0.1) and designated as compound 9 (Reddish brown; Melting point 256 ºC; Yield 0.025 g, 0.05%). These compounds were subjected to physical and spectral studies for confirming their purity and characterization. The remaining fractions were not worked out because of lesser yields and mixtures of compounds.

Isolation by solvent-solvent extraction of macerated methanol leaves extract of S. kunthianus
The macerated methanol leaves extract (7 g) was fractionated with petroleum ether (500 ml). The petroleum ether layer was separated and evaporated. A colourless semisolid residue was obtained which on repeated washing with acetone and recrystallization with chloroform gave a colourless precipitate. Its homogeneity was checked with various solvent systems by TLC and designated as compound 10 (Colorless powder; Melting point 136 ºC; Yield 0.01 g, 0.14%). This compound was subjected to physical and spectral studies for confirming its purity and characterization.

Column chromatography of other extracts of S. kunthianus
Among all the extractions, the compounds were isolated only from successive petroleum ether stem and root extracts, successive chloroform stem extract, macerated leaves and flower extracts and crude methanolic flower extract. Other extracts from successive, maceration and crude extraction methods were subjected to column chromatography but couldn't isolate any compounds.

Characterization of compounds 1 and 2
The 1 H NMR spectra displayed a one proton multiplet at δ 3.20 is assignable to H-3 on the basis of biogenetic analogy. The 1  Since all similar groups are in the same environment these groups should be attached to one carbon atom.

-CH-[C-(-CH 2 -CO-N-(CH 3 ) 2 ) 2 ] 2
Two other valencies of this carbon atom have been satisfied by the long chain methylene groups. Further in the DEPT-135 spectra, it showed 12 signals in the positive side for CH 3  .93 strongly supporting that the compound contains a long chain methylene groups. Based on the above facts the structure was proposed as shown in Figure 1.   N or O). The intensity of the peak at δ 63.30 is more than the other two peaks indicating the presence of more than one carbon atom in that environment. Further the DEPT 135 spectrum of this compound showed that only one peak at δ 63.30 (methylene carbons C-2 and C-6) is in the positive side and the other two signals at δ 69.39 (C-4) and at δ 70.98 (C-3 and C-5) are in the negative side. Based on the above facts the structure was proposed as shown in Figure 1.

Characterization of compound 7
The 1 H NMR spectrum displayed signals for two tertiary methyl groups at δ 0. Flavone glycoside 10 Decahydro-1,1,4a,8-tetramethylphenanthren-2(1H,3H,4bH)-one carbonyl carbon appears at δ 173.76. Consistently, its 1 H NMR signal appears as a multiplet at downfield region δ 4.0 (H-3) for one proton. The latter signal at δ 61.19 appears to be of a hydroxy methyl signal. In the 1 H NMR spectrum corresponding methylene protons appears in the downfield region at δ 4.8 (m, 1H, H-24) and 4.5 (m, 1H, H-24) each for one proton. This signal can go down only if the hydroxyl group is hydrogen bonded with the carbonyl group. Due to hydrogen bonding it will reduce the electron density in the C-24 carbon and the signals go downfield. This suggests that the carbonyl group and the hydroxy methyl group are nearby each other. There is no signal in between δ 40 and 60 in the 13 C NMR. The presence of a methyl group in the C-5 position makes the carbon C-4 electron rich and the signal moves towards up field region and appears at δ 39.98. For similar reasons the long hydrocarbon chain which contains the double bond is connected to C-6. Since there is no other functional group is found in the compound the methyl group at C-12 makes it tetra substituted. To avoid the signals of C-10 and C-13 to go downfield region a methyl group was placed at C-9. The DEPT-135 spectra showed 11 signals in the positive side for CH 3 and CH carbons and 12 signals in the opposite side for CH 2 groups. All the other signals are in accordance with the structure. The mass spectrum also confirms the long chain hydro carbon. Based on the above facts the structure was proposed as shown in Figure 1.

Characterization of compound 9
Compound 9  In addition to this a couple of signals between δ 60.66 and 80.55 indicate the presence of three glycosidic moieties. The signals at δ 53.31 (for two carbons) and 59.16 suggest the presence of three methoxy groups. One of the carbonyl group at δ 182.32 may be assigned to the flavanoid nucleus. Apart from this the signals at δ 33.10 along with unassigned carbonyl signal indicates the presence of acetate moiety. The downfield signal at δ 128.04 shows that C-3 is substituted with oxygen function. It is probable that the glucoside moieties are joined one after another at C-3. Absence of phenolic protons in the 1 H NMR spectrum and presence of methoxy protons indicate that all the phenolic hydroxyl groups are substituted with methoxy groups. In the DEPT-135 spectra showed 25 signals in the positive side for CH 3 and CH carbons and 3 signals in the opposite side for CH 2 groups. The normal 13 C-NMR spectra showed signals for 38 carbons and suggested that 10 quartnary carbon atoms are present in the compound. Since all the positions are satisfied with protons and methoxy groups, the probability of the attachment of the acetic acid group may not be possible in the flavanoid nucleus. It can be safely placed at the end of the glucosidic moiety. Hence, the structure was proposed as shown in Figure 1. The compound 10 in its 1 H NMR spectrum exhibits a peak at δ 1.20 characteristic of the secondary and tertiary methyl groups. The strong singlet at δ 1.60 is characteristic of methylene protons in a ring system. The signal at δ 2.20 is due to the methylene groups attached to a carbonyl group. The 13 C NMR spectrum exhibits signals for 18 carbon atoms and a peak at δ 205 indicates the presence of a carbonyl group. The HSQC spectra showed the presence of four methyl groups, seven methylene groups, four methane groups and three quartnery carbon atoms.

Characterization of compound 10
The signal at δ 30.00 showed the presence of many methylene groups inside a ring system. The mass spectrum a molecular ion peak at 262.173 (calculated 262.23) indicating a molecular formula of C 18 H 30 O. Based on the above facts the structure was proposed as shown in Figure 1.

CONCLUSION
All these compounds herein described were isolated for the first time from S. kunthianus. Except lupeol (1 and 2), betulin (3), α-amyrin (5) and β-sitosterol (7), other five compounds (4, 6, 8, 9 and 10) isolated from S. kunthianus were novel, revealed that S. kunthianus extracts containing novel phytoconstituents which may be responsible for its biological properties. In future, in-vitro and in-vivo studies are warranted to conduct on these isolated compounds to identify its safety and efficacy.