Purification, Characterization and Cytotoxic Activity of Two Flavonoids from Oroxylum indicum Vent. (Bignoniaceae)

Uddin, Kawsar; , Abu Sayeed; , Anwarul Islam; , Aziz Abdur Rahman; , Abbas Ali; , G.R.M. Astaq Mohal Khan; , Md. Golam Sadik

ABSTRACT

Phytochemical examination of the ethyl acetate extract of root parts of Oroxylum indicum Vent. has led to the isolation of two flavonoid: 2,5-dihydroxy-6,7-dimethoxy flavone and 3,7,3’,5’-tetramethoxy-4-hydroxy flavone by a combination of column and preparative thin layer Chromatography. The structure of these compounds were determined by spectroscopic analysis as well as by comparison of their spectral data with previously reported values. The cytotoxicity of these two compounds and crude petroleum ether, ethyl acetate and methanol extracts was done by brine shrimp lethality bioassay. The LC₅₀ values of compound 1 and 2 and petroleum ether, ethyl acetate and methanol extracts were found to be 14.12, 3.80, 3.97, 6.02 and 8.70 μg/ml, respectively.

PDF Abstract XML References Citation

INTRODUCTION

Extracts or compounds isolated from extracts have been playing an important role in the development of medicinal agents that afford many opportunities for the design of new therapeutics to treat diseases with fewer side effects or to combat the growing and serious problems of drug resistance (Alfred, 1960; Sofora, 1982; Wallis, 1985). Aspects of these opportunities we selected a medicinal plant Oroxylum indicum Vent. (Family: Bignoniaceae). The plant is an important herbal medicine in many Asian countries and is used in folk medicine as a cure of various diseases (Biswas and Ghosh, 1994). The root bark is used in fever, bronchitis, intestinal worms, leucoderma, asthma, inflammation, anal troubles etc. The fruit and seeds are used as expectorant, purgative and bitter tonic (Kirtikar and Basu, 1996). In hindu medicine the root, bark, stem and leaf are prescribed for snake bite in diarrhea and dysentery (Ghani, 1998).

Keeping in mind the folkloric reputation, the plant was chosen for phytochemical and biological investigation to isolate more potent bioactive compounds for better health cure system of common people of third world countries.

We, herein, report the isolation, purification and structure elucidation of two flavonoids from the root bark of the plant Oroxylum indicum and their cytotoxic activity.

Materials and Methods

Collection of plant materials: The root barks of the plant Oroxylum indicum Vent. was collected from Gobindoganj, Gaibandha, Bangladesh during July-August,2000. The plant was taxonomically identified by Professor A.T.M. Nadiruzzaman, Department of Botany, Rajshahi University, Bangladesh.

Extraction, isolation and characterization: The barks were dried in an oven at 45°C, crushed and then extracted (850 gm) in soxhlet apparatus using petroleum ether (to remove fatty materials) in “Phytochemistry Research Laboratory, Department of Pharmacy, Rajshahi University, Bangladesh”. The residue was further extracted gradually with ethyl acetate and methanol and the amounts of each extract obtained were 32.50 gm and 20.50 gm respectively. A portion of ethyl acetate extract was subjected to column chromatography on silica gel of 60-120-mesh size (Beckett and Stenlake, 1986). The column was first eluted with n-hexane and then n-hexane with increasing portions of ethyl acetate, then with ethyl acetate and finally with methanol which gave 29 fractions.

Compound 1 was obtained from fraction 8-10 when subjected to PTLC (pet ether: ethyl acetate 3:1), while compound 2 was obtained from the fraction 11-16 using same solvent system.

Compound 1 (as 2,5-dihydroxy-6,7-dimethoxy flavone): Fine crystals. R_f value: 0.621 (pet ether : ethyl acetate 3:1).mp. 195-198^oC. ¹H-NMR (500 MHZ, CDCl₃): δ_H 2.498 (1H, d, J=17 Hz, H-3), 3.312 (1H, dd, J=17.2 Hz, H-3), 3.673 (3H, s, -OCH₃), 3.861 (3H, s, -OCH₃), 6.291 (H-8, s), 7.393 (1H, m, H-4’), 7.481 (2H, m, H-3’/5’), 7.619 (2H, m, H-2’/6’), 7.745 (1H, d, J=2.0 Hz, -OH at C-2), 11.802 (1H, s, -OH at C-5); ¹³C-NMR (125 MHZ, CDCl₃): δ_C: 102.113 (C-2), 48.392 (C-3), 196.014 (C-4), 153.682 (C-5), 128.611 (C-6), 160.355 (C-7), 92.873 (C-8), 155.712 (C-9), 101.934 (C-10), 142.195 (C-1’), 125.453 (C-2’, 6’), 128.162 (C-3’, 5’), 128.235 (C-4’), 60.122 (-OMe), 56.271(-OMe).

Compound 2 (as 3,7, 3’, 5’-tetramethoxy-4-hydroxy flavone): Needle shape crystals, R_f value: 0.721 (pet ether: ethyl acetate, 3:1). Mp. 210-211^oC. ¹H-NMR (500 MHZ, CDCl₃): δ_H 7.572 (1H, d, J=8.0 Hz, H-5), 6.905 (1H, dd, J=8.2 Hz, H-6), 7.235 (1H, d, J=2.0 Hz, H-8), 7.291 (1H, d, J=2.0 Hz, H-2’), 7.293 (1H, d, J=2.0 Hz, H-6’), 3.842 (3H, s, -OCH₃ at C-3), 3.925 (3H, s, -OCH₃ at C-7), 3.944 (3H, s, -OCH₃ at C-3’), 3.946 (3H, s, -OCH₃ at C-5’); ¹³C-NMR (125 MHZ, CDCl₃): δ_C: 158.132 (C-2), 140.001(C-3), 180.401 (C-4), 125.023 (C-5), 111.722 (C-6), 163.715 (C-7), 98.799 (C-8), 159.263 (C-9), 116.122 (C-10), 121.061 (C-1’), 105.203(C-2’), 146.91 (C-3’), 136.133 (C-4’), 146.921 (C-5’), 105.192 (C-6’), 58.124 (OMe at C-3), 57.025 (OMe at C-7), 56.801 (OMe at C-3’), 56.802 (OMe at C-5’).

Determination of cytotoxic activity: The cytotoxic activity of petroleum ether, ethyl acetate and methanol extracts and isolated compounds (1 and 2) were determined by brine shrimp lethality bioassay (Mayer et al., 1982; Persoone et al., 1980).

Preparation of seawater: 38 g of sea salt was weighted, dissolved in one liter of distilled water and filtered off.

Hatching of shrimps: Seawater was kept in a small tank and Artemia salina Leach (brine shrimp eggs) was added to the divided tank. Constant oxygen supply was provided and constant temperature (37±1°C) was maintained for 48 hrs to hatch and mature the shrimp as nauplii (larvae).

Preparation of sample: 1 mg of each sample (petroleum ether, ethyl acetate and methanol extract and compound 1 and 2) were initially dissolved in 200 μl of dimethyl sulfoxide (DMSO) to get a concentration of 5 μg/μl. Forty clean vials were taken for the 4 samples in five concentrations (two vials for each concentration) and two vials also taken for control test for each sample. 5 ml seawater containing 10 brine shrimp nauplii was given to each of the 5 vials and specific volume of samples were transferred from the stock solutions to the vials to get final concentration of 5, 10, 20, 40 and 80 μg ml^-1. Control vials contain 5 ml of seawater and same volume of DMSO as in the sample vials. After 24 hrs the number of survivals in each vial was counted. The percentage of mortality of the brine shrimp was calculated for each concentration and the median lethal concentration (LC₅₀) values were determined.

RESULTS AND DISCUSSION

The ethyl acetate extract of the root barks of the plant Oroxylum indicum yielded two pure compounds 1 and 2 by column and preparative thin layer chromatography. The isolated compounds were identified by spectroscopic method of analysis as well as by comparison of their spectral data with previously reported values.

The ¹³C-NMR spectrum of compound 1 displayed seventeen carbon peaks including one intra molecularly hydrogen bonded carbonyl carbon (δ 196.014), eleven aromatic carbon (δ 142.195, 125.453 (2 C), 128.162 (2 C), 128.235, 153.682, 128.611, 160.355, 92.873, 155.712 and 101.934), two methoxyl carbon at δ 60.122 and 56.271 and two methylene carbon at δ 48.392 and 102.113 (attached with a transdiaxial coupled hydroxyl proton).

The ¹H-NMR spectrum showed signals for two methoxyl group at δ 3.673 and 3.86 (each 3H, s) attached with C-6 and C-7, one aromatic singlet at δ 6.291 for H-8 and five aromatic protons at δ 7.39 (1H), 7.481 (2H) and 7.619 (2H) assigned to H-4, H-3΄/5’ and H-2’/6’ respectively. The spectrum included a broad singlet at d 11.802 attributed to the aromatic hydroxyl proton intra molecularly hydrogen bonded with carbonyl group in ortho position (Sharma, 1997) and a singlet at δ 7.745 due to the presence of a hydroxyl proton which showed a small transdiaxial coupling with δ 3.3 (dd, J=17.2 Hz, H-3).

All the proton peaks in the ¹H-NMR data are in good agreement with the compound 2-Hydroxy flavanone isolated from Collinsonia canadensis (Jan et al., 1999). The ¹³C-NMR shifts are also in accord with the structure assigned.

On the basis of ¹H-NMR and ¹³C-NMR spectral data and comparison with aurhontic flavonoidal compound (Hase et al., 1995; Barua, 1989; Jan et al., 1999, Table 1) the structure of Compound was determined as 2,5-dihydroxy-6,7-dimethoxy flavone (Fig. 1). Although it is a known natural product but this is the first report from this plant.


Fig. 1:	2, 5-dihydroxy-6, 7-dimethoxy flavone (1)

Table 1:	¹H and ¹³C-NMR spectral data of compound 1 and comparison with authentic flavanone

Table 2:	¹³C-NMR and ¹H-NMR spectral data of compound 2


Fig. 2:	3, 7, 3', 5'-tetra methoxy-4 hydroxy flavone (2)

The ¹³C-NMR spectrum of the compound 2 showed nineteen aromatic carbons (δ 158.132, 140.001, 125.023, 111.722, 163.715, 98.799, 159.263, 116.122, 121.061, 105.203, 146.91, 136.133, 146.921, 105.192) and four methoxyl carbon (δ 58.124, 57.025, 56.801, 56.802).

Table 3:	Results of the brine shrimp lethality bioassay of extracts and isolated compounds


Fig. 3:	Determination of LC₅₀ of petroleum ethyl acetate and methanol extract


Fig. 4:	Determination of LC₅₀ of compound 1 and compound 2

The ¹H-NMR spectrum of compound 2 displayed signals for four methoxy group at δ 3.842, 3.925, 3.944 and 3.946 assignable to C-3, C-7, C-3΄ and C-5’ respectively (Table 2). The spectrum included one set of a ABX mutually coupled proton system at δ 7.572 (1H,d, J=8 Hz), 6.905 (1H, dd, J=8 Hz, 2Hz) and 7.235 (1H, d, J=2 Hz) assigned to H-5, H-6 and H-8 respectively and two meta coupled protons at δ 7.291 (1H, J=2 Hz) for H-2 ’ and δ 7.293 (1H, J=2Hz) for H-6’. The ¹H-NMR data was found to be identical to that reported for 3,7, 3’, 5’-tetramethoxy-4-hydroxy flavone by Agarawal (1989). The ¹³C-NMR shifts are also in accord with the structure assigned.

Thus the compound 2 was assigned to be 3,7, 3’, 5’-tetramethoxy-4-hydroxy flavone which is the first report from this plant (Fig. 2).

The result of the brine shrimp lethality bioassay is shown in Table 3. The median lethal concentration (LC₅₀) was determined by extrapolation from the graph (Fig. 3 and 4) and the values were found to be 3.97, 6.02 and 8.70 μg ml^-1 for petroleum ether, ethyl acetate and methanol extract respectively. For the compound 1 and 2 the LC₅₀ values were 14.12 and 3.80 μg ml^-1. respectively.

In this study, all the extracts and compound 1 and 2 showed strong positive results, indicating that the extracts and the compounds are highly cytotoxic as well as biologically active. Each of the test samples showed different mortality rate at different concentrations and was found to be increased with increasing concentration of the sample. Logarithm of concentration versus percentage mortality (Goldstein et al., 1974) was plotted on the graph paper that showed an approximate linear correlation. There was no mortality in the control group. So it is evident that all the test materials were highly lethal to brine shrimp nauplii. However compound 2 and petroleum ether extract were more active with minimum LC₅₀ value and the compound 1 was comparatively less active with maximum LC₅₀ value.

In conclusion, we have successfully isolated, purified and characterized two flavonoids: 2,5-dihydroxy-6,7-dimethoxy flavone and 3,7,3’,5’-tetramethoxy-4-hydroxy flavone from the ethyl acetate extract of root parts of Oroxylum indicum Vent. Both the compounds possess cytotoxic activity. However, a detailed investigation is now under progress for evaluation of it’s therapeutic use.

Acknowledgements

The authors like to thank Dr. Md. Abdur Rashid, Department of Pharmacy, University of Dhaka for spectral data analysis. The authors also wish to thank Professor A.T.M. Nadiruzzaman, Department of Botany, University of Rajshahi, Bangladesh and the Bangladesh National Herbarium, Dhaka, Bangladesh for identification of the plant.

REFERENCES

Agarawal, P.K., 1989. Carbon-13 NMR of Flavonoids. 1st Edn., Elsevier, Amsterdam, pp: 165.
Alfred, B., 1960. Medicinal Chemistry. 2nd Edn., Wiby Publisher Inc., New York.
Barua, A.S., 1989. Two flavonoids from Clerodendrum siphonanthus. Indian J. Chem. Sec. Part B, 28: 198-198.
Ghani, A., 1998. Medicinal Plants of Bangladesh. 1st Edn., Asiatic Society of Bangladesh, Bangladesh, pp: 251.
Goldstein, A., L. Arnow and S.M. Kalkan, 1974. Principles of Drug Action. 2nd Edn., Wiley Biomdical Health Publication, UK., pp: 376-381.
Hase, T., K. Ohtani, R. Kasai, K. Yamasaki and C. Picheansoonthon, 1995. Revised structure for hortensin, a flavonoid from Millingtonia hortensis. Phytochemistry, 40: 287-290.
Direct Link
Jan, F.S., I. Monica, L.D. Max and W. Eckhard, 1999. A novel 2-hydroxy flavanone from Collinsonia canadensis. J. Nat. Prod., 62: 392-394.
Meyer, B.N., N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E. Nichols and J.L. Mclaughlin, 1982. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med., 45: 31-34.
CrossRef PubMed Direct Link
Persoone, G., 1988. The Brine Shrimp Artemia: Proceedings of the International Symposium on Brine Shrimp, Artemia Salina. Vol. 3, Artemia Salina Universa Press, Belgium, pp: 1-3.
Sofowora, A., 1982. Medicinal Plants and Traditional Medicine in Africa. 1st Edn., John Wiley and Sons, Chichester, New York, ISBN-10: 0471103675, Pages: 256.
Vhattacharjee, S., 1387. Chironjib Bonoushodi. Vol. 5, Ananda Publishers Pvt. Ltd., Kolkata, pp: 128.
Wallis, T.E., 1985. Text Book of Pharmacognosy. 5th Edn., CBS Publisher, India, pp: 1-3.
Backett, A.H. and J.B. Stenlake, 1986. Chromatography. 3rd Edn., CBS Publishers and Distributors, New Delhi, India, pp: 75-76.
Biswas, K. and S.E. Ghosh, 1994. Bharater Bonoushodi. 2nd Edn., Calcutta University, Calcutta, India, pp: 858.
Kirtikar, K.R. and B.D. Basu, 1996. Indian Medicinal Plants. Vol. 5, Lalit Mohan Basu Publishers, Allahabad, India, pp: 2422-2423.
Sharma, Y.R., 1996. Elementary Organic Spectroscopy. S. Chanda and Company Ltd., India, pp: 202.

Asian Journal of Plant Sciences

Research Article