Isolation of ( + )-Catechin from Acacia Catechu ( Cutch Tree ) by a Convenient Method

Biologically important polyphenol (+)-catechin was isolated from catechu, the extract of the red heartwood of Acacia Catechu tree (Khoiyer tree). The isolation procedure was developed to get better yield (25%) and to reduce the isolation cost and time. The structure of the isolated (+)-catechin was confirmed with several spectral analyses and chemical studies.


Introduction
Catechu (or cutch), a hot water extract of red heartwood of Acacia Catechu Willd [1] is brown coloured material with bitter taste.It is produced in the cottage industries in good amount in the northern part of Bangladesh, especially in Rajshahi where Acacia Catechu grows in abundance.Unfortunately, only a portion of it is utilized as species with betel leaves for chewing.It has some medicinal values [2].Faruq and co-workers [3] derived a simple method for piece-dying cotton fabric using catechu.
The chief constituents [4] of the red heartwood are catechin and catechu tannic acid along with small proportion of brown coloring matter.It also contains tannin, flavotannin, gallotannin, phloratannin etc [2].(+)-Catechin is a substance, which diminish or arrest the action of some hormones [4]; thus "tyronorman" injected with thyroxin suppresses the rise in basal metabolism.
The tanning property of catechin in human skin may be supposed to be the active ingredient for the treatment of leucoderma (shiti) [5].Catechin has antihormone activity [6].Further its activity has also been correlated with those of vitamin P [6].
It is evident from the literature survey that the catechin is a biologically important polyphenolic compound.Thus to isolate it from natural sources for the welfare of human being will be a welcome effort.The conventional method [7] for the isolation of (+)catechin from catechu is labourious, costly and low yielding (14.2 -17.2%).Therefore, attempt has been made in this work to develop the cost-effective isolation procedure for (+)-catechin from Acacia Catechu.

Materials and Methods
Solvent purification: Solvents used during the work were purified by distillation at the boiling point of the respective solvents.Evaporation of solvents was carried out on a Rotary vacuum evaporator under reduced pressure at bath temperature not exceeding 60ºC.The purity of the compounds was tested by analytical thin layer chromatography (TLC) on silica gel 60 GF 254 cards and the spots were made visible by exposure to UV light.
Melting point: A Reichart micro melting point apparatus was used for recording the melting points.Care was taken to ensure that the heating was done steadily.
1 H-NMR spectra: The 1 H-NMR spectra were recorded on a Bruker AM 300 FT NMR, AM 400 FT NMR and AM 500 FT NMR spectrometers using TMS (Trimethylsilane) as an internal standard.
Mass spectra: The mass spectra were registered on a Varian-MAT 112S and Finnigan MAT-112 and 312A double focusing mass spectrometers connected to DEC PDP 11/34 and IBM-AT compatible PC based system, respectively.Electron Impact (EI), Peak matching and Fast Atom Bombardment (FAB) experiments were performed on MAT-312A mass spectrometers.
Infrared spectra: Infrared spectra were recorded on a PYE-UNICAM SP3 IR spectrophotometer.Spectra were taken in KBr pellets.
UV spectra: UV spectra were recorded on a LKB 4053 Ultraspec K Ultraviolet/Visible spectrophotometer. 13C-NMR spectra: The 13 C-NMR spectra were recorded at 75, 100 and 125 MHz on a Bruker AM 300 FT NMR, AM 400 FT NMR and AM 500 FT NMR spectrometers, respectively.

Preparation of the material
Collection of raw materials: Acacia catechu tree was collected from the botanical garden of BCSIR Laboratories, Rajshahi.

Separation of catechu [8]
One kg of the dried chips of Acacia catechu was taken in an aluminium pot to which ten litres of water were added so that the chips completely immersed under water.It was boiled over an open fire for four hours and allowed to stand for 24 hours so that more catechu might diffuse into the water.The extract was decanted off in a pot and was filtered through a fine muslin cloth to remove wood chips and other suspended materials.
The filtrate was evaporated and the residue obtained was air dried and weighed (180g).Yield of catechu was 18%.

Isolation of (+)-catechin
Isolated catechu (150g) was taken in a five-litre stainless steel beaker containing one litre distilled water.It was boiled with constant stirring for complete dissolution and filtered through a filter paper.Then it was evaporated to 500 ml and allowed to stand for 24 hours.The obtained precipitate was filtered using a filter paper.The aqueous filtrate was rejected.The residue was dissolve in ethanol and filtered.The ethanolic solution was evaporated to dryness and the residue was dissolved into hot water (500 ml).It was allowed to stand for 24 hours.The precipitate was filtered and dried in air (m.p. 95-6ºC, yield 37.5g, 25%).The process of re-crystallization from water was repeated thrice.On drying over phosphorus pentoxide the melting point of isolated (+)-catechin raised to 174-5ºC.Mixed melting point with an authentic sample did not show lowering.
Mass spectra: The mass spectra showed maximum at 290 and minimum at 55.The other fragments were seen at 139, 138, 110, 152, 151 and 123.The molecular mass corresponding to 290 was observed.
UV spectra: UV spectra (λ max [ methanol]) showed maxima at 277nm and 220 nm.[10] The isolated (+)-catechin (0.1g) dissolved in ethanol (10 ml) was refluxed with 50% alcoholic KOH (20 ml) for six hours.The reaction mixture was neutralized with 6N HCl and extracted with ether.A portion of ether extract was treated with 5% sodium bicarbonate solution.Acidification and extraction of the sodium bicarbonate solution with ether gave no detectable substance.The remaining ether extract was treated with 1% sodium hydroxide solution.Neutralization of the alkali layer with 5% HCl followed by extraction with ether gave 0.043g of white precipitate which after crystallization from H 2 O gave 0.039g of phloroglucinol (m.p. 220˚C).

Results and Discussions
The method of isolation and separation for (+)-catechin described in this report was found more convenient compared to others process [7,14,15].The conventional methods involve multi steps process which was not economically feasible.Isolation process of the present work involves lesser steps and yield was found good.
Thin layer chromatographic [9] behavior of the isolated (+)-catechin was in good agreement with the authentic (+)-catechin.The compound showed absorption bands at 220 and 277 nm in the ultraviolet [16].Anthocyanins give an absorption band at 275-280 nm regions.This ultraviolet behavior indicated the possibility of the isolated compound belonging to the flavonoid group.The mass spectra of (+)-catechin with its different fragments are understandable.The molecular mass of (+)-catechin is 290.In the spectra the maxima m/z ratio showed at 290.The IR spectra of (+)-catechin has a broad band around 3400-2600 cm -1 region corresponding to aliphatic and aromatic C-H, phenolic and alcoholic O-H stretching.A band at 1620 cm -1 observed may be due to aromatic C=C stretching.Other stretchings were comparable with IR spectra of authentic (+)-catechin.The 1 H-NMR spectra of +)-catechin was very clear and understandable.The observed signals in NMR spectra were in good agreement with the authentic (+)-catechin [17].(+)-Catechin molecule contains 15 carbons.In the 13 C-NMR spectra, signals appeared at δ 27.7, 66.3, 80.9, 93.9, 95.1, 114.5, 115.1, 118.4 due to C-4, C-3, C-2, C-8, C-2, C-5, C-6́ carbons respectively and others aromatic carbons showed peaks at δ 99. 1, 130.6, 144.6, 144.8, 155.3, 156.1 and 156.4.These data were also in good agreement with the authentic (+)-catechin [17].