The plant Nyctanthes arbor-tristis L. is well known and widely distributed not only in Bangladesh but also in Indo-Pak subcontinent and South East Asia (Anonymous, 1983) having remarkable folk medicinal use. The flowers are used as stomachic, carminative, astringent to bowel, antibilious, expectorant, hair tonic and in the treatment of piles and various skin diseases. The bark is used for the treatment of bronchitis and snakebite (Kirtikar and Basu, 1993; Drury, 1973). It is evident from the existing works that the family Nyctanthaceae is an important source of biologically active compounds. So far literature surveyed, there was no sufficient comprehensive study of biological and phytochemical properties of the plant N. arbor-tristis L. The compound, 4-hydroxy hexahydrobenzofuran-7-one isolated from the chloroform extract of the flowers of N. arbor-tristis L. showed significant antibacterial, larvicidal (mosquito larvae) properties and also found toxic in brine shrimp lethality bioassay (Khatune, 2000). Therefore, it was subjected to anticancer (antitumor) screening on EAC cell to Swiss Albino mice.
The purpose of this study was to evaluate the cytotoxicity of the isolated 4-hydroxy hexahydrobenzofuran-7-one.
Materials and Methods
The fresh flowers of the plant N. arbor-tristis L. were collected
during the month of September-October, (1998) from Rajshahi, Bangladesh. The
plant was identified by Professor A.T.M Naderuzzaman, Department of Botany,
University of Rajshahi and a voucher specimen number has been deposited in Bangladesh
National Herbarium, Dhaka.
Extraction and isolation
The fresh flowers (500 gm) were taken in an aspirator and soaked in 2.5
liters rectified spirit at room temperature for 15 days with continuous changing
the fresh flowers with the old ones every three days interval. The rectified
spirit extract after evaporation of the solvent was fractionated with petroleum
ether, chloroform, ethyl acetate and finally with methanol.
The compound was isolated from chloroform fraction and purified by column chromatography
(Beckett and Stanlake, 1986) followed by preparative thin layer chromatography
(PTLC) (Egon and Stahl, 1969) and obtained as colorless oily mass. The chemical
structure of the compound was determined using [UV, IR (Beximco Pharmaceutical
Ltd., Dhaka), 1H-1H COSY 900, 13C-DEPT and long range correlation (Strathclyde
University, Glasgow, London)] spectral analyses and was identified as 4-hydroxy
hexahydrobenzofuran-7-one (Fig. 1).
For evaluation of cytotoxicity study, an experiment was carried out with
eight adult male Swiss Albino mice (collected from the Animal Resources Branch
of the International Center for Diarrhoeal Research, Bangladesh) of 6-8 weeks
of age were weighed (25±2 gm) and placed into two groups. The mice were kept
in properly numbered iron cages individually and were supplied with basal diet
(Hawk et al., 1954). The rats were acclimatized for 14 days before drug
Transplantation of EAC cells
Ascitic fluids, drawn out from different tumor bearing Swiss Albino mouse,
were diluted with normal saline and the viability of tumor cells were checked
by trypan blue (0.4%) exclusion assay (Hawael et al., 1979). Cell samples
showing above 90% viability were injected intraperitoneally (1 ml each) and
were kept for 24 h for the multiplication of EAC cells.
4-Hydroxy hexahydrobenzofuran-7-one was dissolved in distilled water with
dimethyl sulfoxide (DMSO) and administered intraperitoneally at a dose of 20
mg/kg mouse/day for 5 consecutive days to one group of four mice. The second
group received only water and DMSO and served as the control.
A measured amount of fresh food was supplied daily at 10 h interval and
the general condition of the body and the behavior of the animals were observed
daily, throughout the study. For cytotoxicity study, total intraperitoneal cells
were harvested in normal saline after death at 5th day. Total number of viable
cells per animal of treated experimental groups were compared with those of
control groups and the percentage of cell growth inhibition were calculated
using standard procedures with the reagents supplied by Boehringer Manneim GmbH
Diagnostica. Viable cells were counted by haemocytometer under a microscope
at Sericulture Research Institute, Rajshahi, Bangladesh.
Results are presented as the M±s.d. Students t-test was used for
comparison between the experimental and control groups. P<0.05 was considered
to be statistically significant.
Results and Discussion
Table 1 shows the results of cytotoxicity of 4-hydroxy hexahydrobenzofuran-7-one
on EAC cells. After 5 days of drug administration, the animals of both control
and experimental groups were killed and total intraperitoneal cells were drawn
out and examined under microscope.
||Cell growth inhibition after daily intraperitoneal administration
of 4-hydroxy hexahydrofuran-7-one 20 mg kg-1 mouse day-1 for 5 days in mice
|M±SD= mean±standard deviation. There was no significant difference
between the average no. of cells counted between control and experimental
|M= Simple mean value
SD = Standard deviation.
The compound that has the ability to suppress the growth of tumor cells more
than 75% is considered to be highly anticarcinogenic. The compound 4-hydroxy
hexahydrobenzofuran-7-one was found to inhibit the cell growth only 43.27%.
Non significant difference was observed between the average no. of cells counted
between experimental and control animals. Previously it was reported by Rahman
(1999) that a flavone type compound, pectolinarigenin, isolated from the plant
Clerodendrum indicum L. showed moderate anticarcinogenic (cell growth
inhibition was 66.17%) effect against the same tumor (EAC) cells.
In our study, it was also observed that there were no abnormalities in the behaviour in either control or experimental animals indicating that 4-hydroxy hexahydrobenzofuran-7-one has no adverse effect on central nervous system.
From the study it is concluded that 4-hydroxy hexahydrobenzofuran-7-one has no significant cytotoxicity on EAC cells in mice at the dose and duration used in the study. Further investigation should be carried out with this compound in order to identify the pharmacological mechanism.