Baccaurea ramiflora : Isolation of Aldehydes and in Vitro Biological Investigations

The stem bark of Baccaurea ramiflora was studied. Four aldehydes named as 3 methoxy 4 hydroxy-cinnamaldehyde (coniferyl aldehyde); 3, 4, 5 trimethoxy cinnamaldehyde; 3, 4, 5 trimethoxy benzaldehyde and 3,4 dimethoxy benzaldehyde) (veratraldehyde) have been isolated and then identified by NMR spectroscopy. All of them are first time reported for this plant. Here in vitro biological investigations include antioxidant and cytotoxicity study. Among all fractions, the chloroform soluble fraction exhibited strong free radical scavenging activity having IC 50 value of 12.87 µg/ml compared to BHT (IC 50 value 5.64 µg/ml). On the contrary, aqueous soluble fraction exhibited most toxicity towards brine shrimp compared with vincristine sulphate having LC 50 value of 1.44 and 0.9258 μg/ml respectively.


Introduction
World Health Organization (WHO) claimed that 80% of people still rely on plant-based traditional medicines for their primary health care. Natural origins lead to development of many drugs [1]. So phytochemical research is of paramount importance especially for third world countries where synthetic drug research is highly barricaded due to lack of resources and infrastructures.
Baccaurea is a genus of flowering plant belonging to the family Phyllanthaceae. The term "Baccaurea" is derived from Latin and it refers to the golden yellow color of the fruits [2]. 80 species of this genus have been reported around the world. Based on the fruit character, this is divided into following classes: [3] • Rambai-Thin skinned fruits.
Baccaurea ramiflora belongs to Phyllanthaceae family, which is a slow growing, evergreen, short to medium height shade loving plant. Baccaurea ramiflora is distributed mostly in tropical areas like South East Asia region, the sub-Himalayan tract and Andaman Islands [4]. Locally it is known as latkan and bhubi [4].
According to a report published by Digital Herbarium of Crop Plants, they have the following features: Root: Tap root system. Leaf: Leaves are papery, oblong to obovate-oblong, measuring 9 -18 cm long and 3 -8 cm wide. Adaxial (upper) surface of leaf is green and abaxially (lower) surface is yellowish-green. The base of leaf is cuneate.
Flowers: Flowers are small, borne in clusters on old branches or trunk. Flowers are yellowish-white.
Fruits: Fruits are ovoid or sub-globose, about 2.5 cm in diameter, reddish-yellow or purple when mature.
A wide range of compounds e.g. phenols, esters, sterols etc. have been iso- lated from the stems of Baccaurea ramiflora [6].
As well as analgesic activity from seeds [7], anthelmintic from the whole plant [8], antioxidant activity from fruits [9], cytotoxicity from fruits [10] and hypoglycemic and hypolipidemic activity from the leaves [11] of Baccaurea ramiflora were mentioned. Further research can identify whether there are any unidentified bioactive principles.
Phytochemical profiling of the stem bark of Baccaurea ramiflora has not done extensively. So in this investigation, we have tried to focus on this part, which lead to isolation of aldehydes from the stem bark of Baccaurea ramiflora for the very first time. In vitro antioxidant and cytotoxicity activity of this plant has been also checked.

Collection and Preparation
The stem bark of Baccaurea ramiflora was collected in April 2019 from Kishoreganj district. Later it was identified by an expert from Bangladesh National Herbarium (BNH) and a voucher specimen was deposited (DACB Accession number-55316). After cleaning and shade drying for two weeks, they were crushed into coarse powder using high capacity grinding machine.

Extraction
About 1500 gm of powdered plant material was taken in an amber-colored bottle and soaked with distilled methanol for 15 days with occasional shaking and stirring. The mixture was therefore filtered using a fresh cotton plug. The solvent of the mixture was evaporated using Buchii Rotavapour rotary evaporator at 40˚C temperature and low pressure and the extract was prepared.

Chromatographic Separation
After evaporation we obtained ethyl acetate and methanolic extract which was then subjected to vacuum liquid chromatography (VLC) and it yielded 40 fractions of different polarity [12]. Selected VLC fractions were taken and gel permeation chromatography was done using Sephadex LH 20 for further separation [13]. Later these column fractions were analyzed by thin layer chromatography [14] and compounds of interest were isolated using preparative layer chromatography (PLC) [15].

Determination of DPPH Scavenging Activity
The free radical scavenging activities of the plant extracts on 1,1-diphenyl-2picrylhydrazyl (DPPH), a stable radical, were estimated [16]. 2.0 mL of a methanol solution of the extract at different concentration from 400.0 to 1.5625 μg/mL were mixed with 2.0 mL of a DPPH methanol solution (20 μg/mL). After 30 minutes reaction period at room temperature in dark place the absorbance was measured at 517 nm against methanol as blank by UV spectrophotometer.
The antioxidant potential was assayed from the bleaching of purple colored methanol solution of DPPH radical by the plant extract as compared to that of tert-butyl-1-hydroxytoluene (BHT) by UV spectrophotometer.
Inhibition of free radical DPPH in percent (I %) was calculated as follows: Where, Absorbance of blank is the absorbance of control reaction (containing all reagents except the test material).
Extract concentration providing 50% inhibition (IC 50 ) was calculated from the graph plotted inhibition percentage against extract concentration.

Brine Shrimp Lethality Bioassay
Brine shrimp eggs were hatched in simulated sea water to get nauplii. By the addition of calculated amount of dimethylsulphoxide (DMSO), desired concentration of the test samples were prepared. The nauplii were counted by vis-ual inspection and were taken in vials containing 5 ml of simulated sea water. Then samples of different concentrations were added to the pre-marked vials through micropipette. The vials were then left for 24 hours. Survivors are counted after 24 hours [16]. The median lethal concentration (LC 50 ) value was calculated from the graph plotted percentage mortality rate against extract concentration.
Strong free radical scavenging activity has been showed by the chloroform soluble fraction of the plant extract having IC 50 value of 12.87 µg/mL with compared to BHT (IC 50 value 5.64 µg/mL) while petroleum ether soluble fraction exhibited good antioxidant activity ( IC 50 = 15.47 µg/mL). Aqueous soluble fraction exhibited most toxicity towards Brine shrimp while petroleum ether soluble fraction exerted moderate toxicity compared with vincristine sulphate having LC 50 value of 1.44, 1.831 and 0.9258 µg/mL respectively.

Characterization of Compound 1
VLC fraction of 15 yielded compound 1 by PLC as colorless liquid and molecular formula was found to be C 10 H 10 O. 1 (Table 1) of 1 showed two one proton signals at δ 7.09 (d, J = 1.6Hz) and 6.99 (d, J = 8.0 Hz), which were assigned to aromatic protons H-2 and H-5 respectively; another two proton signals at δ 7.42 (d, J = 16.0 Hz) and δ 7.15 (dd, J = 8.0 Hz, 1.6 Hz) were assigned to α and β protons respectively. They showed trans coupling (J = 16.0 Hz) with each other and the β proton showed additional coupling (J = 8.0 Hz) with the aldehyde proton (γ). The most deshielded one proton doublet at δ 9.68 was accounted for the aldehydic proton. The three proton singlet at δ 3.98 was characteristic for a methoxy group, located at 4 of the benzene ring. The spectral data confirmed compound 1 as 3 methoxy 4 hydroxy cinnamaldehyde (coniferyl aldehyde) [16].

Characterization of Compound 3
Compound 3 was also isolated from the VLC fraction of 15 by PLC as colorless liquid and molecular formula was determined as C 10  which were assigned to aromatic protons H-2 and H-6. The most deshielded one proton singlet δ 9.845 was accounted for the aldehydic proton. The nine protons singlet at δ 3.99 was characteristic for three methoxy group located at 3, 4, 5 of the benzene ring. Based on the above features, the compound 3 was identified as 3, 4, 5 trimethoxy benzaldehyde [18].

Characterization of Compound 4
VLC fraction of 17 + 18 yielded compound 4 by PLC as yellow liquid and its molecular formula was found to be C

Free Radical Scavenging Activity
Antioxidant activity of plant extracts can be accurately measured using DPPH assay method [15]. Table 2, Figure 2 showed % inhibition values of different solvent fractions of Baccaurea ramiflora stem bark at variable concentration, while Table 3, Figure 3 depicted their IC 50 value. Table 4 provided their summative antioxidant activity. Probably phenolic compounds are responsible for their antioxidant property. Their antioxidant activity was also previously mentioned [5] [9].

Brine Shrimp Lethality Bioassay
Brine shrimp lethality bioassay has been utilized as a primary screening method of lethality of different plant extracts. All the samples having LC 50 value < 1000 µg/mL are considered for further pharmacological analysis [16].