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.

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.

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].

Finally their structures were elucidated using ¹H NMR spectroscopy (400 MHz, CDCl₃).

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:

I % = ( 1 − A b s o r b a n c e   o f   s a m p l e A b s o r b a n c e   o f   b l a n k ) × 1 0 0 %

Where, Absorbance of blank is the absorbance of control reaction (containing all reagents except the test material).

Extract concentration providing 50% inhibition (IC₅₀) was calculated from the graph plotted inhibition percentage against extract concentration.

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 visual 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 (LC₅₀) value was calculated from the graph plotted percentage mortality rate against extract concentration.

VLC fraction of 15 yielded compound 1 by PLC as colorless liquid and molecular formula was found to be C₁₀H₁₀O. ¹H NMR spectrum (400 MHz, CDCl₃)

(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].

VLC fraction of 15 yielded compound 2 by PLC as light yellow liquid. Molecular formula was determined to be C₁₂H₁₄O₄. In ¹H NMR spectrum (400 MHz, CDCl₃) (Table 1) of 2, two protons singlet at δ 6.84 protons was assigned to H-2 and H-6. Two one proton signals at δ 7.42 (d, J = 16.0 Hz) and δ 6.63 (dd, J = 16.0 Hz, 8.0 Hz) were assigned to α and β protons respectively. The most deshielded one proton doublet at δ 9.78 was indicated aldehydic proton (H-γ). The nine proton singlet at δ 3.97 was characteristic for three methoxy groups located at 3, 4, 5 of the benzene ring. So the compound 2 was identified as 3, 4, 5 trimethoxy cinnamaldehyde [17].

Compound 3 was also isolated from the VLC fraction of 15 by PLC as colorless liquid and molecular formula was determined as C₁₀H₁₂O₄. ¹H NMR spectrum (400 MHz, CDCl₃) (Table 1) of 3 displayed a two protons singlet at δ 7.718, 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].

VLC fraction of 17 + 18 yielded compound 4 by PLC as yellow liquid and its molecular formula was found to be C₉H₁₀O₃. Three proton signals at δ 7.45 (d, J = 1.6 Hz), δ 7.07 (d, J = 8.4 Hz) and δ 6.95 (dd, J = 8.4 Hz, 1.6 Hz) were displayed in ¹H NMR spectrum (400 MHz, CDCl₃) (Table 1) of 4, accounted for an ortho & para substituted aromatic ring assigned as H-2, H-5 and H-6 respectively. H-2 and H-6 showed meta coupling (J = 1.6 Hz) to each other while H-5 and H-6 showed ortho coupling (J = 8.4 Hz) to each other. The most deshielded one proton singlet at δ 9.857 was characteristic for aldehyde proton while the six protons singlet at δ 4.00 indicated presence of two methoxy groups located at 3, 4 of the benzene ring. The compound 4 was identified as 3, 4 dimethoxy benzaldehyde (veratraldehyde) [19].

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 IC₅₀ 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 has been utilized as a primary screening method of lethality of different plant extracts. All the samples having LC₅₀ value < 1000 µg/mL are considered for further pharmacological analysis [16]. Table 4, Figure 4 depicted mortality rate of different fractions of Baccaurea ramiflora while Table 5, Figure 5 showed different degree of lethality of plant extracts of Baccaurea ramiflora to Brine shrimp. Among the fractions, chloroform soluble fraction was found to be most toxic to brine shrimp and petroleum ether soluble fraction showed moderate toxicity compared to anticancer drug vincristine sulphate, which support the previous data about this [9] [10] [20]. Further in vivo acute oral toxicity study can confirm whether this toxicity level is harmful for susceptible biological systems or not.

BHT = Tert-Butyl-1-hydroxytoluene, BRA = Aqueous soluble fraction, BRE = Ethyl acetate soluble fraction, BRC = Chloroform soluble fraction, BRP = Petroleum ether soluble fraction, BR = Baccaurea ramiflora; Absorbance of blank = 0.3018.

VS = Vincristine sulphate.