Isolation and Cytotoxic Investigation of Flacourtin from Oncoba spinosa

Background: Oncoba spinosa, an endangered medicinal plant whose secondary metabolites have not been extensively profiled, and which is hitherto yet to be examined for cytotoxicity, is being investigated in this study. Methods: Leaves of Oncoba spinosa (800 g) were extracted with 95% aqueous methanol. The crude extract was partitioned with n-hexane and the resultant defatted extract was extensively chromatographed on silica gel to yield compound 1 which was subjected to spectroscopic analysis. A brine shrimps lethality test was used to establish the cytotoxicity potentials of the isolated compound and the plant extracts. Results: Compound 1 was elucidated as flacourtin, 3-hydroxy-4-hydroxymethylphenyl-6-O-benzoyl-β-d-glucopyranoside. The LD50 values obtained were less than 1000 µg/mL for flacourtin and the plant extracts. Conclusion: Flacourtin is being reported for the first time in the O. spinosa. The preliminary toxicity assay indicated that flacourtin and the plant extracts were not cytotoxic; thus, the tradomedicinal uses of the plant may portend no danger.


Plant Collection and Purification
Fresh leaves Oncoba spinosa were collected at the Botanical Garden University of Ibadan, Oyo State, Nigeria in the month of November, 2014. The plant was identified by the taxonomist and curator, Mr A. Owolabi in the Botanical Garden. A voucher copy of the plant with herbarium number FHI 108806 was deposited at the Forestry Research Institute of Nigeria, Ibadan.
The air-dried and pulverized leaves (800 g) were extracted with 95% aqueous methanol to give a crude extract (140 g). The crude was reconstituted in methanol and defatted with n-hexane to afford 80.60 g of defatted extract. Twenty grams of the defatted extract was chromatographed on silica gel using gradient of dichloromethane and methanol to give a sub-fraction (1.22 g) which was further purified on silica with gradient of hexane, ethyl acetate and methanol. Compound 1 (6 mg) was obtained as an off-white powder which was recrystallized in ethylacetate and thereafter subjected to spectroscopic analysis.

Cytotoxicity Assay
Cytotoxicity of the crude extract, defatted fraction and compound 1 were investigated using brine shrimp lethality assay. A spoonful quantity of the egg of brine shrimps (Artemia salina Leach) was sprinkled into a partly covered crucible containing seawater in order to allow partial illumination. The eggs hatched into matured nauplii after about 48 h and 10 matured nauplii were added to varying concentration of the test samples (1000, 100 and 10 µg/mL). All the experiments were carried out in triplicate and distilled water was used in place of the test samples for negative control. Numbers of survivors were counted after 24 h and the LC 50 was computed at 95% confidence limit using the Finney program.

Discussion
The NMR spectra of compound 1 showed a pattern characteristic of a phenolic ester glycoside. Clusters of peaks at δ 3.5-5.0 indicated the presence of a sugar moiety while the aromatic region showed the presence of eight protons with J values typical of an aromatic system, thus suggesting two substituted benzene rings (Figure 1). The IR spectrum showed absorption bands (cm −1 ) at 3000 Three aromatic protons of ring B with J values attributable to a 1,2,4-trisubstituted aromatic system showed at δ 7.03, 6.77 and 6.48. The two protons each at ortho and meta position in ring A are in the same chemical environment, thus both protons at ortho position resonated at δ 8.05 with Jo and Jm of 7.2 and 1.5 Hz respectively. Similarly, symmetrical protons at meta positions were observed at δ 7.53 with coupling constant of 7.2 and 7.8 Hz, showing that the protons were doubly ortho-coupled, which connoted that ring A is monosubstituted. All the spectroscopic data and their assignments were in good agreement with the report of Bhaumik et al [4]. The compound (Figure 1), which was isolated for the first time from leaves of O. spinosa, was elucidated as 3-hydroxy-4hydroxymethylphenyl-6-O-benzoyl-β-D-glucopyranoside, previously named flacourtin.
The brine shrimps lethality test indicated that crude extract, methanol fraction and flacourtin had LD50 values greater than 1000 μg/mL ( Table 1) which implied that they exhibited no cytotoxic activity on the nauplii.

Conclusions
Flacourtin is being reported for the first time in the O. spinosa. The preliminary toxicity profile as indicated by brine shrimp lethality test showed that the compound and the plant extracts were not cytotoxic; therefore, the tradomedicinal uses of the plant may portend no danger. Three aromatic protons of ring B with J values attributable to a 1,2,4-trisubstituted aromatic system showed at δ 7.03, 6.77 and 6.48. The two protons each at ortho and meta position in ring A are in the same chemical environment, thus both protons at ortho position resonated at δ 8.05 with J o and J m of 7.2 and 1.5 Hz respectively. Similarly, symmetrical protons at meta positions were observed at δ 7.53 with coupling constant of 7.2 and 7.8 Hz, showing that the protons were doubly ortho-coupled, which connoted that ring A is monosubstituted. All the spectroscopic data and their assignments were in good agreement with the report of Bhaumik et al. [4]. The compound (Figure 1), which was isolated for the first time from leaves of O. spinosa, was elucidated as 3-hydroxy-4-hydroxymethylphenyl-6-O-benzoyl-β-D-glucopyranoside, previously named flacourtin.
The brine shrimps lethality test indicated that crude extract, methanol fraction and flacourtin had LD 50 values greater than 1000 µg/mL ( Table 1) which implied that they exhibited no cytotoxic activity on the nauplii.

Conclusions
Flacourtin is being reported for the first time in the O. spinosa. The preliminary toxicity profile as indicated by brine shrimp lethality test showed that the compound and the plant extracts were not cytotoxic; therefore, the tradomedicinal uses of the plant may portend no danger.