Fig 1.
Two color staining and scoring system of the bones, teeth and cartilages in treated zebrafish larvae at 6 dpf.
(A) target craniofacial bones and cartilages related to a sample of the stained zebrafish larvae: blue parts represent ceratohyal and red parts demonstrate mineralized sections, (B) ceratohyal scoring system: 0 when the target area was just blue without bone mineralization, 1 for bone mineralization of one part of target cartilage (one row of osteoblasts), 1.5 for red stained one part of cartilage, but two parts of the paired one (one and two rows of osteoblasts), 2 for red stained two parts of cartilage (two rows of osteoblasts), 2.5 for red stained two parts of cartilage, but three part of the paired one (two and three rows of osteoblasts), and 3 was for completely mineralized bone(three rows of osteoblasts), (C) ceratobrancial 5 as location of teeth scoring system: 1 for samples with just one tooth (4V1) on both posterior ceratobranchial arches, 2 for calcified 4V1 and 3V1, 2.5 for 4V1-3V1 and 5V1, 3 for 4V1, 3V1 and 5V1, 3.5 for 4V1, 3V1, 5V1, 4V2, and 4 for four and more calcified teeth, (D) cartilage of ceratohyal angle scoring: 0 for Ch-angle ≤ 70, 1 for 70 < Ch-angle < 90, 2 for Ch-angle ~ 90, and 3 for Ch-angle > 90, (E) the destination between Meckel’s cartilage and ceratohyal (M-Ch), the destination between two Opercles (head wide)the destination between Meckel’s cartilage and notochord (head length) to calculate M-Ch: Wide, M-Ch: length, wide: length. Circles showed the calcified cells.
Fig 2.
The schematic process of column chromatography of the root extract of Ferula. ovina.
(A) Shoots of F. ovina, (B) Root of F. ovina, (C) extract of the root of F. ovina, (D) Thin Layer Chromatography (TLC) of methanol extract of the root of F. ovina compared to Ferutinin (Four fractions), Red allows for the result of extract samples, blue arrows for Ferutinin, purple dashed lines for the situation of fractions, (E) column chromatography of methanol extract of the root of F. ovina: red shows Stylosin, yellow represents Tschimgine, blue indicates Ferutinin.
Fig 3.
The samples of alcian blue-alizarin red staining of the zebrafish larvae (6dpf) exposed to Fenoferin, Ferutinin and root extract of Ferula ovina compared to control.
Scale bars represent 500 μM. The blue parts represent ceratohyal and red parts indicate mineralized sections. (A) larvae exposed to 0.1 μg/ml of Fenoferin from 2dpf, (B) larvae exposed to 0.5 μg/ml of Fenoferin, (C) larvae exposed to 1 μg/ml of Fenoferin, (D) larvae exposed to 1.25 μg/ml of Ferutinin (E) larvae exposed to 5 μg/ml of root extract of Ferula ovina, (F) larvae exposed to negative control (DMSO).
Fig 4.
Effect of concentration of Fenoferin, Ferutinin and root extract of Ferula ovina on bone mineralization, tooth formation and cartilage changes in treated zebrafish larvae.
Bars represent mean +SEM. Samples were analyzed in triplicate. (A) Craniofacial cartilage ratio: the gray bars represent M-Ch: Length; the yellow bars represent Wide: Length; the Green bars represent M-Ch: Wide, (B) Ceratohyal angle: the light blue bars indicate ceratohyal angle, (C) Ceratohyal mineralization: The orange bars represent ceratohyal mineralization (D) Tooth formation: The blue bars represent ceratohyal mineralization (E) The blue part represents cartilage changes including M-Ch: Length, Wide: Length, M-Ch: Wide and ceratohyal angle; the red part indicates bone mineralization and tooth formation. The circles represent groups with significant difference in mean, which were statistically different (* p < 0.05), by multiple comparison of means using one-way ANOVA and Tukey’s post hoc test (gray circles for M-Ch: Length, yellow circles for Wide: Length, Green circles for M-Ch: Wide, light blue circles for ceratohyal angle, blue circles for tooth formation, orange circles for ceratohyal mineralization).