Botanical and Chemical Fingerprinting of Medicinal Roots of Justicia gendarussa Burm f.

Macro-microscopic analysis

Macroscopic characters of dried roots and powder were keenly observed under naked eyes to record the specific botanical characters. The external features of the test samples were documented using Canon IXUS digital camera.

Dried roots were preserved in formalin-acetic acid-alcohol preservative solution (5% formalin – 5 ml, 5% acetic acid – 5 ml and 50% ethyl alcohol – 90 ml).[23] After 48 h, very thin transverse sections of root were obtained using sharp blade followed by safranin staining[24] for microscopic visualization. Features were photographed using Zeiss AXIO trinocular microscope attached with Zeiss AxioCam camera under bright field light. Magnifications of figures are indicated in scale bars.

A pinch of coarse powder sifted through 80 pore size mess was mixed with drops of choral hydrate on microscopic slides and mounted with a drop of glycerin-water. Slides were observed and characterized under Zeiss AXIO trinocular microscope. Magnifications are indicated by scale bars.[25]

Physicochemical analysis

Physicochemical characterization such as loss on drying (LOD) at 105°C, total ash (TA), acid-insoluble ash (AIA), water-soluble ash (WSA), alcohol-soluble extractive (ASE) value (ASE) and water-soluble extractive (WSE) value were determined as per Quality Standard of Indian Medicinal Plants.[26]

Preliminary phytoconstituents screening

Preliminary phytoconstituents screening was done to detect the presence of active constituents in the ethanolic extract of J. gendarussa.[27]

High-performance thin layer chromatography fingerprinting

One gram of powdered roots was extracted with 10 ml ethanol and kept for cold percolation for 24 h and filtered. 4, 8 and 12 μl of the plant extract were applied on a precoated silica gel F254 on aluminum plates to a bandwidth of 7 mm using Linomat 5 (CAMAG, Muttenz, Switzerland) TLC applicator. J. gendarussa plate was developed using toluene:ethyl acetate:formic acid (5.0:4.0:1.0) as mobile phase in CAMAG twin trough chamber. The developed plate was visualized under short UV, long UV in CAMAG TLC photodocumentation unit, then derivatized with anisaldehyde-sulfuric acid reagent,[28] and scanned under UV 254, 366 and 620 nm postderivatisation. R_f, color of the spots and densitometric scan were recorded using CAMAG Scanner 4.[2930]

Gas chromatography-mass specrtometry analysis

Gas chromatography-mass specrtometry (GC-MS) analysis was carried out using Thermo Scientific Mass Spectrophotometer equipped with Triple Quad XLS. The column used was HP-5ms Ultra Inert (length: 30.0 m; diameter: 0.25 mm), with a film thickness of 0.25 μm. The carrier gas used was helium at a flow rate of 1.3 ml/min at a constant rate. Two microliters sample injection volume was utilized. The inlet temperature was maintained as 280°C. The oven temperature was programed initially at 60°C for 3.5 min and then programed to increase to 300°C at a rate of 10°C. Total run time was 22 min. The MS transfer line was maintained at a temperature of 240°C. MS was recorded using electron impact at fixed electron energy of 70eV and data were evaluated using total ion count for compound identification and quantification. The spectra of the components were compared with the spectral database of known components in the GC-MS library (NIST-11). Measurement of peak areas and data processing were carried out by Turbo-Mass-OCPTVS-Demo SPL software.[31]

Macro-microscopic observations

Macroscopically, the dried roots of J. gendarussa were about 10 cm long with a diameter of 0.5 cm. Dried roots were yellowish brown in color with rough and wrinkled surface and root scars. Powder of root was yellowish in color [Figure 1c–e] with pleasant odor. Transverse section of dried root showed elongated, compactly arranged cork cell and cortex of root was well developed under epidermis. Outer and inner region of root showed the presence of xylem vessels with some intracellular spaces. Xylem vessels were spherical and oval in shape. Large xylem vessels were found toward the outer region and their size was gradually decreased toward the inner region near pith. Parenchymatic rays were arranged in a uniseriate manner [Figure 1f–k]. Powder microscopy showed the presence of pitted lignified parenchymatous cells with lobed projection, sclereids of various dimensions were scattered and fiber sclereids with vessel elements were found. Group of stone cells were also observed in powder microscopy [Figure 2a–l].

Figure 2

(a-l) Powder microscopy of dried roots of Justicia gendarussa

Physicochemical analysis

Physicochemical characters such as LOD, TA value, AIA, water-insoluble ash, ASE and WSE are expressed in %w/w [Table 1].

Table 1

Physicochemical analysis of dried roots of Justicia gendarussa

Preliminary phytochemical analysis

According to Harborne's methodology, phytoconstituents analysis revealed the presence of carbohydrates and some secondary metabolites such as alkaloids, steroids, flavonoids, phenols, saponins and quinone [Table 2].

Table 2

Preliminary phytochemical screening

High-performance thin layer chromatography fingerprinting

R_f values and color of the spots in chromatogram developed in toluene: ethyl acetate:formic acid (5.0:4.0:1.0) for ethanolic extract of dried roots were recorded [Table 3]. TLC photodocumentation revealed the presence of many phytoconstituents at different R_f values. High-performance thin layer chromatography (HPTLC) densitometric scan of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatization). On photodocumentation, under short UV, six spots were observed; under long UV, there were nine spots and under 620 nm on postderivatization with anisaldehyde-sulfuric acid spray reagent, six spots were recorded [Figure 3a–c]. Densitometric scan at 254 nm revealed ten peaks corresponding to ten different compounds in the ethanolic extract, compounds with R_f0.04 (40.01%), 0.17 (11.99%), 0.23 (26.47%), 0.40 (1.95%), 0.42 (1.71%), 0.53 (0.93%), 0.62 (9.59%), 0.67 (1.77%), 0.80 (1.96%) and 0.90 (3.62%) are shown in Figure 4a. Densitometric scan at 366 nm [Figure 4b] showed six peaks such as R_f – 0.05 (3.71%), 0.15 (4.05%), 0.25 (2.20%), 0.58 (5.97%), 0.64 (2.61%) and 0.96 (81.46%). Figure 4c depicts seven peaks with R_f – 0.03 (23.91%), 0.23 (22.20%), 0.26 (14.84%), 0.48 (2.98%), 0.72 (6.91%), 0.81 (26.22%), and 0.86 (2.93%) after postderivatization at 620 nm.

Table 3

R_f values of all the samples

Figure 3

(a-c) High-performance thin layer chromatography photodocumentation of ethanolic extract of Justicia gendarussa

Figure 4

(a-c) HPTLC densitometric scan of ethanolic extract of Justicia gendarussa

Gas chromatography-mass specrtometry analysis

GC-MS of J. gendarussa indicated the presence of five constituents. Out of five constituents, one was unidentified. The remaining four constituents were found in trace amounts [Table 4]. Compounds identified from GC-MS analysis were fatty acids (oleic acid, 9,12-octadecadienoic acid and 6,9,12-octadecatrienoic acid) and steroids (estra-1,3,5(10)-trein-17-β-ol), which correlate well with the results of phytochemical screening. Mass spectrum of ethanolic extract of J. gendarussa indicated the similarity of identified compounds and structures with different retention times as expressed in Figures 5a–d and 6.

Table 4

Details of compounds identified from ethanolic extract of Justicia gendarussa Burm F. root

Figure 5

(a-d) Mass specrtometry of compounds identified from ethanolic extract of Justicia gendarussa root

Figure 6

Gas-liquid chromatogram of ethanolic extract of Justicia gendarussa

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Nil.

Conflicts of interest

There are no conflicts of interest.