The genus Artocarpus belongs to family Moraceae and consists of more than 50 species. The species are either evergreen or deciduous trees, found in India, southern China, Malaysia and the Solomon Islands. Fruits of specific Artocarpus species are edible such as jack fruit and bread fruit, whereas other species of this genus (notably, A. heterophyllus Lam., A. altilis (Parkinson) Fosberg, A. hirsutus Lam., A. lakoocha Roxb. and A. camansi) usually find broad application as traditional medicines for treating diseases like diabetes, diarrhoea, dermatitis, malarial fever, asthma, tapeworm infection and anaemia.1 Studies on leaf extracts of A. lakoocha Roxb. clearly demonstrate its ability in protection against liver damage, lowering elevated blood pressure and managing the blood sugar.2 Major components in the bark, leaves and heart woods of the genus Artocarpus were found to be flavonoids with isoprene side chains3-6 possessing antioxidant, anti-inflammatory, anti-diabetic, tyrosinase and melanin inhibition properties.3,6-9
A. hirsutus commonly known as wild jack, is mainly distributed in South Western Ghats of Peninsular India.10 Like other species of the genus Artocarpus, A. hirsutus also acquires similar plant biographies. Notably, it’s leaves are used traditionally in treating burboes and hydrocele whereas the leaves, fruits and barks help to cure diarrhoea, skin disease and haemorrhage.11 The paste of bark ash in coconut oil is used for Tinea curas (dhobi’s itch). Topical application of stem bark infusion is found to be effective in healing sores, cracks and pimples,1 whereas the fruit juice of A. hirsutus induces appetite and relives the pains of haemorrhage.10 Studies on pylorus ligated rats demonstrates that the A. hirsutus stem bark extract reduces the gastric secretory volume, acidity and ulceration.12
Nutritional natural supplements are often superior to synthetic supplements as they have minimal adverse effects, even in use for longer period. However, despite the benefits, there is still a relatively low commercial demand for these supplements. This could be due to non-availability of authentic raw material; inferior quality of raw material supply as well as lack of reproducible analytical techniques on herbal research.13 These technical complications can be resolved by conducting pharmacognostic studies of the raw material, which includes organoleptic characters, macroscopic analysis, microscopic analysis and powder study, physicochemical analysis, phytochemical analysis, HPLC and HPTLC finger print analysis.14
Marker compounds of natural origin are mostly used for confirming the correct botanical identity of the respective starting material. It is difficult to identify the correct marker compound for all traditional medicines because of the presence of unidentified multiple active constituents. By using chromatographic fingerprints, the authentication and identification of herbal medicines can be accurately achieved even if the amount and/or concentration of the components are not same in different plant extracts. Consequently, the lack of reliable chromatographic fingerprints in the literature demonstrates the need to develop authentic techniques to reproduce pharmacologically active and chemically characteristic component of the herbal medicine.
The genus Artocarpus has been monographed by Jarrett.15 However, no pharmacognostic study has been reported on A. hirsustus except its stem bark by Dibinlal and Seethadevi.12 The present study is thus designed and executed in order to incorporate microscopic, phytochemical, HPLC and HPTLC finger print analysis of leaf and stem wood of A. hirsutus as an additional source of analytical techniques on this species.
MATERIALS AND METHODS
General experimental procedures
HPTLC finger printing studies were performed on CAMAG LINOMAT 5 using pre-coated TLC plates [10 x10 cm, silica gel 60 F254 TLC plates (Merck, India)]. The images were captured by using photo-documentation chamber (CAMAG REPROSTAR 3). The HPLC analysis was performed with Shimadzu Lab Solution HPLC system using LC-20AD software. Morphological evaluation was carried out using Nikon eclipse Ci microscope and the photographic images were captured by using a digital Nikon camera DS-Ri 2 fixed with the microscope.
Collection of plant materials
Fresh matured leaves and stem wood (Figure 1A and 1B) were collected from Udupi district, Karnataka, India, by the author in January 2015 and authenticated by Dr. M.D. Rajanna (Professor, Botanical Garden, University of Agriculture Sciences, GKVK, Bangalore, India). The voucher specimens were deposited in the departmental herbarium and assigned the voucher numbers (RD/HU-AH/10 and 12).
Fresh samples of leaf and stem wood were preserved in formalin-acetic acid-alcohol (40% formalin: 5 mL: 50% ethanol: 90 mL; glacial acetic acid: 5 mL). Transverse sections were taken by using razor blade and were stained with Toluidine Blue O 0.05% in benzoate buffer (0.25g of benzoic acid in 200 mL water pH 4.4),16 washed with water, observed under bright field as well as the epifluorescence optics using Nikon eclipse Ci microscope and the photographic images were captured using a digital Nikon camera DS-Ri 2 fixed with the microscope. The images were processed on Image NIS elements BR.
Preparation of plant extract
The fresh leaves and stem wood materials were dried and pulverized to a coarse powder. The powdered materials were individually extracted with three volumes of ethanol at refluxing condition (65–70°C) for three times. The ethanolic extracts were dried completely under vacuum and subjected to phytochemical analysis, high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC) finger print analysis to identify the chemical nature of the secondary constituents present in it.
The preliminary phytochemical screening for sterols, triterpenoids, alkaloids, flavonoids, lactones, tannins, saponins and carbohydrates were carried out as described by Evans.17
HPTLC fingerprinting analysis
HPTLC finger printing studies were performed following the methods of Wagner and Baldt,18 and Harborne.19 Various concentrations of both leaf and stem wood extracts (1, 2.5, 5, 10 and 20 mg/mL) were prepared in HPLC grade methanol (SD Fine chemicals, India) filtered through Whatman filter paper No. 1 and were used as test solutions for HPTLC analysis. The test solutions (2.0 μL each) were then loaded on pre-coated TLC plates [10 x10 cm, silica gel 60 F254 TLC plates (Merck, India) with aluminium sheet support] with band length of 6 mm using a CAMAG LINOMAT 5 Automatic Sample Spotter (Camag Muttenz, Switzerland) in duplicate. Similarly, another TLC plate (5 x 10 cm) was loaded with 2.0 μL each of ethanolic stem wood extract (concentration: 20 mg/mL), leaf extract (20 mg/mL), oxyresveratrol (1mg/mL) and artocarpin (1 mg/mL). All loaded TLC plates were placed in twin trough glass chamber saturated with mobile phase (chloroform: methanol, 8:2) for 20 minutes and eluted to a distance of 9 cm. The developed plate was positioned in the photo-documentation chamber (CAMAG REPROSTAR 3) and images were captured at 254 and 366 nm. Furthermore, these plates were scanned at 200 – 400 nm by using HPTLC Scanner 3. To visualize the spots, the developed plates were exposed to iodine vapours as well as sprayed with vanillin sulphuric acid reagent followed by drying at 100 °C in hot air oven for 10 minutes. The images of vanillin sulphuric acid reagent treated plates were recorded in daylight and at 366 nm.
The HPLC analysis were performed with Shimadzu, LC-20AD, prominence using a BDS Hypersil C18 (Thermo), 250 x 4.6 cm, 5 μM column and mobile phase A: 0.05% acetic acid in water; B: 100% acetonitrile. The gradient program is depicted in box 1.
Total flow rate: 1.0 mL/min; detector: PDA at 254 nm; injection volume: 20 μL; run time: 30 min; diluent: methanol (HPLC grade)
Sample solutions: Dissolved 100 mg each of the ethanolic leaf and stem wood extracts with 25 mL of diluents in 50 mL volumetric flasks. After sonication for 15 minutes the volume was adjusted up to 50 mL with diluent and then filtered through Whatman No. 42 (ash less, diameter 125 mm, Cat. No. 1442-125).
Standard solutions: oxyresveratrol and artocarpin solutions were prepared with diluents at concentration of 0.1 mg/mL.
RESULTS AND DISCUSSION
Leaf lamina is dorsiventral and the upper epidermis is mucilaginous. A few hairy trichomes are present near the midrib region. Occasionally, the outer cuticle envelope is interrupted by hydathode openings. The mesophyll cells are entirely of palisade parenchyma (2-4 cells thick) type and filled with numerous chloroplasts. It forms a complete network of cells with several secretory glands, called pearl glands, which is a significant feature of the leaf lamina surrounded by narrow bridges that contains vascular bundles. The upper epidermis is single layer thickness consisting of cystoliths here and there with calcified cell walls. The hairy trichome present in the lower epidermis consists of solitary prismatic calcium carbonate crystals at its base. The midrib contains 8-10 vascular bundles. Vascular bundle is amphicribral type i.e. xylem surrounded by phloem. A sclerenchymatous cap surrounds bundle on the upper epidermal side (Figures 2A-F and 3A-D).
Transverse section of Leaf of A. hirsutus A. The entire view of leaf stained with TBO (4x); B. Transverse section of leaf viewed under 10x enlarged view of midrib; C. Transverse section of leaf blade; D. Enlarged view of leaf blade viewed under 10x; E. Transverse section shows the complete network of cells with secretory cells; F. Presence of trichome in the lower epidermis layer of leaf blade.
Transverse section of the leaf viewed under UV. A. Leaf blade reflects the blue and red colour resembling the lignified cell wall and vascular bundles (blue) and chloroplast filled palisade parenchyma cells (red); B. Enlarged view of chemical content present in the leaf (arrowed); C. Midrib region fully lignified; D. Palisade parenchyma cells shows the presence of chloroplast and the leaf blade covered with the thin layered cuticle.
The stem wood is brown to light black in colour, diffuse-porous, with solitary pores (occasionally grouped with two or three) and distinct growth rings. Tylose formation occurs in vessels in the matured wood. Vessel elements are simple perforation plates. Intervessel pits are alternate. The medullary rays are 2-3 cells thick. Ray cells are procumbent with one row of upright or squared marginal cells, axial parenchyma cells are paratracheal (Figure 4A-B).
The preliminary phytochemical analysis (Table 1) of extracts of leaf and stem wood revealed that the presence of sterols, terpenoids, flavonoids, tannins and saponins. Vinay et al,20 reported the presence of alkaloids, flavonoids, saponins and terpenoids in A. hirsutus fruit whereas similar results described by Lakshmi et al,21 in root. Azeem et al,22 demonstrated the presence of flavanoids, glycosides, saponins, carbohydrates, proteins and amino acids, tannins and alkaloids in ripped fruits.
HPTLC finger prints were developed for extracts in various solvent systems with changing polarities to verify the chemical constituents present. Our study revealed that the solvent system, chloroform:methanol (8:2) was superior among the other combinations for both leaf and stem wood extracts. The HPTLC images, as shown in figures 5, 6, 7 and 8, indicate the clear separation of constituents of both extracts. The Rf values are tabulated in table 2.
HPTLC photo documentation of ethanolic extract of wood of A. hirsutus: A. at 254 nm and B. at 366 nm light. Where, Tracks 1and 2: 1 mg/mL, Tracks 3 and 4: 2.5 mg/mL, Tracks 5 and 6: 5 mg/mL, Tracks 7 and 8: 10 mg/mL, Tracks 9 and 10: 20 mg/mL.
HPTLC photo documentation of ethanolic extract of wood of A. hirsutus after A. Iodine vaporization; B. Vanillin sulphuric acid treatment recorded in visible light; C. Vanillin sulphuric acid treatment recorded at 366 nm light. Where, Tracks 1and 2: 1 mg/mL, Tracks 3 and 4: 2.5 mg/mL, Tracks 5 and 6: 5 mg/mL, Tracks 7 and 8: 10 mg/mL, Tracks 9 and 10: 20 mg/mL.
HPTLC photo documentation of ethanolic extracts of leaf of A. hirsutus A. at 254 nm and B. at 366 nm Light. Where, Track 1and 2: 1 mg/mL, Track 3 and 4: 2.5 mg/mL, Track 5 and 6: 5 mg/mL, Track 7 and 8: 10 mg/mL, Track 9 and 10: 20 mg/mL.
HPTLC photo documentation of ethanolic extracts of leaf of A. hirsutus after A. Iodine vaporization; B. Vanillin sulphuric acid treatment recorded in visible light; C. Vanillin sulphuric acid treatment recorded at 366 nm light. Where, Tracks 1and 2: 1 mg/mL, Tracks 3 and 4: 2.5 mg/mL, Tracks 5 and 6: 5 mg/mL, Tracks 7 and 8: 10 mg/mL, Tracks 9 and 10: 20 mg/mL.
HPTLC photo documentation of ethanolic extracts of leaf and stem wood of A. hirsutus along with oxyresveratrol and artocarpin after A. at 254 nm; B. at 366 nm light. Where, Track 1: 20 mg/mL stem wood extract; Track 2: Oxyresveratrol (1 mg/mL); Track 3: Artocarpin (1mg/mL); Track4: 20 mg/mL leaf extract.
HPTLC of stem wood extract
HPTLC of ethanolic extract of stem wood illustrated 7 compounds when observed under 254 nm light (Figure 5A). The spot having Rf at 0.60 was found to be the major component, which was navy blue in colour. When the same TLC plate was monitored under UV light at 366 nm (Figure 5B), 13 compounds were noted. Of these, the spot with Rf at 0.30 was found to be major component, which appeared as bluish fluorescent in colour. The intensity of the major compound at 254 nm was reduced when it was observed at 366 nm.
There were 6 compounds with yellow to yellowish brown colour when the TLC plate was exposed to iodine vapours (Figure 6A). Both compounds with Rf 0.30 and Rf 0.60 were found to be major components.
When the TLC plate was treated with vanillin sulphuric acid and heated at 100 °C for 10 minutes, 6 compounds were detected at day light (Figure 6B). The spots with Rf below 0.30 showed pink colour whereas Rf at 0.60 and Rf at 0.73 were yellow in colour. The spot with Rf at 0.30 was found to be major compound. Interestingly when the same TLC plate was visualized under 366 nm (Figure 6C), the spots of Rf of 0.60 and Rf of 0.73 were illuminated with pale yellow and blue fluorescence respectively. The spots at Rf of 0.30 and Rf of 0.60 were found to be major compounds.
HPTLC of leaf extract
HPTLC finger print of ethanolic leaf extract displayed 4 spots (Figure 7A) with Rf at 0.28, 0.58, 0.63 and 0.82 when the TLC plate was analysed at 254 nm and the intensities of all spots were low. There were 8 spots observed when the TLC plate was visualized at 366 nm (Figure 7B). The spot having Rf at 0.80 was found to be a major component at 366 nm, which was blue fluorescent in colour. The spot with Rf at 0.88 when visualized in 366 nm was pink in colour and expected to be a triterpenoid.18
Five spots with yellow to yellowish brown colour were identified when the TLC plate was exposed to iodine vapours (Figure 8A). The intensity of the compound with Rf at 0.65 was highest compared to the other compounds present in the extract. The intensity of the major compound (Rf 0.80) at 366 nm was diminished, similarly it was observed that on exposure to iodine vapours, it’s yellow colour disappears. When the TLC plate was treated with vanillin sulphuric acid and heated at 100 °C for 10 minutes, 5 spots were identified at day light (Figure 8B). The spot above Rf at 0.80 appeared as pink in colour with maximum intensity. Interestingly when the same TLC plate was visualized under 366 nm (Figure 8C), more than 10 spots with different colour intensities were detected. The spots having Rf at 0.18 and Rf at 0.23 were bluish fluorescent in colour. The spots with Rf at 0.55 and Rf at 0.60 were yellow and greenish yellow in colour respectively. The spots at Rf of 0.80 and Rf of 0.85 were pink in colour. The intensities of all these spots were same and could be isolated individually.
Comparison of HPTLC finger prints with standard compounds
Further the HPTLC finger prints (Figures 9A, 9B, 10A, 10B and 10C) of both stem wood and leaf extracts (20 mg/mL) were compared with two standard materials notably oxyresveratrol23 and artocarpin.24 Oxyresveratrol is a stilbene derivative and found in species of the same genus, viz. A. lakoocha Roxb.; A. champlasha Roxb.; A. heterophyllus Lam.; A. gomezianus Wall. (7, 24-26).
HPTLC photo documentation of ethanolic extracts of leaf and stem wood of A. hirsutus along with oxyresveratrol and artocarpin after A. Vanillin sulphuric acid treatment recorded at visible light; B. Vanillin sulphuric acid treatment recorded at 254 nm light; C. Vanillin sulphuric acid treatment recorded at 366 nm light. Where, Track 1: 20 mg/mL stem wood extract; Track 2: Oxyresveratrol (1 mg/mL); Track 3: Artocarpin (1mg/mL); Track 4: 20 mg/mL leaf extract.
Oxyresveratrol spot was illuminated with bluish fluorescence at 366 nm with Rf at 0.30 (Figure 9B) and this spot was appeared as pink in colour when the TLC plate was sprayed with vanillin sulphuric acid and heated at 100 °C for 10 minutes (Figure 10A; 27) whereas it was detected as purple in colour when it was visualized at 366 nm (Figure 10C). Artocarpin is a flavonoid and reported to be found in species of the same genus mainly A. heterophyllus, A. incises, A. nitidus and A. hirsutus.24,28-30 The HPTLC of artocarpin displayed as dark spot at 254 nm (Figure 9A) and navy blue spot at 366 nm (Figure 9B). The compound also appeared as yellow spot with vanillin sulphuric acid reagent when detected at visible light and at 366 nm (Figure 10A, 10C).
HPTLC finger print of ethanolic stem wood extract showed the presence of both oxyresveratrol as well as artocarpin as the major compounds. Whereas appearances of these compounds were absent in HPTLC finger prints of ethanolic leaf extract. The constituents of stem wood and the leaves were totally different and cannot be interchanged. Hence it is essential to isolate the actives from the leaf extract and explore their pharmacological benefits. Recently, it was reported by us that oxyresveratrol isolated from wood of A. hirsutus possessed promising anti-inflammatory, antioxidant and skin lightening properties.23
HPLC chromatogram of stem wood extract (Figure 11) represented the presence of 4 major peaks, with retention time (RT) at 11.206 (17.928%), 20.114 (4.001%), 23.092 (52.368%) and 25.180 (7.001%) minutes. Of these, the peak eluted at 23.092 min was found to be the major component and contains more than half of the total weight. The second highest peak was found with RT at 11.206 min. These two peaks correspond with artocarpin (RT 23.040 min) and oxyresveratrol (RT, 11.004 min) respectively.
The HPLC chromatogram of leaf extract (Figure 12) showed the presence of 5 peaks with retention time (RT) at 7.533 (10.849%), 9.910 (11.874%), 19.544 (52.368%) 20.197 and 20.592 (7.001%). However, the corresponding peaks for oxyresveratrol and artocarpin were not detected in the HPLC chromatogram. It further reconfirms with the HPTLC results and represents the absence of oxyresveratrol and artocarpin in the leaf extract.
Present study describes the micromorphology, phytochemical analysis, HPLC and HPTLC finger print analysis of the leaf and stem wood which are in use as the traditional medicine. Determination of cell structural organisation and analysis of the tissues system are some of the pharmacognostic properties that are important for identifying the correct species of the plant and for distinguishing between closely related species of the same genus. The leaf shows the distinct network of mesophyll cells with the pearl glands and the palaside parenchyma cells possessing 2 to 4 cells thick with several chloroplasts. The axial parenchyma cells of stem wood are paratracheal and vessels are filled with tylose and vessels elements show the pits with simple perforation plates.
The preliminary phytochemical analysis of ethanolic extracts of leaf and stem wood of A. hirsutus showed that the presence of various secondary metabolites like sterols, terpenoids, flavonoids, lactones, tannins and saponins. The mobile phase, chloroform: methanol (8:2) was found to be superior solvent system for HPTLC and the plates were displayed with maximum separation of the components having distinct Rf values. This solvent system can be used during the isolation of constituents from both extracts. HPTLC finger prints of stem wood extract demonstrated the presence of oxyresveratrol and artocarpin, whereas they were absent in leaf extract and it was further confirmed by HPLC analysis. Hence the stem wood extract cannot be replaced with leaf extract for commercial purpose.
In conclusion, the micromorphological parameters, HPLC and HPTLC finger prints presented in this report can be used as diagnostic tools for the correct identification of the raw material of similar species, as well as to distinguish the admixture of known/or unknown material and to check the quality of the raw material. Both oxyresveratrol and artocarpin could be used as reference compounds for standardization of the stem wood extract of A. hirsutus.