Macro-microscopy and HPTLC Atlas of Heartwood of Erythroxylum monogynum Roxb. (Indian Bastard Sandalwood)

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Macro-microscopy
The heartwood are submerged in water for two days and free hand sections were taken following standard procedures. [33]Heartwood was polished with silicon carbide waterproof paper 320 grit and scanned with canoscan LiDE 300 (3500dpi) and micro-morphological characters were examined under Zeiss Axiolab 5 trinocular microscope fitted with Axiocam 208 color camera.Powder characters were drawn under 200X magnifications with the help of Olympus BX43 trinocular microscope having drawing tube.

Chemicals, Solvents and Reagents
All the chemicals and solvents used were AR grade (Merck).For visualizing the developed spots in TLC, reagent containing vanillin (1 g) sulphuric acid (5%) in ethanol (VSA) was used.The mobile phases used were toluene: ethyl acetate: formic acid (7:2.5:0.5, v/v/v) for ethanol extract.This mobile phase offered a good separation of phytoconstituents while running the trial TLCs.Similarly, chloroform: methanol (6:2, v/v) lifted the alkaloids in the alkaloid fraction.6] Instrument for HPTLC Automatic sampler ATS4 was used for application of extracts on TLC plate; twin trough chamber (10×10 cm) was used for plate development; visualizer was used for photo documentation under UV-Visible conditions; Scanner 4 with winCATS software was used for obtaining densitograms; TLC plate heater was used for derivatization (all from CAMAG, Switzerland).

HPTLC Procedure
Powdered stem heartwood (1 g) was soaked overnight in ethanol (25 ml).Boiled over water bath, filtered and concentrated to 10 ml.For alkaloid fraction preparation, the drug (5 g) was soaked overnight with 5% acetic acid.Then filtered, neutralized with sodium hydroxide and pH was adjusted to 8. Then repeatedly extracted with chloroform, concentrated to 10 ml.Ethanol extract and alkaloid fraction (10 μl on each track) were applied as 8 mm bands on silica gel 60F 254 coated aluminium plate (8x10 cm) using ATS4 applicator from 10 mm from left side and 10 mm from bottom of the plate.The plate was developed in the mobile phases after pre-saturation of the twin trough chamber (10×10 cm) with mobile phases.The plates were developed up to 90 mm from the bottom.The developed plates were air dried, viewed under 254 nm, 366 nm and the images were documented using visualizer followed by dual wavelength scanning with the aid of Scanner 4 at λ 254 nm (D 2 lamp, absorption mode) and λ 366 nm (Hg lamp, fluorescence mode) with a slit dimension of 6×0.45 mm and scanning speed of 20 mm/s.One plate was dipped in a dip tank containing VSA reagent and heated at 100°C or till the appearance of coloured spots, the derivatized TLC plates were photo documented under white light followed by scanning at λ 520 (W lamp, absorption mode) and other plate was derivatized in Dragendroff 's reagent to detect alkaloids.

Macroscopy
Heart wood is very hard and heavy, difficult to break; longitudinally cut pieces are 10 to 15 cm long, 1 to 4 cm wide and 1 to 2 cm thick; surface smooth, fracture sharp and splintery, cut surface shows dark narrow line of late wood and light coloured wide zone of early wood (annular rings), dark brown in color but later on turns slightly reddish-brown (Figure 2 and 3); specific gravity 0.83; odour pleasant, taste slightly bitter and astringent.

Microscopy
TS of heartwood shows simple perforation; diffused porous vessels are mostly arranged in radial multiplication groups of as many as 6 to 8 vessels, minimum 2 and maximum 11 celled group; few are isolated, circular or elliptical in outline, 95 to 120 per mm 2 , vessel length 270 to 1200 µm, width 40 to 90 µm, few are up to 120 µm; vessels contain non-lignified thin-walled tyloses with reddish brown content, crystal like dry gum substance and oil globules; xylem rays are storied, filled with brownish content and starch grains; mostly uni and bi-seriate, rarely tri-seriate, running almost straight and parallel, except near to vessels it get slightly bent, inter ray distance 30 to 150 µm; axial parenchyma partially vesicentric (paratracheal) embedded with reddish brown content, gum like substances and oil globules; isolated and irregularly running 3 to 6 cells of metatracheal parenchyma connects adjacent tracheids; fibres, vessels/vessel groups also present the growth ring margin, a few isolated metatracheal parenchyma are embedded with prismatic crystals of calcium oxalate; ground tissue consist non septate, radially arranged, thick walled, narrow lumened fibres which are 600 to 1600 µm in length, 7 to 11.5 µm thick wall and up to 27 µm wide, mostly angular to rectangular and few are oval in shape (Figure 4).RLS shows heterogeneous xylem rays composed of linear rectangular shaped body ray cells (procumbent), mostly 2 to 14 cells rows having single rows of square shaped marginal (upright) cells on both side; thick walled tailed pitted vessels have tyloses with yellow to reddish brown content and white colour dry gummy (crystal mass like, but not glittering under polarizer light) substances; diffused to diffused in aggregate paratracheal and irregularly storied metatracheal axial parenchyma have two distinct sizes with circular and elliptical pits; square shaped metatracheal cells arranged in vertical columns of 10 to 28 cells, maximum 36 cells high, embedded with prismatic crystals of calcium oxalate, rectangular shaped metatracheal cells contain reddish brown content, gum like substances and oil globules (Figure 5 A and B).
TLS shows thick-walled tailed pitted vessel, inter pits are simple round to oval and at few places elliptical; lenticular shaped ray cells 120 to 350 µm high and 15 to 35 µm wide; rays groups are 38 to 48 per mm 2 , uni and bi-seriate, mostly 4 to 16 cell rows high, storied, embedded with reddish brown content, starch grains, oil globules and adjacent to arranged axial parenchyma, pitted vessels with tyloses, fibre-tracheids, fibres (Figure 6 A and B).

Powder Microscopy
Fibres are thick walled, narrow lumened, crossing with thin line like middle lamellae between adjacent walls, mostly non storied, few are embedded with brownish content, ends pointed, length mostly 0.6 to 1.2 mm and few are up to 1.8 mm, width 9 to 40 µm, cell-wall thickness 4 to 19 µm; simple perforated, cylindrical with circular or elliptical opening tailed vessels, tails on both side and few with tails on single side but a little projection is there on the other side, end wall slightly oblique, inter-vessel pits are simple and opposite, mostly oval to circular, vessel ray pits have two distinct sizes, similar to inter-vessel pits and same ray cell 2 to 5 µm in width and elliptical, pits up to 19 µm, vessels have two type of tyloses; prismatic crystal fibre; fibre tracheids having thick-walled simple pits, length 600 to 1050 µm and width 30 to 60 µm; fragment of xylem ray parenchyma having reddish to yellowish brown content embedded with starch grains;

HPTLC fingerprint of Ethanol extract of E. monogynum
The TLC profile of ethanolic extracts of stem heartwood of E. monogynum was developed in the solvent system toluene: ethyl acetate: formic acid (7:2.5:0.5 v/v/v).The solvent system ratio is chosen by trial and error method to obtain distinguishable band separation.TLC finger print showed numerous phytochemicals appeared as bands at 254 nm, 366 nm and vanillin-sulphuric acid reagent, showed 5 bands at 254 nm, 6 bands at 366 nm and plate derivatized with vanillin-sulphuric acid showed 11 bands (Table 1).The HPTLC finger print profile of ethanol extract of the stem heartwood E. monogynum showed 12 spots at UV 254 nm with 2 spots appeared major with area of more than 10% the R f values (Figure 8); 10 spots with one spots appeared major with an area of more than 10% the R f values shown at UV 366 nm (Figure 9); after derivatization with VSA the plate scanned at 520 nm showed 13 spots with five spots appeared major with an area of more than 10% (Figure 10).The alkaloid fraction shows two orange spots at R f 0.35 and 0.85 presence of alkaloid (Figure 11).

DISCUSSION
Incorrect identification of plants species has been resulting in adulteration and substitution of raw drugs in herbal drug industry. [37]Drug adulteration has to be developed as a major area of study under herbal drug standardization and studies on adulteration practices have to be taken up along with identification of the crude drugs.Plant anatomy is an important basic tool for authentication of dried heartwood in various levels (chemically processed, burnt, etc).DNA barcoding is a powerful supplement tool for identification heartwood but due to presence of tannins, resins, gums, essential oils, pigments etc. poses challenges. [38]Microscopic characters could be significantly used for authentication at various levels.Though researchers feels anatomical features are difficult to differentiate in close genera in certain family, thorough analysis at cellular level or powder  microscopy might help further down solving the authentication issues. [40,41]e vernacular and scientific names differ for E. monogynum, S. album and C. deodara, but, in the raw drug markets E. monogynum heartwood and oil are traded as S. album and C. deodara in the name of sandal wood, Davadaram wood and sandal wood oil. [31]It indicates that the availability of authentic wood for crude drug markets is dwindling or it is not available at all.Some of the researches published on the authentic plant has covered the anatomical and DNA barcoding, but not about the adulterations practices in these wood and powder microscopy work. [8,29,30] the present study on stem heartwood external morphology and internal anatomical characters TS, TLS RLS and powder microscopy characters are documented.Quantitative and qualitative macro-micro-morphological characters of stem heartwood can strengthen the taxonomic decisions within the market adulteration.TLC/HPTLC studies are crucial for identification of any herbal drug in addition to microscopic identification.Pharmacopoeias on herbal drugs emphasis the use of TLC for the identification of raw drugs procured from market before using for formulations.The research work to resolve the adulteration available in the herbal market.For the HPTLC, ethanol was used for extracting the phytoconstituents as it is the high polar solvent capable of extracting high polar compounds like glycosides, saponins, tannins, etc.However, for alkaloids which are present in the form of salts, the alkaloids are extracted by soaking in the acidified aqueous solution, neutralization with a base and extracting with an organic solvent which is immiscible with water. [42]is study will be helpful for differentiating other controversial sources of C. deodara and sandalwood; the C. deodara wood is non-porous and presence of fibre tracheids with bordered pits is one of the main distinguishing characteristics. [39]Sandalwood can be anatomically differentiated by the presence of vessels arrangement mostly solitary diffused porous, even 2 or 3 together λ=254 nm λ=366 nm λ=520 nm (Derivitized)  µm in long, stored with brownish content. [28,29]E. monogynum has diffused porous vessels arranged in radial multiplication groups of as many as 6 to 8 vessels, but minimum 2 and maximum 11 celled group, 95 to 120 per mm 2 , length 270 to 1200 µm, ray cells uni and bi-seriate, 38 to 48 ray groups per mm 2 , 120 to 350 µm height, 15 to 35 µm wide and 4 to 16 cell rows high; fibres lumen non-storied, middle lamellae present between adjacent walls, 600 to 1600 µm in length, 7 to 11.5 µm wall thickness and up to 27 in width.

CONCLUSION
This study sets specific macro-microscopic protocol on stem heartwood of E. monogynum and also to differentiate it from its substitute and adulterant.The difference in TLC and HPTLC spot in the ethanol extract which can be used for the identification and differentiate the authentic heartwood from the adulterant available in crude drug market will be helpful in differentiating their identity purity self-discipline and also focus on quality control standardization of the herbal drug.
differentiating their identity purity self discipline and also focus on quality control standardization of the herbal drug.

Figure 7 :
Figure 7: Powder microscopy of E. monogynum.a: fibres, b: fragment of xylem ray parenchyma, c: tailed pitted vessels, d: crystal fibre; e: fibre tracheids, f: oil globule, g: starch grains, h: radial longitudinally cut xylem ray crossing with vessels, fibres and fibre tracheids, i: prismatic crystals of calcium oxalate, j: different shape and size of xylem parenchyma cells, k: tracheids, l: tangential longitudinally cut xylem ray associated with fibre and axial parenchyma.