Morpho-anatomical , Phytochemical and TLC / HPTLC Studies on Whole Plant of Plectranthus vettiveroides ( Jacob )

Plectranthus vettiveroides (Jacob) N.P. Singh & B.D. Sharma (Syn. Coleus vettiveroides Jacob) is an aromatic perennial herb belongs to family Lamiaceae (earlier classified under Labiatae).1,2 The plant is small, bushy, profusely branching, succulent with quadrangular purplish stems and grows upto a meter height.3-5 Though being very shrinking population now, it is cultivated at present in Kollidam, a village on the bank of the river Kollidam, Nagai district, Tamilnadu, India. P. vettiveroides is an endemic plant to Tamil Nadu and Kerala and is extinct in wild.4,6

Morpho-anatomical studies are helpful to authenticate the botanical source. Preliminary phytochemical evaluation provides information about presence of phytoconstituents in the plant. Physico-chemical parameters indicate the purity and identity of the plant material. In the present investigation, morpho-anatomical, preliminary phyto-chemical, physico-chemical parameters and TLC/HPTLC fingerprint of the whole plant were carried out and reported. The finding of present study will be helpful in standardization of formulation consisting of whole plant of P. vettiveroides as ingredient in their powdered form and also for authentication/identification of dried whole plant of P. vettiveroides.
Thirubuvanaimangalam, Kollidam, Nagai district, Tamilnadu, India (GPS data 11ᵒ19'35.4''N 79ᵒ 43'06.5''E). The voucher specimen of the whole plant of P. vettiveroides (Reference code No. C/298A Wp15) were deposited in the department of Pharmacognosy, CSMRADDI, Arumbakkam, Chennai, India for future reference. The collected plant material was washed with water and dried under shade. The shade dried whole plant of P. vettiveroides was coarsely powdered (10/44) and utilized for macro-microscopical, physico-chemical, phytochemical and TLC/HPTLC finger print analyses. The morphological, micromorphological and microscopical studies were carried out by standard methods as described in literature. [13][14][15] Quantitative analysis for total ash, acid insoluble ash, water and alcohol soluble extractive values, loss on drying at 105°C and other solvent extractive values were carried out in triplicate from powder of whole plant according to the standard method recommended. 16

Preparation of extracts for phytochemical studies
The shade dried whole plant material was coarsely powdered and 15 g of plant powder was soaked with n-hexane for 48 hr. Then the extract was filtered by using Whatmann No.1 filter paper and concentrated. Similarly, the remaining plant material was successively extracted with chloroform, ethyl acetate and alcohol. All the extracts were qualitatively tested for the presence of various phytochemical constituents of plants by conventional methods. 17 Method for developing TLC/HPTLC Sample preparation 4 g of powdered plant material was soaked with alcohol for overnight. Then extract was boiled for 10 minutes, concentrated, filtered by using Whatmann No.41 filter paper and made up to 10 ml volumetric flask. 18,19

Instrument
The CAMAG (Switzerland) HPTLC instrument was operated at relative humidity 53 ± 5 % and temperature was maintained at 20 ± 5ºC. The solvent system was optimized with different ratio for mobile phase by trial and error. Qualitative HPTLC analysis was carried out on 10×10 cm TLC aluminium plate pre-coated over silica gel 60F 254 with 0.2 mm thickness. 10 µl of samples solution was applied on the plate with 6 mm band width fitted with a 25 micro liter syringe using CAMAG HPTLC ATS 4 sample applicator. The plate was developed in ascending mode by using the mobile phase ratio toluene: ethyl acetate: Formic acid (8: 2: 0.2 v/v) up to 90 mm in a twin-trough CAMAG glass chamber previously saturated with mobile phase for 15 minutes at 25°C. The dried plate visualization was carried out through CAMAG Visualizer at UV 254 nm and 366 nm, photo-documented and scanned up to 90 mm plate width by deuterium lamp using a CAMAG TLC scanner 3 with Win CATS Software version 1.4.4. Then the plate was derivatized with 10 % alcoholic-sulphuric acid reagent and heated at 105°C when the compounds appeared as colored spots. Photo-documented and scanned at 540 nm up to 90 mm by tungsten lamp. 18,19

Macroscopy
The detailed macroscopic characters observed are reported in Table 1 and Figures 1 and 2.

Root
Detailed TS of the root shows outer cork consists of compressed cork of rectangular cells filled with dark brown content; followed by cortex consists of up to 8 rows of parenchymatous cells with a few starch grains and sandy crystals; Phloem is narrower zone with obliterated cells; central wider xylem composed of vessels, isolated or occur in radially arranged groups of two or three, parenchyma are mostly vesicentric and contain a few starch grains, fibres are thick walled and many in number, medullary rays are uni-multiseriate medullary rays which run almost parallel to each other and widening towards the phloem (Figure 3).

Stem
Detailed TS of mature stem shows outer suberised cork with dark brown content; cortex consists of 10-15 rows of tangentially running

S.No.
Macroscopic character Leaves Stem Root and rootlets 1.
Colour, odour and taste Gray, aromatic and bitter Brownish black, aromatic and bitter Brownish black, aromatic and bitter 2.
Size and shape Upto 7 cm long with petiole Upto 7 mm width, angular with furrow Tap root upto 5 mm lateral roots upto 1mm width 3.
Texture and fracture Dull and soft furrowed and splintery Smooth and short    parenchymatous cells, embedded with group of cortical fibres, followed by narrow zone of phloem consists of obliterated cells; followed by wide zone of xylem consists of isolated or radially arranged rows of vessels, parenchyma which are mostly vesicentric, thin walled fibres occupying the major area of the xylem (Figure 4).

Leaves
Petiole Detailed TS of the petiole is oval in outline with a concavity on upper surface, covered with numerous covering and glandular trichomes, a single layer of epidermis, 3 to 6 rows of collenchymatous hypodermis; the ground tissue is parenchymatous, wide; a circle of discontinuous vascular bundles and parenchymatous pith ( Figure 5).

Midrib
Detailed TS of midrib shows single layer of upper and lower epidermis covered with thin cuticle, bearing numerous covering and glandular trichomes, covering trichome uni and multicellular, glandular trichomes sessile, 2-4 rows of collenchymatous hypodermis, parenchymatous ground tissue embedded with group of vascular bundles ( Figure 6).

Powder microscopy
The powder microscopic characters are shown in Figure 7.

Physico-chemical and preliminary phyto-chemical analysis
The physico-chemical and preliminary phyto-chemical analyses of whole plant of P. vettiveroides are presented in Tables 2 and 3, respectively.

TLC/HPTLC fingerprint
The HPTLC finger print profile of alcohol extract of the whole plant showed 15 spots at UV 254 nm with 2 spots appeared major with an area of more than 10 % the R f values, 18 spots with three spots appeared major with an area of more than 10 % the R f values shown at UV 366 nm and after derivatised with alcoholic sulfuric acid reagent the plate          scanned at 540 nm showed 17 spots with four spots appeared major with an area of more than 10 % (Figures 8-10).

DISCUSSION
In Tamil Nadu both P. vettiveroides and Vetiveria zizanoides are referred and sold in the same names, Kuruver or Vettiver. The roots of Vetiveria zizanoides are being used as an adulterant due to the non-availability of P. vettiveroides. It was noticed that the raw drug being sold as 'Kuruver' in the market was actually the roots of V. zizanoides. Thus, the confusion between this two roots exists in literature and as well as in the market. Scarcity in availability of P. vettiveroides roots is the reason for adulteration, the plenty of available roots of V. zizanoides which looks similar in appearance is used as adulterant.
Pharmacognostic characters of herbal drugs play an important role since particular macro-microcopic features are unique for each plant.
The macroscopic and microscopic studies of the herbs should be Pharmacognosy Journal, Vol 12, Issue 1, Jan-Feb, 2020 the first and fundamental step to authenticate the botanical source. Proceeding for chemical methods of standardization, preclinical and clinical evaluation will bear no value if authentic drugs are not used. Macro-microscopic evaluation is simple and cost effective. 20,21 The high values of loss on drying denote the presence of high water and volatile content. Water soluble extractive value shows the presence of more amounts of polar compounds. 16 The high values of acid insoluble ash and powder microscopy is the indicative of high level of silica present in the plant material as it is grown in sandy river bed, the plant absorbs silica and store it in inner cells. TLC/HPTLC fingerprint of alcoholic extract of whole plant of P. vettiveroides provides a suitable method for monitoring the identity, purity and also standardization of the drug.
From the results of this study, it has been found the dried whole plant of P. vettiveroides can be easily identified macro-microscopically and phyto-chemically. This study sets specific morpho-anatomical, preliminary phytochemical and physico-chemical protocol and TLC/ HPTLC fingerprint on dried whole plant and powder of P. vettiveroides and could also be helpful in standardization of formulation containing whole plant of P. vettiveroides as ingredient in powdered form.

CONCLUSION
Morpho-anatomical, preliminary phytochemical, physico-chemical and TLC/HPTLC studies on whole plant of P. vettiveroides has been carried out and reported. Findings of this study may be useful for