Physicochemical Evaluation and Pharmacognostical Standardization of Pellionia heyneana Wedd

Introduction: Pellionia heyneana Wedd. Leaves have long been employed as a traditional remedy by the Cholanaikan tribe of South India to treat various ailments. Methods: Pharmacological and physicochemical evaluation of P. heyneana leaf has been carried out to determine its macro and microscopic characters, and also some of its quantitative characters as per standard procedures. Results: The pharmacognostical evaluation of P. heyneana leavesrevealed the presence of characteristic microscopic features of the crude drug like cystoliths in upper epidermis, helicocytic stomata in lower epidermis, large number of peculiar shaped, huge (200-400 μm) foliar sclereids, absence of palisade tissue in the lamina etc. Powder microscopy showed the presence of calcium oxalate crystals, stone cells, multicellular trichomes, resinous blocks, spiral vessels, xylem fibre, starch grains, simple fibre etc. Conclusions: All the parameters evaluated in the study will aid to identify the authenticity of P. heyneana leaf even from the crushed or powdered form.


INTRODUCTION
Pharmacognostic evaluation of drugs has assumed immense significance in the realm of pharmaceutical industry due to the realization that reproducibility and quality of herbal drugs are directly related to the authenticity of the plant material.Qualitative or quantitative uniqueness of different drugs with respect to the morphological, anatomical and bio chemical parameters are acknowledged as one of the reliable tools in distinguishing allied drug samples in the herbal industry.Lack of proper documentation and the adoption of stringent quality control measures continue as a major setback in the use of herbal drugs.To mitigate this, quality assurance by standardization of the drug employing different pharmacognostic parameters has become the need of the hour. 1 This is particularly important since these parameters are not easily amenable by the environment.Hence, this might help not only to authenticate and identify the drug, but also for its safe and efficacious use.The plant Pellionia heyneana Wedd. is an erect or decumbent herb which belongs to Urticaceae family.It is widely distributed in Peninsular India, Sri Lanka, Cambodia, Indonesia, Thailand, and China.Tradi tionally, its leaves are used by the Cholanaikan tribes to enhance general health and immunity and also to treat various liver ailments. 2The genus Pellionia as well as the species, P. heyneana is a very scarcely studied groupand there have been only a couple

Physicochemical Evaluation and Pharmacognostical Standardization of Pellionia heyneana Wedd. Leaf
of attempts made to study the pharmacognostic aspects of the species.The plant leaf is very difficult to distinguish from other allied species in dried or crushed form.Thus the present study has been carried out to evaluate the detailed pharmacognostical and physicochemical properties of P. heyneana leaf.

MATERIALS AND METHODS
Collection of plant material P. heyneana Wedd.plants were collected from Kallar, Thiruvananthapuram district and authenticated by the plant taxonomist of the Institute.Voucher speci men was deposited at the Jawaharlal Nehru Tropical Botanic Garden and Research Institute Herbarium (TBGT 57060 dated 10/12/2010).

Macroscopic and organoleptic characterization
The following macroscopic and organoleptic char acters for the fresh leaves were noted: phyllotaxy, size, shape, colour, venation, presence or absence of petiole, apex, margin, base, lamina, texture, surface, odour and taste. 3,4croscopic characterization Anatomical studies of the leaf Free hand transverse sections of leaf lamina and midrib were prepared, stained with safranin, mounted on glass slides using glycerine and observed under light microscope with camera attachment and photo micrographs were taken. 3harmacognosy Journal, Vol 8, Issue 6, Nov-Dec, 2016

Quantitative leaf microscopy Stomatal number and stomatal index
A small piece of leaf was cleared by boiling with sodium hypochlorite solution.The upper and lower epidermis were peeled separately.The peeled epidermis was placed on slide and mounted with glycerine water.Average number of stomata per mm 2 of the epidermis of the leaf (stomatal number) is calculated from the microphotographs taken using camera attached microscope. 10Values for upper and lower epidermis were deter mined separatelyusing the equation: Where, S= the number of stomata per unit area and E = the number of epidermal cells in the same unit area of leaf.

Determination of vein-islet number and vein-let termination number
The veinislet number is the average number of veinislets per mm 2 of a leaf surface midway between midrib and margin and the average number of terminated veinlet islets per mm 2 of a leaf was taken as veinlet termination. 3

Powder analysis
The fresh leaves were separated from the collected plants, thoroughly washed with fresh water, shade dried and powdered.The leaf powder is boiled with chloral hydrate for 510 min, and then stained with phloro glucinol, safranin, glycerine and iodine solution to determine the presence of lignified cells, calcium oxalate crystals, starch grains etc. 5,6

Fluorescence analysis
The fluorescence character of the plant leaf powder (40 mesh) was studied both in daylight and UV light (254 and 366 nm) after treatment with different reagents like sodium hydroxide, picric acid, acetic acid, hydro chloric acid, nitric acid, iodine, ferric chloride etc. 7,8 The colour changes were noted using Methuen handbook of colour. 9

Physicochemical analysis
The following physicochemical parameters of P. heyneana leaf powder were determined according to the quality control methods for medicinal plant materials. 10termination of pH pH 1% solution: Dissolved 1 g of the leaf powder in 100 mL of distilled water, filtered and checked the pH of the filtrate with a standardized glass electrode.
pH 10% solution: Dissolved 10 g of the leaf powder in 100 mL of distilled water, filtered and checked pH of the filtrate with a standardized glass electrode.

Determination of alcohol soluble extractive
Take 5 g of the air dried leaf powder in100 mL of ethanol in a closed flask, shaken frequently during 6 h and allowed to stand for 18 h.Filtrate is collected and evaporated to dryness in a tared flat bottom shallow dish, dried at 105˚C and weighed.The percentage of alcoholsoluble extractive is calculated with reference to the air dried drug.

Determination of water soluble extractive
Methodology followed as directed for the determination of alcohol soluble extractive using water instead of ethanol.

Determination of petroleum ether soluble extractive
Methodology proceeded as directed for the determination of alcohol soluble extractive, using petroleum ether instead of ethanol.

Loss on Drying (LOD)
About 23 g of leaf powder is accurately weighed in a China dish and kept in a hot air oven maintained at 105˚C for 5 h.After cooling in a desiccator, the loss in weight was recorded.This procedure was repeated till constant weight was obtained.

=
Loss in weight Loss on drying (%) LOD 100 Weight of the druging

Swelling index
Leaf powder (1 g) was taken in a measuring cylinder (25 mL) and suspended in 25 mL distilled water for 1 h by thorough mixing every 10 min.After 3 h, volume in mL occupied by the plant material including any sticky mucilage was measured.The experiment was repeated three times for accuracy and the swelling index was calculated.

Foaming index
Finely divided (sieve No. 1250) plant powder 1 g was kept in to a 500 mL flask containing 100 mL of boiling water for 30 min.Then cooled and filtered into a 100 mL volumetric flask and added sufficient water to makeup the volume.The prepared decoction was poured in to 10 stop pered test tubes each 1 mL, 2 mL up to 10 mL.The volume of the liquid in each tube was adjusted to 10 mL with water.The tubes were duly stop pered and shaken in a lengthwise motion for 15 sec (two shakes per second) and allowed to stand for 15 min.The foam height in each tube was measured.
Foaming index =1000/a 'a' is the volume of the plant decoction for foaming foam of height 1cm.

Determination of total ash
About 23 g weighed crude drug powder in a tared silica dish was ignited and weighed.Scattered the powder drug on the bottom of the dish and incinerated by gradually increasing the heat not exceeding dull red heat until free from carbon, cooled and weighed.The % w/w of total ash with reference to the airdried drug was calculated.

Determination of acid insoluble ash
Boiled the ash for 510 min with 25 mL of diluted hydrochloric acid, collected the insoluble matter in a Gooch crucible, washed with hot water, ignited and weighed.Percentage of acid insoluble ash is calculated with reference to the air dried drug.The % w/w of acid insoluble ash with reference to the airdried drug was calculated.

Determination of water-soluble ash
To the crucible containing the total ash, add 25 mL of water and boil for 510 min.Collect the insoluble matter in a Gooch crucible, wash with hot water and ignite in a crucible for 15 min at a temperature not exceeding 450°C.Subtract the weight of insoluble matter from the weight of the ash.The difference in weight represents the water soluble ash.Percentage of water soluble ash is calculated with reference to the air dried drug.The % w/w of watersoluble ash with reference to the airdried drug was calculated.
Percentage extractive and characteristics of fractions P. heyneana leaf powder was first extracted with hexane using Soxhlet apparatus, powder was then dried and again extracted with chloroform and finally with ethanol to get; 1. Hexane fraction (PHHF), 2. Chloroform fraction (PHCF) and 3. Ethanolic fraction (PHEF).Yield in g/100 g leaf powder of PHHF, PHCF and PHEF of P. heyneana leaf powder was cal culated.Consistency, colour and odour were also evaluated.

Macroscopic and organoleptic characterization
Macroscopic and organoleptic characters of the fresh leaves were noted and the results were presented in the Table 1.

Microscopic characterization of P. heyneana leaf Anatomical studies of leaf
Transverse section of the leaf shows two distinguished regions, midrib region and laminar region.
Midrib: A prominent projection on the abaxialside (Figure 1A).Upper epidermis is of single layered large rectangular cells with outer thin layer of cuticle and some epidermal cells with cystoliths (Figure 1B).Single layered lower epidermis has small rectangular cells (Figure 1C) with uniseriate multi cellular trichomes with tapering end (Figure 1D).Ground tissue is represented with 68 layers of collenchyma seen below the upper epidermis and 46 layers above the lower epidermis.In between the collenchyma is seen the thin walled thickly packed parenchyma cells with cluster of calcium oxalate crystals.Single layers of sclerenchyma cells seen below the upper collenchyma, vascular bundles arehorseshoe shaped (Figure 1E).Lamina: Narrow lamina, upper epidermis is single layered large rectan gular cells with an outer thin layer of cuticle.Upper epidermis is followed by two layers of sclerenchymatus hypodermis.Hypodermis is followed by 56 layers of mesophyll tissue; palisade absent (Figure 1F).Single layered lower epidermis of small rectangular cells is seen with numerous helicocytic stomata.Large number of huge foliar sclereids (200400 µm length) with peculiar shape was found throughout the leaf (Figure 1G).

Quantitative leaf microscopy
Quantitative leaf characteristics were observed and the results were shown in the Table: 2 and Figure 2.

Powder analysis
Analysis of P. heyneana leaf powder revealed the presence of prismatic calcium oxalate crystals, stone cells, multicellular trichomes, helicocytic stomata, epidermal cells, spiral vessels, xylem fibre, starch grains, simple fibreand abundance of special shaped large foliar sclereids (Figure 3).

Fluorescence analysis
Chemical tests of P. heyneana leaf powder were conducted with different reagents and observed under UV 254 nm and UV366 nm.The results were compared with their respective observations in visible light and they were represented in Table 3.

Physicochemical analysis
Physiochemical parameters of P. heyneana leaf powder were evaluated and the observations are presented in Table 4.

Percentage extractive and characteristics of extract and fractions of P. heyneana leaf
Physical characteristic and percentage yield of PHHF, PHCF and PHEF were shown in the Table 5.

DISCUSSION
The macroscopic characters of leaf (Table 1), the species possess simple leaf with a mean length 14 ± 4.5 cm and breadth 5.25 ± 1.25 cm.The plants, in general have an obliquely elliptic oblong entire leaf with cuneate base and reticulate venation.Leaf colour is typically green, being dark green in the adaxial and pale green in the abaxial sides.Phyllotaxy is alternate or sub opposite.A reduction in the leaf size is observed in the successive pairs of leaves.This rather gives an alternate appearance for the leaf.T.S of leaf showed the presence of upper and lower epidermis with barrel shaped or rectangular shaped cells, typical of most leaves.Presence of cystolithis evident in the epidermis.Ground tissue is predominantly made of collenchyma.Patches of parenchyma with calcium oxalate crystals embedded in it has also been noted.Horseshoe shaped vascular bundle with xylem and phloem in the midrib is a key characteristic of the species.Presence of large sclereids (200 -400 µm) is typical to the species.Perhaps, the roughness of the leaves is due to the presence of sclereids.Stomata in the species is helicocytic characterised by a helix of four or more subsidiary cells surrounding the guard cells.This is the first report of the presence of helicocytic stomata in the species.Payne stressed that helicocytic stomata is an unrecognised stomatal pattern in dicotyledonae and he observed this type of stomata in many species including its close ally, Pellionia daveauana. 11The present observation of helicocytic stomata in P. heyneana is in broad consensus with Payne (1970), whereas Ushakumari et al.(2004) reported the presence of cyclocyticstomatata in P. heyneana. 12Total absence of stomata in the adaxial surface is a feature of importance in the identification of the drug.Absence of palisade tissue in the lamina is yet another feature specifically confronting the species.These features might offer authenticity in the identification of the species which would be largely helpful in distinguishing it from adulterants.Fluorescence analysis is important in distinguishing the drug from its adulterants in its powdered form. 13,14Comparative account of the fluo rescence analysis treated with different chemical reagents and observed at 254 and 366 nm UV are depicted in Table 3. Evaluation of physicochemical parameters aids in the formulation of pharmacopoeial standards.Among the various physicochemical charac teristics evaluated in the present study (Table 4), ash value is one of the common features used to determine the identity and purity of the drug. 15he extract left behind after exhausting the crude drug is indicative of the chemical constituents of the drug in question.Total ash usually contains carbonates, phosphates, silicates and silica. 3,16High total ash value of P. heyneana (18.61% w/w) noted in the study may be due to high quantity of calcium crystals of the plant.This could be well corroborated with the results of T.S. of lamina in which are seen patches of parenchyma with calcium oxalate crystals.The water soluble ash gives an estimate of the amount of inorganic elements in the sample.Acid insoluble ash in P. heyneana amounts to 11.63% w/w, which indicates that almost half of the total ash is soluble in acid.The extractive values are useful to evaluate the nature of chemical constituents present in crude drug and also to help in estimation of specific constituents soluble in a particular solvent. 17The extractive value of the three serial fractions revealed that the quantity of extract is maximum in ethanolic fractions which means the plant contains more polar compounds.

Figure 1 :
Figure 1: Leaf anatomy of P. heyneana leaf.A -T. S. of leaf midrib, B -T. S. of midrib showing single layered upper epidermis with large rectangular cells, C -T. S. of midrib showing single layered lower epidermis with small rectangular cells, D -Uniseriate multicellular trichomes, E -Leaf vascular bundle, F -T. S of lamina (UPE -upper epidermis, LEP -lower epidermis, HP -hypodermis) G -T. S. of lamina showing foliar sclereid in mesophyll tissue.

Figure 2 :
Figure 2: Quantitative leaf characteristics of P. heyneana A -Leaf peeling of lower surface showing helicocytic stomata, B -Leaf peeling of upper surface without stomata, C -Stomatal impression of lower surface, D -Vein inlet number in 1mm2, E -Helicocytic stomatal complex showing guard cells (GD) and subsidiary cells (SC).

Table 1 : Macroscopic and organoleptic characters of P. heyneana leaf Macroscopic parameters Observation
Results are out of 25 observations ± SD.

Table 2 : Quantitative leaf microscopy of P. heyneana
Values are expressed as mean ± SD of ten values.