Pharmacognostic Evaluation of Epilobium hirsutum Linn .

Background: Epilobium hirsutum Linn. (Onagraceae) is used widely as traditional medicine for its various medicinal values. Despite the popular utilization of this plant, still no conclusive study has been reported so far regarding the pharmacognostical standardization. Aim: Thus, the present study was aimed to scientifically establish a standard monograph of E. hirsutum on the basis of pharmacognostical aspects. Material and methods: Macroscopic, qualitative and quantitative microscopic characters of E. hirsutum were analyzed. Results: The diagnostic characters of the E. hirsutum were evaluated based on the macroscopical and microscopical characters. Conclusion: The obtained qualitative and quantitative standards will provide referential information for correct identification, purity, standardization and preparation of monograph of E. hirsutum.


INTRODUCTION
Epilobium hirsutum Linn. is a flowering plant belonging to family Onagraceae, commonly known as great willowherb and great hairy willowherb or hairy willowherb. 1 E. hirsutum is a large, annual, densely covered with white hair.The leaves are 1-3 in number, 2-12 cm long, 0.5-3.5 cm wide, lanceolate, stem-leaves usually opposite, sessile, stem clasping and small teeth shaped.The flowers are 10-16 mm long, pink purple, axillary in long and short leafy racemes at the end of stem and branches, calyx-tube liner, 4 angled, lobes 4 short pointed, soon falling off.Petals 4, erect or spreading, notched, stamens 8, alternate ones longer, stigmas 4, distinct, erect at first, recovered afterwards.The capsule is 4-9 cm long.4] Common habitats include marshland, ditches and the banks of rivers and streams.Earlier claim shows that the E. hirsutum has antinociceptive, 5 anti-inflammatory, 6 antioxidant, 7 it alters the protein synthesis by alteration in enzyme and mRNA, 8 in-vitro iron chelation activity. 9The plant is also used as an antimicrobial, antitumor and in the treatment of enlarged prostate, prostatitis, cystitis, burning feeling when urinating, and burning feeling after prostate operation. 10Despite the popular utilization of this plant, still no conclusive study has been reported so far regarding the pharmacognostical standardization.Hence, the present cram was focused to investigate the pharmacognostical properties of E. hirsutum.

Chemicals and instruments
All reagents and chemicals used for pharmacognostic screening were analytical grade.Compound microscope was used for the study.The photography was done by using microscope camera using Toupview software.Camera lucida was used for determination of quantitative microscopical characters.

Morphology and Microscopy
The morphological studies were carried out by using simple determination technique like shape, size, colour, odour, taste, surface and fractures.Microscopic studies were carried out by preparing thin hand section of leaf, stem and root.The sections were cleared with alcohol and stained.Histochemical reactions were applied with concentrated hydrochloric acid and phloroglucinol for identification of lignified elements, iodine solution for starch grains, Sudan red-III for cuticle layer and oil globules, Ruthenium red for mucilage and acetic acid for calcium oxalate crystals.The leaf constants like stomatal number, stomatal index, vein termination number, vein islet number and palisade ratio were measured using camera lucida. 11

Morphological characteristics
The morphological studies revealed (Figure 1) that the leaves was simple, sessile, green in colour, oblong-lanceolate in shape, 2-12 cm long, 0.5-3.5 cm wide, aromatic odour with characteristic and aromatic taste.Leaves had sharply serrulate margin, acute apex, symmetrical base, reticulate pinnate venation, thin texture and pubescent surface.The stem was green in colour, cylindrical in shape, about 0.5-1 m long, aromatic odour   with characteristic and aromatic taste.The surface was pubescent with short fracture.The root was brownish in colour, cylindrical in shape, about 8 cm long and 0.5-1 cm in diameters, the root was odourless and tasteless.Longitudinal ridges and rootlets with short fracture were present on surface of root.The flowers were large and have 4 notched petals.The size of flower was 10-16 mm long and purple pink in color.The sepals were green; stigma was white with 4 lobes.The fruits were capsular and slender 5-8 cm long, brownish in color and containing numerous seeds embedded in fine silky fluff.The seeds were oblong and flattened in shape, 1 mm long and had nearly white hairs.

Qualitative microscopical characteristics
Transverse section of leaf The transverse section of leaf was shows that upper and lower epidermis of lamina consists of single layered, rectangular cells, distinct cuticle, it also contain uniseriate, unicellular covering trichomes (Figure 2 A-B), whereas Figure 2 C shows anomocytic type of stomata, lower epidermis contents more stomata.Mesophyll consist palisade single layered, compact cells, with several layered spongy parenchyma, oil globules and vacuoles were present throughout mesophyll.Midrib was consists of upper and lower epidermis same as that of lamina, collenchyma present below upper epidermis and above lower epidermis, vascular bundles were arc shaped hydrocentric and were present more towards dorsal side, rest of midrib was filled with normal parenchymatous cells (Figure 2 D).It also contains brownish matter, needle shaped calcium oxalate crystals and starch grains (Figure 2 E-F).

Transverse section of stem
Transverse section of stem shows that epidermis consists of single layered, quadrangular cells and thick cuticle.It also contains uniseriate, unicellular covering trichomes.Hypodermis was present below epidermis consist of few layer of collenchyma and clorenchyma cells.Cortex consists of several layers of thin walled cellulosic parenchyma cells.Mature stem contains of brownish matter in cortex.Unlignified group of fibers were present outside the phloem.Vascular bundle was closed collateral type; phloem contains of sievetubes and companion cells.Xylems were well developed, consists of xylem parenchyma and vessels.Pith was large, thin walled, polygonal parenchyma cells (Figure 3 A-B).Cortex and pith also contains needle shaped calcium oxalate crystals and starch grains (Figure 3 C-D).

Transverse section of root
The transverse section of root revealed that cork consists of several layers of thin walled, polygonal cells with brown content.Cortex consists of several layers of thin walled, polygonal parenchymatous cells, brownish matter and needle shaped calcium oxalate crystals.The phloem shows phloem parenchyma, sievetubes and unlignified phloem fibers.Xylem studied for their specific characters.The present study will provide useful informa tion for correct identification of plant.In addition the results of the present study could be useful for prepara tion of a monograph of the E. hirsutum.consists of xylem parenchyma, vessels and medullary rays.Pith was very small and consists of parenchyma cells (Figure 4 A-B).

Powder characteristics
The shade dried powder of E. hirsutum shows greenish brown colour having characteristic odour and sweet, bitter, sharp and sour taste.Epidermis consists of thin walled, irregular parenchymatous cells with wavy outline.Trichomes were uniseriate, unicellular.Parenchyma shows thin walled polyhedral cells with intercellular space.Cork shows polygonal isodiametric cells with brownish matter.Mesophyll consists of spongyparenchyma with vascular strands.Xylem vessels were lignified spiral shaped.Fibres were lignified, thick walled and cylindrical in shape.
Starch was simple, oval and rounded shaped.It also contains brownish matter whereas the stomata were of anomocytic type (Figure 5 A-J).

Quantitative microscopical characteristics
These values were especially useful for identifying the different species of genus and also helpful in determination and authenticity of the plant.The results were shown in Table 1.

DISCUSSION
The pharmacognostic evaluation is the first step towards the confirmation of identity and to assess the purity and quality of plant.Before the use of plant it is necessary that standards should be available to control and check the identity of the plant along with its quality.As per WHO the macroscopic and microscopic description of medicinal plant is the initial step towards establishing its identity, it should be carried out before any tests are undertaken. 14

CONCLUSION
The present work deals with the pharmacognostic study of E. hirsutum.
The morphological and microscopical study of plant and powder were