Pharmacognostical and Physico-chemical Standardization of Euphorbia neriifolia Leaves

Objective: To investigate pharmacognostical and physico-chemical standardization of Euphorbia neriifolia leaves. Materials and Methods: Fresh and dried leaves with powder samples of E. neriifolia were examined macroscopically and microscopically. As per Ayurvedic Pharmacopeia of India and World Health Organization guidelines on quality control methods for medicinal plants materials suggested parameters were determined for standardization of E. neriifolia leaves. Physico-chemical, primary phytochemical, fluorescence and quantitative screenings along with primary HPTLC fingerprinting assessment were performed. Results: Macroscopic examination demonstrated that fresh leaf of E. neriifolia has dark green in colour, herbaceous odour with characteristic taste. Dried leafs are grey brownish in colour, characteristic odour with broken crumpled and papery fracture. Microscopy of leaf showed the single layered thick rectangular or tubular adaxial epidermal cells. Mesophyll tissue was differentiated into two or three layered adaxial zones of radially elongated palisade cells and wider abaxial spongy mesophyll cells revealed the differentiated dorsiventral lamina. Mid-rib composed of epidermis, collenchymas and spongy parenchyma cells. Physico-chemical parameters like, foreign matter was found to be 0.46%. Total ash, acid insoluble ash and water soluble ash was found 6.33%, 1.23% and 6% respectively. Loss on drying was found to be 4.69%. Swelling and foaming index was found 11.7 ml and 333 ml respectively. Quantitative screening suggested that the leaf powder has indicated alkaloid and saponin estimation as 0.26% and 3.67% respectively. The HPTLC fingerprinting of EN6 extract fraction was showed the Rf values at 254 nm with their respective UV-visible spectrum wavelengths scanned in between 200-400 nm. They are 0.01 (265 nm), 0.05 (369 nm), 0.09 (263 nm, 264 nm), 0.18 (400 nm), 0.20 (279 nm), 0.31 (400 nm), 0.44 (378 nm), 0.45 (382 nm), 0.54 (377 nm), 0.55 (383 nm), 0.62 (400 nm), etc. at different concentrations of sample application. The HPTLC plate was also scanned at 366 nm and 540 nm. Conclusion: The present investigation is an additional standardization research in support with previous reports and will be helpful for qualitative and quantitative standardization of herbal formulations containing E. neriifolia. Further investigations are going on this extract fraction in reference to identification, quantification and validation of HPTLC methods using various standard marker compounds along with exploration of its pharmacological activities.

India has a glorious tradition of usage of therapeutic preparations from natural herbs which have protecting and healing properties with negligible side effects. 37ue to emerging interest 80% of world's population is

Primary phytochemical screening
Primary phytochemical screening of extract fractions of E. neriifolia leaves were performed to identify the presence of class of phytoconstituents like, alkaloids, amino acids, carbohydrates, flavonoids, glycosides, proteins, saponin, steroids, tannins and phenols. 43,44uorescence analysis Dried powder of E. neriifolia leaves was studied for fluorescence study as such and besides after treating independently with water, 1 N of HCl, HNO 3 , H 2 SO 4 , NaOH, KMnO 4 , KOH, alcoholic NaOH and KOH and ammonia using normal and ultra-violet light (254 nm).Colour effect of petroleum ether, toluene, chloroform, ethyl acetate, n-butanol and distilled water extract fractions were also observed using normal and ultra-violet light (254 nm). 45,46antitative assessment of phytoconstituents Alkaloid determination Alkaloid content was studied with reference to procedure mentioned by Harbone. 47

Saponin determination
Saponin content was determined with reference to procedure mentioned by Obdoni and Ochuko. 48

Primary HPTLC fingerprinting analysis
Based on the percentage yield and presence of phyto-constitutents in the different extract fractions, EN6 extract fraction was selective for studied by HPTLC technique for normal phase separation of components.

Preparation of solution of EN6 extract fraction
An accurately weighted 500 mg of EN6 extract fraction and dissolved into a 5 ml of methanol in volumetric flask respectively.It was then sonicated for 20 min.The solution was kept a side for 30 min to settle down the aliquots.The aliquots of stock solution of EN6 extract fraction was transferred to 10 ml volumetric flask and volume of was adjusted with methanol to get the final concentration 100 µg/µl of extract fraction.Further this solution was used for identification of extracted phytocompounds.

Chromatography and HPTLC fingerprinting analysis
A chromatographic study was performed on prewashed and preactivated 20.0 x 10.0 cm aluminum Lichrosphere HPTLC plates pre-coated with silica gel 60 F 254 of 0.2 mm thickness layer (Merck KGaA, Darmstadt, Germany).Spots of extract fraction EN6 was applied at 2, 4, 6, 8, 10 and 15 μl at application position 8.00 mm with band length 8.00 mm by a CAMAG Linomat-V automatic TLC sample spotter (Camag Muttenz, Switzerland) equipped with a 100 µl syringe (Hamilton) under a continuous drying stream of nitrogen gas at a constant application speed of 150 nl/s.The linear ascending development with the above mobile phase in a 20.0 x 10.0 cm twin trough glass chamber (CAMAG) previously saturated with mobile phase for 15 min at room temperature (25± 2 °C) and relative humidity 40%.The development distance was 80.00 mm (development time 20 min with filter paper) and 20 ml mobile phase was used.After development, the plate was dried with a stream of hot air and densitometric scanning was performed at 254 nm in absorption-reflectance mode and 366 nm in absorption-fluorescence mode by using a CAMAG TLC scanner 3 and CAMAG visualizer with automatic digital camera linked to win-CATS software (Version 1.4.6).adopting traditional medicine.Government of India has initiated several attempts to explore ethanopharmacology and traditional uses of plant for evaluation of their therapeutic potential as well as help to generate data to put these botanicals in international market of public healthcare domain. 38,39A noticeably small amount of profitable phytochemical entities have entered on facts based therapeutics but hard works are still required to recognize bioactive molecules in herbal drugs. 40The present investigation provides outline of up-to-date information on pharmacognostical and physico-chemical standardization of E. neriifolia leaves.

Authentification of E. neriifolia plant and its leaves
Fresh, well grown E. neriifolia plant and its leaves were collected from local region of Bhopal, Madhya Pradesh, India in August-September month of 2012 and was authenticated by taxonomist, Dr. Vijay V. Bhadane, Department of Botany, Pratap College, Amalner-425401, Maharashtra, India.Voucher specimen (No.PCA/Bot-P1637) was deposited in the same.The photograph and herbarium of E. neriifolia as shown in (Figure 1)

Macroscopic evaluation
Macroscopic evaluation of fresh and dried E. neriifolia leaves were studied as per the method of Brain and Turner. 41The colour, odour, taste, condition, fracture, texture, size and shape parameters were considered for the evaluation.

Microscopic evaluation
Microscopic evaluation of fresh leaf was performed according to the procedure mentioned in the Ayurvedic Pharmacopoeia of India. 6The vascular bundle, stomata types, calcium oxalates, xylem, mesophyll and epidermal cells, trichomes, starch grains, etc. were studied.The dried powder of leaf was cleared with sodium hydroxide and mounted in glycerin medium after staining.The various staining reagents such as toluidine blue, safranin, fast green and iodine were used to study calcium oxalates crystals, starch grains, stomata, trichomes, epidermal cells, parenchyma, xylem parenchyma and lignified xylem fibers.The photographs of different magnifications were taken by Leica photographic microscope.

Extraction and fractionation of E. neriifolia leaves
Freshly collected E. neriifolia leaves were dried in shade.Course powder of dried leaves (300gm) was extracted successively using petroleum ether, toluene, chloroform (Non-polar), ethyl acetate, n-butanol and distilled water (Polar) solvents by maceration process of extraction for the period of seven days for solvent with occasional stirring.The macerate mixtures were filtered each two times with muslin cloth separately and thereafter extract fractions obtained were concentrated, dried and designated as EN1 (Petroleum ether), EN2 (Toluene), EN3 (Chloroform), EN4 (Ethyl acetate), EN5 (n-butanol) and EN6 (Aqueous).The percentage yields and consistencies of extract fractions were recorded.

Physico-chemical analysis
The percentage of foreign matter, 42 total ash, acid insoluble ash, water soluble ash, 6 loss on drying, 43 swelling 6 and foaming index 42 were examined.
The slit dimension of scanner was set at 6.00 × 0.45 mm (Micro) with 100 μm / step data resolution and 20 mm/s scanning speed.Latter, the plate was dipped in anisaldehyde sulfuric acid derivatising reagent and plate was dried with a stream of hot air, scanned at 540 nm in absorptionreflectance mode with the same software system.The volume of sample applied, color of the resolved band, peak number, R f value, peak height and peak area were noted.The UV-visible spectra's were also scanned at 254 nm under D 2 lamp with 200-400 nm start and end wavelengths.

Macroscopic evaluation of E. neriifolia leaves
The plant is herbaceous.The fresh leaves are dark green in colour with herbaceous odour, characteristics taste, rough fracture, leathery texture with size approximately 2-7 cm wide and 8-20 cm long.The dried leaves are grey brownish in colour with characteristic odour, tasteless, broken crumpled and papery fracture, thin and papery texture with size approximately 1.5 to 6 cm wide and 15-20 cm long.Both fresh and dried leaves are cuneate shape, base is acute and apex is sub-acute.Margins are entire even, smooth throughout with pointed and acute tip.Venation is pinnate, anastomosing near the margin.Veinlets are prominent at lower surface.Lamina is entire and flat with carks.The macroscopic photographs of E. neriifolia leaves are shown in (Figure 2).

Microscopic evaluation
T. S. of E. neriifolia leaf showed the single layered thick rectangular or tubular adaxial epidermal cells.Single to double layered abaxial epidermis with circular to rectangular epidermal cells.Mesophyll tissue was differentiated into two or three layered adaxial zones of radially elongated palisade cells and wider abaxial spongy mesophyll cells revealed the differentiated dorsiventral lamina.The spongy mesophyll had wide air chambers and partition filaments formed by lobed and interconnected 6-9 layered spongy parenchyma cells.Midrib region slightly rose on the adaxial side whereas broadly semi-circular on the adaxial side.Mid-rib composed of epidermis, collenchymas and spongy parenchyma cells.In certain regions of epidermis, five celled uncreated trichomes with blunted tip and glandular trichomes with bi-celled head were embedded.Vascular bundles are prominent towards the ventral side and covered with endodermis.The vascular bundles consist of lignified xylem and non-lignified phloem.Mesophyll cells were called idioblasts.Spongy parenchyma showed the presence of starch granules.The spongy mesophyll had wide air-chambers.E. neriifolia leaf surface showed the anomocytic type of stomata that were covered with guard cells surrounded by 2-3 subsidiary cells followed by polygonal epidermal layers.Adaxial surface contains more stomata in comparison to abaxial surface of leaf.Leaf surface also indicated presence of glandular as well as unicellular blunted trichomes.The microscopic photographs of E. neriifolia leaves are shown in (Figure 3).The microscopic examination of powder of E. neriifolia leaves has indicated the presence of numerous idioblastic, rosette, square, prismatic and acicular shaped calcium oxalate crystals and starch grains both simple and compound, well arranged annular vessels, anomocytic stomata, unicerrate multicellular trichome, epidermal cells, spongy parenchyma, xylem parenchyma, starch grains, lignified xylem fibres and oxalate crystals.Microscopic photographs of powder of E. neriifolia leaves are shown in (Figure 4).

Extraction and fractionation
The scheme for extraction and fractionation of E. neriifolia leaves are shown in (Figure 5).The percentage yields and consistencies of all the extract fractions are shown in (Table 1).

Physico-chemical analysis
The foreign matter was found 0.46%.Total ash, acid insoluble ash and water soluble ash was found 6.33%, 1.23% and 6% respectively.The moisture content or loss on drying was found to be 4.69%.Swelling and foaming index was found 11.7 ml and 333 ml respectively.

Primary phytochemical screening
The results for primary phytochemical screening of various extract fractions of E. neriifolia leaves are shown in (Table 2).

Fluorescence analysis of extract fractions and powder of E. neriifolia leaves
The results of fluorescence analysis of extract fractions of leaves as well as powder of E. neriifolia are shown in (Table 3) and (Table 4) respectively.

Quantitative assessment of phytoconstituents
Quantitative assessment of phytoconstituents in leaf powder indicated alkaloid and saponin estimation as 0.26% and 3.67% respectively.

DISCUSSION AND CONCLUSION
In spite of the accessibility of hyphenated critical techniques, identification and assessment of plant drugs by pharmacognostical and physicochemical consideration is still more reliable, precise and economical.According to WHO, macroscopic and microscopic evaluation of plant is the first step towards establishing its identity and purity.It must be performed prior to any other tests to be carried out. 43In line with above statement, we have performed the macroscopic and microscopic studies of fresh and dried leaf along with the powder of E. neriifolia.The results of macroscopic studies will be helpful for identifying it from the substitutes or adulterants.Microscopic evaluation of drug helps to categorize the ordered cellular organization of drug substance by their known histological features like epidermal cells, mesophyll cells, vascular bundles, stomata, trichomes, starch grains, etc.The use of a variety of reagents or stains helps to differentiate cellular structure depending on their chemical character.The results suggest that powder of E. neriifolia leaves have higher aqueous extractive value followed by petroleum ether, ethyl acetate, toluene, chloroform and n-butanol.The physico-chemical parameters help in judging the purity and quality of the drug.Foreign matter was present in trace amount due to first hand procurement of plant material from non-polluted location.Ash values are useful to identify the occurrence of any siliceous infectivity and water soluble salts.They are the chief quantitative standards values and useful in shaping accuracy as well as purity of drugs. 48The results of total ash, acid insoluble ash and water soluble ash were 6.33%, 1.23% and 6%, , respectively.Low level of total ashes is indication of little amount of carbonates, phosphates and silica in the selected plant leaves.There is a chance of presence of nonphysiological substances in the crude drugs so total ash value is not always reliable.Therefore, the authentication of acid insoluble ash was also studied.It showed ash in leaves.Loss on drying was found to be 4.69% which signify the significant amount of mucilage in leaves.Percent active chemical constituent is usually expressed on air-dried basis in crude drugs.Moisture content of drug was studied for making the solutions of exact strength.It should be minimizing for preventing the decay of crude drug because of microbial infectivity or chemical alteration.Swelling and foaming index was found in the range of 11.7 ml and 333 due to presence of mucilage in the leaves.Results of primary phytochemical study showed that the presence of a variety of phytocompounds in the leaves which are known to have a choice of therapeutic value for curing disease ailments.EN6, the aqueous extract fraction has shown the presence of amino acids, alkaloids, flavonoids, carbohydrates, proteins, glycosides, saponin, tannins, steroids and phenols.Previous research reports suggested that saponins, flavonoids, tannins, alkaloids and phenols have anti-inflammatory activities whereas flavonoids, glycosides, alkaloids and tannins have hypoglycemic effects. 49,50Results of fluorescence analysis of extracts of E. neriifolia leaves showed its characteristic fluorescent colours in tested inorganic and organic chemicals.The fluorescent phenomenon of drug powder determines the purity and quality of plant material.Fluorescence features exhibited by many chemical constituents which indicate fluorescence at visible in day light.U.V     light shows fluorescence in various herbal drugs.As per literature report, alkaloid such as berberine is not visible in day light.It is due to decomposition of drugs due to fluorescent derivatives after treating with various chemical reagents. 51Results of quantitative estimation of phytoconstituents indicates that E. neriifolia leaves have more percentage of saponin content (3.67%) than alkaloid content (0.26%).Hence, quantitative and fluorescence standard of powder provides valuable information to substantiate and authenticate the phytomedicine.Primary HPTLC fingerprinting analysis results showed there is a presence of huge number of phytoconstituents with respect to their Rf values and UV-visible spectra's (200-400 nm) scanned at 254 nm, 366 nm and after derivatization at 540 nm.These phytoconstituents may includes, flavonoids, terpenes, steroidal saponins, alkaloids, glycosides, tannins and phenolic acids, etc. with promising biological actions which can be utilized to expand prospective drugs.Based on the results, there is a recommendation to co-relate these phytoconstituents present in the EN6 extract fraction with various standard marker compounds for its identification, confirmation and validation by analytical research.Similarly, for other chemical constituent's isolation technique are required to be developed.Based on reported biological activities E. neriifolia extracts and isolates can be explored for their therapeutic potential by use of modern assay methods. 52he present investigation is an additional standardization research in support with previous reports and will be helpful for qualitative and quantitative standardization of herbal formulations containing E. neriifolia as E. neriifolia leaves might be utilized as a potential therapeutic agent.Further investigations are going on this extract fraction in reference to identification, quantification and validation of HPTLC methods using various standard marker compounds along with exploration of its pharmacological activities.