Pharmacognostic, Phytochemical and Physicochemical Investigations of Hypericum hookerianum Wight & Arn. (Hypericaceae) of Palni Hills, India

Introduction: Hypericum hookerianum Wight and Arn. (Hooker’s wort) is a lesser known unfamiliar yet critically endangered native therapeutically active native medicinal plant. It is being characterized by the presence of various secretory glands. Methodology: In this present study, characterization of the plant in terms of morphology, anatomy and histochemistry of tissues and phytochemicals and antimicrobial potentials were made. Results: The epidermal layer contains oil cells while histochemistry showed the presence of the secondary metabolites. Qualitative analysis indicated presence of maximum phytocompounds in the high polar ethanolic extract; flavonoids, anthocyanin and phenol are prominently present and quantified. Conclusion: The results suggest that the less studied herb, H. hookerianum is a multifaceted high value species having a wide range of phytochemicals with abundant medicinal properties.


INTRODUCTION
Hypericum is a large genus of herbs, shrubs and sometimes trees comprising 490 species of 36 sections. 1hey represent a large pool of natural resource for phytochemical, pharmacological and product development investigations as they accumulate pharmacologically important secondary metabolites.Many of the species contain structurally and functionally diverse phenolics, flavonoids and therapeutically important phenolic derivatives the dianthrone, hypericins and the prenylated phloroglucinol, hyperforin.The highest levels of the chemicals are found at full flowering stage when almost all the plants of the genus are widely used in folk medicine in different parts of the world.The growing interest in the usage of Hypericum in traditional medicine is due to several reasons the principal one being that modern medicine has invariably side effects and its costs are prohibitive.Among an estimated 27 species reported from India including the most popular and productized, H.perforatum and the recently investigated H. hookerianum, a high altitude shrub of the Himalayas and the Western Ghats.Hypericum hookerianum Wight and Arn.Also known as Norysca hookeriana Wight and Arn.commonly known in Nepal as Mehandiphul, is a round topped shrub with weakly spreading, non-erect branches with golden-yellow flowers also distributed in Sikkim, Khasiand Jaintia hills of Himalayas and in the high altitudes (>2300 m) of the Nilgiris and Palni hills of the Western Ghats. 2 The terete shrubs are 2-2.5 m in height, with rather flaccid ovate leaves, set bifarious, mucronate to mucronate at apex. 2 The tribal people of the Shola forest (Tamil Nadu) in Palni hills use the aerial parts of the plant for treating burns and wounds demonstrated by Mukherjee and Suresh. 3to have wound healing properties.The Toda tribe of the Palni hills uses it as a folklore medicine for its different therapeutic potentials, including antidepressant, spasmolytic, stimulant, hypotensive and antifungalactivities. 4 Leaf anatomy of hypericum species differ greatly and is the basis for not only identifying the species but also grouping them under different taxonomic criteria.Anatomical features in terms of types of glands present, secretions of the glands, interconnecting and extension of the ducts, quality of the products of the secretory glands and phytochemical profiles of the secretion play an important in differentiating the species.The anatomy and ultra-structure of the cavities in the internodes, leaves and petals varied extensively between species of Hypericum, especially in H. hookerianum.The secondary growth is more extensive in shrubs (H.inodorum and H. forestii).The petals also differ between species in the mesophyll structures and the occurrence and location of secondary structures. 5It is against this background, the pharmacognostic profile, anatomy of the secretory

Photomicrographs
Microscopic descriptions of tissues are supplemented with micrographs wherever necessary.Photographs of different magnifications were taken with Nikon LabPhoto 2 microscopic Unit.For normal observations, bright field was used.For the study of crystals, starch grains and lignified cells, polarized light was employed.Since these structures have birefringent property, under polarized light they appear bright against dark background.Magnifications of the figures are indicated by the scale-bars.Descriptive terms of the anatomical features are as given in the standard anatomy book. 13

Fluorescence Evaluation
The fluorescence analysis of the dried leaf powder was carried out by the method of Kokoski et al. 14 A small quantity of the sample treated using fluorescence reagents (such as 1 N NaOH, 1 N HCl, 50% H 2 SO 4 , FeCl 3 , Iodine Solution, Acetic Acid Glacial, 0.1N NH 4 OH, 1 % CH 3 COOH) and analyzed under visible light and ultra violet light (Wavelength 254 nm).

Phytochemical determination
The dried leaf powder of H. hookerianum was extracted successively with different polarity solvents such as Hexane, Ethyl Acetate and Ethanol.After 24 hours, the dried extracts were obtained and were analyzed using chemical reagents for both qualitative phytochemical evaluation following standard procedure. 15

Estimation of total phenol, anthocyanin and flavonoid
The Folin-Ciocalteu method was used to determine total phenolic content (TPC) of the various solvent extracts as described by Singleton and Rossi. 16Gallic acid was used as a reference standard (20-100 μg/mL) for plotting calibration curve.A volume of 0.5 mL of the plant extract (100 μg/mL) was mixed with 1.5 mL of Folin-Ciocalteu reagent (diluted 1:10with de-ionized water) and were neutralized with 3 mL of sodium carbonate solution (7.5%, w/v).The reaction mixture was kept in dark at room temperature for 30 min with intermittent shaking for color development.The absorbance of the resulting blue color was measured by using double beam UV-Vis spectrophotometer at fixed wavelength of 765 nm.The TPCs were determined using linear regression equation obtained from the standard plot of gallic acid.The content of total phenolic compounds was calculated as mean±SD (n=3) and expressed as mg/g gallic acid equivalent (GAE) of dry extract.Total anthocyanin content of the extract was determined by the pH differential method. 17A pH 1.0 buffer solution was prepared by mixing 125 ml of 0.2 N KCl with 385 ml of 0.2 N HCl and 490 ml of distilled water.The pH of the buffer was adjusted to pH 1.0 with 0.2 N HCl.A pH 4.5 buffer solution was also prepared by mixing 440 ml of 1.0 M sodium acetate with 200 ml of 1M HCl and 360 ml of distilled water.The pH of the solution was measured and adjusted topH 4.5 with 1M HCl.The sample extract (0.5 ml) was diluted to 12.5 ml in the pH 1.0 and 4.5 buffers and allowed to equilibrate in the dark for 2 h.The absorbance of the samples at 512 nm (A 512 nm) and 700 nm (A 700 nm) was measured.The difference in absorbance (∆A) between the anthocyanin extract diluted in pH 1.0 and pH 4.5 buffers was calculated and total anthocyanin content was also estimated.The total flavonoid content (TFC) of each extract was investigated using the aluminum chloride colorimetry method. 18The calibration curve was prepared by diluting quercetin in 95% ethanol (0 -100 µg/mL).The diluted extract or quercetin (2.0 mL) was mixed with 0.1 mL of 10% (w/v) aluminum chloride solution and 0.1 mL of 0.1 mM potassium acetate and 2.8 mL of distilled water in a test tube.The mixture was kept at room temperature for 30minutes to complete the reaction.Then the maximum absorbance of the mixture was measured at 415 nm with double beam structures, phyto chemical constituents, anti-bacterial activity of H. hookerianum were studied.

Collection and authentication of the plant
Leaves of H. hookerianum were collected from Pambar Shola forest in the outskirts of Kodaikanal Town of Palni hills in the Western Ghats of Indian the month of June, 2015 (Figure . 1).The taxonomical identification of the plant was authenticated by Prof P. Jayaraman, Institute of Herbal Science, Plant Anatomy Research Centre, Chennai, Tamil Nadu.The voucher specimen (voucher no.CNBT/15/HH/01) was preserved in our laboratory for future reference.

Reagent and Chemicals
All the chemicals and reagents used were of analytical grade, purchased from Sigma chemical co.(St Louis, MQ, USA) and Merck (Darmstadt, Germany).

Macroscopic Investigation
For morphological observations, fresh leaf approx.3-5 cm in length was used.The macro-morphological characteristics of the leaf were observed under magnifying lens. 6

Pharmacognostic studies
Pharmacognostic studies include organoleptic characters and microscopical features of the plant.

Morphological investigation
The morphology evaluation is used to find the closely related species and used to study the external texture and sensory characters such as color, odor, taste, size, shape etc. Fresh leaves of H.hookerianum were used for the morphological studies and reported. 7,8

Microscopical investigation of fresh leaf and dried powder
The transverse and longitudinal sections of the fresh leaves were prepared using standard procedures. 9The normal leaf samples of H. hookerianum were cut and detached from the plant and fixed in F.A.A. (Formalin -5ml+ Acetic acid -5ml + 70% Ethyl alcohol -90ml).After 24 hrs of fixing, the specimens were dehydrated with a graded series of tertiary-Butyl alcohol as per the schedule given by Sass. 10 Infiltration of the specimens was carried by gradual addition of paraffin wax (melting point 58-60°C) until TBA solution attained super saturation.The specimens were cast into paraffin blocks.The paraffin embedded specimens were sectioned with the help of Rotary Microtome.The thickness of the sections was 10-12 μm.Dewaxing of the sections was by customary procedure. 11The sections were stained with Toluidine blue as per the method published by O'Brien et al.. 12 Since Toluidine blue is a polychromatic stain.The staining results were remarkably good; and some cytochemical reactions were also obtained.The dye rendered pink color to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies, etc. Wherever necessary sections were also stained with safranin and Fast-green and IKI.For studying the stomatal morphology, venation pattern and trichome distribution, paradermal sections as well as clearing of leaf with 5% NaOH or epidermal peeling by partial maceration employing Jeffrey's maceration fluid. 10were prepared.Glycerine mounted preparations were made and the different cell components were studied and measured.The dried powdered material of leaf was used to evaluate the powder analysis following the method of Brian and Turner. 7V-Vis spectrophotometer against blank.A typical blank solution contained all reagents except aluminum chloride which is replaced by the same amount of distilled water.The amount of flavonoid was calculated from linear regression equation obtained from the quercetin calibration curve.TFC was expressed as mg/g quercetin equivalent (QE) of dry extract.All the values were expressed on dried weight basis and each value represents mean ± SD (n=3).

Physicochemical parameters
The proximate analysis to assess the physicochemical parameters like total ash value, loss on drying and water soluble extractive values, pH value, solubility, etc. were determined as per WHO guidelines. 19

Antibacterial activity
The plant extracts were analyzed for their antibacterial activity against selected human pathogens including P. vulgaris (MTCC -426), E. coli (MTCC -443), B. subtilis (MTCC -441) and M.lutens (MTCC -1538).In this method, discs of 4 mm were loaded with different solvent extracts of H. hookerianum.The discs were placed on the solidified Mueller -Hinton Agar (MHA) plates spread with test bacterial strains to which the sensitivity was measured (Figure .3).The plates were incubated at 37ºC for 24 h and after incubation the zones of inhibition were measured. 20

Statistical analysis
All experiments were repeated at least three times.Results are reported as Mean ± S.E.M. (Standard Error of Mean).

Macroscopical investigation
The classification of this plant through the process of identification was done at the Institute of Herbal Science, Plant Anatomy Research Centre, Chennai, and Tamil Nadu as follows:

Microscopical investigation of the fresh and dried powder
Leaf: The transverse section of the leaf found that the presence of anamocytic stomata, epidermal cells often contain oil cells, guard cell with many chloroplast in lower epidermis and absence of stomata in upper epidermis.Palisade cells are arranged to sub adjacent to upper epidermis with very long cells, in midrib upper surface consists of one layered epidermal cells, cuticles are colored, collenchymatous are less greenish with less contents and innermost colorless.Xylem vessels are radially arranged, phloem cells are arranged with more dark contents.The leaf consists of a planoconvex midrib and thick smooth lamina.The midrib is flat on the adaxial side and conically thick on the abaxial side.The midrib is 750 µm thick and 700 µm wide (Figure .2.1).The epidermal layer of the

Epidermal cells and stomatal morphology
The epidermal structure was studied from the paradermal sections of the lamina.The adaxial epidermis is apostolates; the cells are rectangular to polyhedral and have thick straight anticlinal walls.The abaxial epidermis is stomatic ferous (Figure 2.5).The epidermal cells small, polygonal and have slightly wavy ad thick walls.The stomata are thick and elliptical with narrow stomatal aperture.The stomata are actinocytic type.The guard cells are surrounded by six or more radiating rectangular subsidiary cells.In surface view the stomatal complex appear star shaped (Figure 2.6).

Venation pattern:
The venation is densely reticulate; the thickness of the vein is gradually reduced from primary veins to secondary and tertiary veins.The vein-islets are wide and polygonal in outline.The vein boundaries are thick and straight.The vein boundaries are thick and straight.The vein terminations are short and simple (unbranched) or repeatedly branched to form dendroid outline (Figure .2.7).

Powder characteristics
The powder preparation of the plant exhibits the following inclusions, i) Fragments of small epidermal peelings are common in the powder.The epidermal peelings are usually adaxial epidermis.The epidermal cells are polygonal in outline and their anticlinal walls are straight.The cells possess prominent nuclei and Vessel elements (Figure .2.8) Thin, long, cylindrical vessel elements are occasionally dense bordered pits and oblique narrow end wall perferatious.The vessel elements are up to 470µm long and 40µm thick.

Fluorescence Evaluation
The fluorescence analysis is adequately susceptible and enables the precise and accurate determination of the analyte over a pleasing concentration range without several time-consuming dilution steps prior to analysis of pharmaceutical samples. 21The fluorescence analysis of the powdered leaves of H. hookerianum is presented in the Table 1.

Phytochemical Determination
The phytochemical evaluation of the different solvent extracts of the leaves of H. hookerianum showed the presence of various phytochemical constituents which contribute to the medicinal activity of the plant listed in Table 2.The presence of various bioactive compounds justifies it might be utilized for the development of traditional medicines and further investigation is needed to elute novel active compounds from the medicinal plants which may create a new way to treat many incurable diseases.

Phenolic, anthocyanin and flavonoid contents
The total phenolic content, total anthocyanin content and total flavonoid content of the ethanolic leaf extract, calculated from the calibration curves were 14.15 ± 0.43 gallic acid equivalents/g, 13.59 ± 0.45 cyanidin equivalent/g and 28 ± 1.37quercetin equivalent/g respectively (Table 3).Phenolic compounds have redox properties, which allow them to act as antioxidants.As their free radical scavenging ability is facilitated by their hydroxyl groups, the total phenolic concentration could be used as a basis for rapid screening of antioxidant activity.Flavonoids, including flavones, flavanols and condensed tannins, are plant secondary metabolites, the antioxidant activity of which depends on the presence of free OH groups, especially 3-OH.Among three organic extracts, maximum amount of phenol, anthocyanin and flavonoid content were found in ethanol followed by ethyl acetate and hexane extracts.

Physicochemical Parameters
The proximate analysis to assess the physicochemical parameters like total ash value, loss on drying and water soluble extractive values, pH value, solubility, etc.The percentage yield (% w/w) with color and consistency are mentioned in Table 4.

Antibacterial activity
Among the different solvent extracts of H. hookeranium leaves tested for antibacterial activity, ethanol extract showed better activity when compared to other solvent extracts.Proteus vulgaris showed highly sensitive to plant extract followed by M. luteus, B. subtilis and E.coli.   .The physicochemical evaluation using percentage yield (w/w) color and consistency parameters is useful in the identification and authentication of crude extract.The antibacterial investigation revealed the potency of active principles in each extract which may be attributed to the high content of flavonoids, which have been reported to be involved in inhibition of nucleic acid biosynthesis and other metabolic processes. 24he present study is a preliminary data for further studies.Moreover, this data was first time reported from this high altitude plant of Palni Hills, Western Ghats of India.

CONCLUSION
Pharmacognostic, phytochemical, physicochemical investigation were carried out for the first time in the high altitudes (>2300 M) plant of the Palni Hills, H. hookerianum and the results were computed to predict the utility of the species to treat common human values.The macroscopical, anatomical and powder microscopical studies provides the basis for further identification and to lay down standards for leaves.The preliminary phytochemical screening of the solvent extracts reveals the presence of flavonoids, anthocyanins, phenols, etc.The results suggest that, H.hookerianum is found a prospective versatile high value species having a wide range of phytochemicals with immense medicinal properties.Further exploration may reveal many novel features and its extraction may bring out novel products of specialized kind which needs to be upgraded / popularized before the remaining species disappear from the face of the Earth.

Table 4 : Physico-chemical parameters of the leaves of H. hookerianum Wight & Arn.
23ants possess contain numerous phytochemical constituents, many of which are known to be biologically active compounds and are responsible for exhibiting diverse pharmacological activities.22According to the previous records of pharmacognosy sciences plants have been used in native medicinal systems globally.Medicinal plant based research are rapidly increasing worldwide and further study with emphasis on efficiency, safety and quality of plant secondary metabolites is the need of the hour.9Macroscopiccharactersinvolvesize, arrangement, venation, texture, surface characters, markings and hardness of the plant materials.The microscopical studies (anatomical and histochemical) are often necessary to establish the botanical identity of commercial samples of medicinal plants and may play an important part in checking adulteration and substitution.It involves longitudinal and transverse sectional views of the leaves of the plant.23Theleaves of H. hookerianum is characterized by the presence of different types of internal secretory oil cells present in the epidermal region.The lamina of the leaves has prominent wide circular secretory cavities responsible for secretion of secondary metabolites.The bioactive secondary metabolites have been shown to decrease the risk and development of many human diseases through various biological mechanisms.The results of preliminary chemical testing confirmed thepresence of various classes of bioactive chemical constituents in ethanolic extract of H. hookerianum leaves including phenols, flavonoids, anthocyanin's, steroids, alkaloids, anthroquinones, glycosides and terpenoids.Therefore, based on the quantitative phytochemical analysis results, the total phenolic and flavonoid contents of different extracts of H. hookerianum leaves were estimated.The ethanol extract, was found to contain the highest content of total phenol (14.15 mg/g), anthocyanin (13.59mg/g) and flavonoid (28.47 mg/g) as compared to hexane/ethyl acetate extracts (9.14/8.65/19.47mg/g and 12.33/11.18/23.28mg/g, respectively) Standard deviation (SD) = ±SD; Number of readings (N) =3DISCUSSION