Himalayan region is the treasure of biodiversity and consists of diverse flora and fauna. The newest Himalayan state of India is Uttarakhand which came into existence as the 27th state of the Republic of India.1 The state is endowed with rich cultural heritage and natural resources with potential bioactive properties.
Several less explored plants are available in the Himalayan region. One such plant is Rhododendron arboreum Sm. (family Ericaceae) which is a popular plant especially for its beautiful flower and flower juice which is consumed as a popular beverage in the Himalayan region. The plant is commonly known as “Burans” and “Laligurans ” and is an important contributor to the economy of the rural areas of Uttarakhand. R. arboreum is the national flower of Nepal and state tree of Uttarakhand.2 The plant is supposed to be described in Ayurveda as Kurabaka which is kept in the Kashaya group.3 and sold in the local market of Nepal in the name of “Rohitaka”.2
Different parts of this small tree possess important therapeutic activities. The leaves of this plant showed hepatoprotective effects in rats.4 Apart from the leaves of this plant, other parts like flowers and bark are also used for various therapeutic purposes. The plant contains numerous phytoconstituents like ursolic acid, lupeol, sitosterol, taraxerol and betulin (Figure 1).5
The pharmacognostic evaluation parameters are useful in the identification and standardization of a crude drug.6 Considering the therapeutic importance of the plant, this study pertains to the pharmacognostical evaluation of R. arboreum leaves. To the best of our knowledge, the leaves of R. arboreum from Kumaun region of Uttarakhand were investigated for its morphological and cytological investigation with the help of scanning electron microscopy for the first time.
MATERIAL AND METHODS
The fresh leaves and flowers of R. arboreum were collected from Ramgarh region of Nainital district, Uttarakhand, India. The herbarium specimens were prepared and authenticated from Regional Ayurveda Research Institute (RARI), Jhansi and the voucher specimen was deposited in the respective Herbarium for future reference (Accession no. 25277).
Organoleptic study of R. arboreum was carried out to evaluate its sensory characters such as colour, odour, taste, shape, size as per the reported method.7,8 The leaves of the plant material were placed separately on a watch glass and observed carefully for their apparent characteristics like general appearance, colour, odour and taste. The observations were recorded as accurately as possible that provided information on obvious physical properties.7,8
The leaves of the R. arboreum were subjected to microscopical studies. The material was dipped in FAA (Formalin–acetic acid-alcohol) solution.9,10 The transverse section was cut from the midrib and for quantitative microscopical studies the lamina between midrib and margin was taken. Leaves were soaked in 70% v/v ethanol followed by free hand section cutting and then viewed under the microscope. Moreover, quantitative microscopical studies such as determination of stomatal index, vein islet number and veinlet-termination number were also carried out as per the standard protocols.11,12 The material was placed on the slide and stained with different staining reagents and examined under the microscope (Leica DM 2500 LED).12
Scanning electron microscopy
Leaf sample for R. arboreum was subjected to scanning electron microscopy (SEM) with the help of scanning electron microscope. Electron microscope (ZEISS EVO 40 EP) was used and images were analyzed by QUANTAX 200 analytical software. The sample was properly dried with an electric lamp and then separately mounted on stubs using double-sided adhesive tape. After that the samples were sputtercoated with 5 nm cathodic spraying (Polaron gold) using a separate coating machine to make the sample more conductive and suitable for SEM analysis and then observed under the scanning electron microscope (ZEISS EVO 50). SEM was carried out under the following analytical conditions: EHT = 20.00 kV, WD = 9.5 mm and signal A = SE1. The SEM photographs were captured on the Zeiss EVO 50 scanning electron microscope at the required magnification (ranging between 100x to 8000x) at room temperature.13
Preliminary phytochemical screening
Different extracts (methanolic, ethyl acetate and petroleum ether) of R. arboreum were subjected to qualitative evaluation for the presence or absence of different groups of phytoconstituents.11,12 such as alkaloids, flavonoids, saponins, carbohydrates, glycosides, lipids, phenols and tannins.
Fluorescence analysis is an important qualitative evaluation parameter of herbal drugs that is helpful in the detection of the adulteration in the raw material or finished products. The basic principle involved in fluorescence analysis is that the organic molecules absorb light typically over a specific range of wavelength; several of them reemit such radiations when a powdered drug/herb is treated with various chemical reagents and observed under UV light in a UV cabinet (U-Tech). Varying range of coloured fluorescence produced in short-wavelength (254 nm) and long-wavelength (366 nm) were prudently scrutinized for the detection of fluorescent compounds as per the suggested method.16
RESULTS AND DISCUSSION
Organoleptic evaluation is an important initial step for the identification of the plant material. R. arboreum was evaluated for its basic botanical and organoleptic characteristics. The plant has dark glossy green, broad leaves with size ranging between 6-20 cm, with a brown coating beneath. The leaves are oblong-lanceolate, 4-6 cm wide, glabrous, white or rusty brown-tomentose beneath. The leaves are present in abundance towards the ends of the branches. Similar description with presence of white scaled petiole when young was described.17 The flowers of R. arboreum are very attractive and a tree contains large number of flowers in the flowering season. Flowers of Rhododendron exhibit coloration ranging from deep scarlet, to red with white markings, pink to white. The plant species collected for the present study had dark red flowers in dense globose cymes as presented in Figure 2 and the details of the organoleptic characters are presented in Table 1. Forests with Rhododendron tree covers are of astonishing sight when in full bloom.
The transverse section of R. arboreum leaf through midrib depicted a normal dicot leaf. The outer most layer of cuticle was followed by upper epidermis and then collenchyma. Long palisade layer was present. The inner circle consisted of the xylem and phloem cells. Vacuole was also present mainly in the lower epidermis part. The cells of upper epidermis were tightly closed as compared to the lower epidermis. Spongy parenchyma also contained vacuole. Microscopical features of the R. arboreum leaf are presented in Figure 3.
Stomatal index, vein-islet number and veinlet-termination number were determined as a part of quantitative microscopic evaluation. The mean values were calculated from different observations. Measurements of different tissues of leaf and stem were carried out, and the results are presented in Table 2.
Scanning electron microscopy
Use of powerful microscopical tools for the anatomical studies is gaining importance these days. SEM is one of the dominant methods for the surface structure elucidation of the plant based drugs. This technique is proving as one of the most exciting and momentous tool for the study and elevation of surface topography of solids.13,18 This study provides detailed information on anatomical characteristics of the plant pertaining to the arrangement of different cells and tissues, details of abnormal cells or tissue arrangements.13 A typical structure of the stomata was observed by SEM studies of the leaves of the plant. The paracytic stomata were found in cluster form. The epidermal cells were long and spongy. The cells also contained some grooves and papillae throughout the surface. The details of the SEM studies of R. arboreum are presented in Figure 4.
The physicochemical eavaluation of R. arboreum was carried out and the results are expressed in Table 3.
Preliminary phytochemical screening and fluorescence analysis
The preliminary phytochemical study revealed the presence of different phytoconstituents in different extracts of R. arboreum (Table 4). Highest chemical identities were present in the methanolic and ethyl acetate extracts whereas the petroleum ether extract showed the presence of only lipids and major constituents. The methanolic and ethylacetate extract revealed the presence of alkaloids, tannins, reducing sugar and phenolic compounds. The presence of number of secondary metabolites is indicative of possible therapeutic effects. Observations presented in Table 5 indicate identifiable traits of the plant material tested. Colour variations from pale green to dark green and black were observed on treatment with the mentioned solvents and reagents under day light and UV light (short and long wavelength). Fluorescent study of R. arboreum using different chemical reagents exhibited various colouration under visible light and UV light. UV light induces several fluorescent components in natural products.
|Organoleptic charecters||R. arboreum leaf||R. arboreum flower/juice|
|Odour||Odourless||Similar to that of beetroot|
|Taste||Bitter/Acrid||Acrid followed by astringent|
|Quantitative microscopy||R. arboreum|
|Vein islet number||34-42-67|
|Veinlet termination number||32-36-45|
|Palisade layer||397.35±29 µm|
|Xylem vessels||148.9±36 µm|
|Parameters||R. arboreum leaf|
|Acid insoluble ash||0.32|
|Water soluble extractives||12%|
|Alcohol soluble extractives||9.65%|
Rhododendrons are believed to be amongst the widely grown ornamentals of great horticultural interest. The genus comprises of about 1000 species worldwide.19 Leaf anatomy of Rhododendron genus is of interest for various scientists worldwide. A critical comparison of the leaf structures in two Rhodendron species viz. R. catawbiense and R. ponticum propagated in USA was carried out using scanning electron microscopy. The results of the study indicated that R. ponticum has higher stomatal density than R. catawbiense leaves. The study also demonstrated the thermonasty (i.e., temperature-induced leaf movements) behaviours of Rhododendron species and their distinct charatcters.20
The distribution of the species R. arboreum Sm. is restricted to a small number of South East Asian countries like India, Bhutan, Nepal, Sri Lanka, Northern Myanmar, Northern Thailand, South Western China, and Northern Vietnam. Different species of Rhododendron have been extensively studied by Indian researchers but most of the studies were focused on the pollen morphology.21,22 Sufficient studies have not been carried out on the pharmacognostical aspect of R. arboreum leaves. Although, one extensive study was recently carried out by Panda and Kirtania.23 on the leaf anatomy and pollen morphology of R. arboreum based on herbarium and field based exomorphological data to evaluate the intraspecies variations in R. arboreum complex. In the study several leaf samples from Arunachal Pradesh, Sikkim, Meghalaya, Darjeeling, Shilong, Uttaranchal, Bhutan, Nepal, Himachal Pradesh, Tamil Nadu and China were presented and the stomata complex was evaluated on Arunachal Pradesh, Sikkim, Bhutan, Sikkim, China, Kerala, and Tamil Nadu samples.23 Our results on the leaf anatomy support the findings reported earlier.23
The results of the present study provide an insight on the pharmacognostical evaluation of the R. arboreum leaves from Uttarakhand region. The details presented in the study confirms the qualitative presence of various secondary metabolites in the leaves and flowers of R. arboreum. Moreover, the data of this study could be useful in the preparation of the pharmacopoeial monograph of R. arboreum. Further phytochemical and pharmacological studies are required to unravel the safety and bioefficacy of R. arboreum.
Rhododendron arboreum Sm. commonly known as Burans is an important plant of the Himalayan region.
R. arboreum from Uttarakhand region was investigated for its morphological and cytological characteristics with the help of scanning electron microscopy
The results of the present study provide an insight on the pharmacognostical evaluation of the R. arboreum leaves from Uttarakhand region.