Microscopic Leaf Constant Numbers of Chromolaena odorata in Thailand

Introduction: Chromolaena odorata (L.) R.M. King and H. Rob. (syn. Eupatorium odoratum L.), commonly known as Siam Weed, Christmas Bush, or Common Floss Flower, is a species in family Asteraceae. It has been widely used as medicinal plants for a long time. In Thailand, C. odorata is locally used to treat skin diseases and insect bites. Leave juice from the crushed leaves can be used as a haemostatic and anti-inflammatory in the treatment of skin wounds. Objective: This study aimed to investigate the leaf constant values of microscopic characters of leaf in term of stomatal number, stomatal index, epidermal cell number, trichome number, trichome index and oil gland number of C. odorata in Thailand. Method: Microscopic evaluation of leaf constants are frequency used for the medicinal plant samples. Leaf constant numbers used to identify between some closely related species. Light microscope (LM) attached a digital camera and scanning electron microscope (SEM) were used in this study. Results and Conclusion: The results showed anomocytic stomata and multicellular non-glandular trichomes on both upper and lower epidermis of C. odorata, while oil glands were found on only lower epidermis of the leaf. The microscopic leaf constant numbers of C. odorata were established and could be used for the species identification of C. odorata in Thailand.


INTRODUCTION
Chromolaena odorata (L.) R.M. King and H. Rob.(syn.Eupatorium odoratum L.), commonly known as Siam Weed, Christmas Bush, or Common Floss Flower, is a species in family Asteraceae.C. odorata is a fast-growing perennial shrub distributed in Asia, Africa and the Pacific.It may reach 1 meter or more as a free standing shrub.The leaves are opposite, deltoid to ovate-lanceolate with three-nerved leaves and aromatic when crushed.It has a long-pointed tip and dentate margin.The inflorescences are corymbs with cylindrical heads located on the terminals of lateral branches.It presents in blue, pink, lavender, or white color.The seeds are a brownish gray to black achene with a pale brown pappus.It is sometimes grown as an ornamental and medicinal plant. 1 In Thailand, C. odorata is locally used to treat skin diseases and insect bites.5] The pharmacological activities of C. odorata have been shown a wide biological activities such as anthelmintic activity, insecticidal activity, analgesic activity, anti-inflamma-tory, antimicrobial, blood coagulating, insecticidal and antioxidant activities and many other medicinal properties. 1,6he microscopic examination is one of the quality control process for herbal material to determine the characteristics, identity and degree of purity of medicinal plant materials, and should be carried out before any further tests are undertaken. 7Microscopic leaf constant numbers are frequently used to identify between some closely related species.It has great value for a quality of the medicinal plants based on these specific characters. 8The leaf constant numbers of C. odorata in Thailand has never been established.Therefore, the present study aimed to investigate the leaf constant values of microscopic characters of leaf in term of stomatal number, stomatal index, epidermal cell number, trichome number, trichome index and oil gland number of C. odorata in Thailand.Light microscope (LM) attached a digital camera and scanning electron microscope (SEM) were used in this study.

Plant materials
The fresh mature leaves of C. odorata were collected from different locations throughout Thailand.Plant samples were authenticated by one of the authors, Ruangrungsi N. The voucher specimens were deposited at College of Public Health Sciences, Chulalongkorn University for future references.

Leaves preparation
Fresh mature leaves were cleaned.The leaf portion between midrib and margin from the middle region was cut into small pieces (10 x 10 mm 2 ).Tissues were disintegrated by poaching leaf samples in 10% hydrochloric acid under low heat for 1 hr.They were immersed in Haiter TM bleach solution containing 6% sodium hypochlorite (50% in water) until chlorophyll was removed.When bleaching was completed, leaf samples were washed with water and then cleared by gently warming with chloral hydrate solution (4 g of chloral hydrate / 1 ml of water) until it was transparent.After rinsing with water, the leaf sample was mounted with glycerin and observed under a light microscope (Zeiss Axio Imager A2) with an attached digital camera (Cannon Power shot A640) for the determination of leaf constants.

Microscopic analysis
Microscopic evaluation of leaf constants such as stomatal number, stomatal index, epidermal cell number, trichome number, trichome index and oil gland number were examined.Thirty fields of plant sample were carried out and determined using Axio Vision program.

Determination of stomatal number and stomatal index
Stomatal number is an average number of stomata per square millimeter (mm 2 ) of epidermis of the leaf.Stomatal index (SI) is a percentage of stomata from the total number of epidermal cells in a same unit area of leaf, 9 which can be calculated as:

SI S S E T = + + ×100
Where, S = number of stomata per mm 2 in a given area of leaf E = number of epidermal cells per mm 2 in the same area of leaf T = number of trichomes per mm 2 in the same area of leaf

Determination of epidermal cell number
Epidermal cell number is an average number of epidermal cell per mm 2 of leaf surface.The number of epidermal cell on the upper and lower surface of leaf in 1 mm 2 in each field were counted.

Determination of trichome number and trichome index
Trichome number is an average number of trichome per mm 2 of epidermis of leaf surface.The number of trichome on the upper and lower surface of leaf in 1 mm 2 in each field were counted.Trichome index (TI) is a percentage of trichome from the total number of epidermal cells in a same unit area of leaf, which can be calculated as: Where, S = number of stomata per mm 2 in a given area of leaf E = number of epidermal cells per mm 2 in the same area of leaf T = number of trichomes per mm 2 in the same area of leaf

Determination of oil gland number
Oil gland number is an average number of oil gland per mm 2 of leaf surface.The number of oil gland on the lower surface of leaf in 1 mm 2 in each field were counted.

Scanning electron microscope (SEM) evaluation
The leaf samples were examined by scanning electron microscope (JEOL, model JSM-5410LV, Japan).The leaf was cut about 5 mm 2 and mounted on the stub with double sided adhesive tabs.After that, the specimens were coated with gold in sputter coater, then examined with scanning electron microscope at 15 kV. 10

Data analysis
All leaf constant parameters were determined at least in thirty fields of plant sample.The results expressed as mean ± standard deviation (SD), minimum and maximum values.

RESULTS AND DISCUSSION
Microscopic leaf constant values are possibly used to distinguish between some closely related species of which cannot clearly characterized by general microscopy. 9Microscopic evaluation should be the first step to identify the plants. 11The results of leaf constant numbers consisting of stomatal number, stomatal index, epidermal cell number, trichome number, trichome index and oil gland number were shown in Table 1.
In this study, the epidermal cell numbers of C. odorata on lower epidermis surface could not be counted to calculate the constant values of the stomatal and trichome indices because all of the oil gland cells in this species shielded some epidermal cells around these oil glands.
The stomatal number and stomatal index are very specific criteria for identification and characterization of herbal crude drug. 12Although the stomatal number varies greatly with the age of the leaf, the stomatal index remains highly consistent.The one of microscopic evaluation which can distinguish plant species is stomatal type by their form and arrangement in the surrounding cells.The different types of stoma are often available for matured leaves that are distinguished by their form and arrangement in the surrounding cells.In recent study, the stomata type of C. odorata was classified as anomocytic or ranunculacious type of stomata (Figure 1).The stomata is surrounded by varying number of cells, which are generally not different from those of the epidermis which supported in characteristics of C. odorata. 7The stomata were found on both upper epidermis and lower epidermis which called amphistomatic leaf as distribution of stomata.][15] Trichomes are epidermal protuberances located on aerial parts of plant, that protect plant from excess transpiration, high temperature, ultraviolet light, and herbivore attack.Trichome number has been used for identification of some plants that have trichomes covering their leaves. 16he results showed that there were multicellular uniseriate trichomes on both upper and lower epidermis of C. odorata (Figure 2, 3).Micrographs of the trichome characteristic were taken from scanning electron microscope were demonstrated in Figure 4.The trichome number on the upper epidermis was found to be 5.17 ± 1.12, which was less than that of the lower epidermis (47.40 ± 3.04).] Essential oils are complex, volatile mixtures of certain secondary plant metabolites.A wide range of plant species produce essential oils.In addition, essential oils are most commonly found in special secretory structures either on the surface of the plant or within the plant tissues.The type of structure is specific to the family or species.This can be useful to identify plant material and verify the authenticity of the plant source in the case of suspected adulteration.The secretory tissues can be examined using scanning electron microscopy, which enables detailed observation of major stages in the secretory cells including their membrane system and the overall size of the gland. 17In this study, the glandular trichomes were found on only lower epidermis of C. odorata (Figure 3B).The scanning electron micrograph of glandular trichome from C. odorata leaf was demonstrated in Figure 5.The circular structure is embedded in the shallow pits of epidermis is an oil gland, a modified glandular hair which secretes oils to give the plant its aroma.C. odorata has such oil glands lying in the epidermis of the leaf.This also explains that the leaves are aromatic when crushed.The previous study also reported that there were glandular trichome located in shallow pits, mostly on the abaxial side.The gland is sessile, broadly bowl shaped and consist of two vertical rows of cells. 13Moreover, the powder microscopy of C. odorata leaf revealed presence glandular trichomes. 18

Figure 1 :
Figure 1: Light microscope photographs of stomata on the (A) upper and (B) lower epidermis of Chromolaena odorata.

Figure 2 :
Figure 2: Light microscope photographs of trichome on the (A) upper and (B) lower epidermis of Chromolaena odorata.

Figure 4 :
Figure 4: Scanning electron micrographs of the trichome characteristic on both (A) upper and (B) lower epidermis of Chromolaena odorata (multicellular uniseriate trichome type).

Table 1 : Microscopic leaf constant numbers of Chromolaena odorata in Thailand.
*nd: not detected.