Macro-Micromorphological, Antioxidant and Preliminary Phytochemical Studies on Two Terrestrial Orchids Eulophia Nuda Lindl. and Geodorum Densiflorum (Lam.) Schltr.

An attempt for study of two terrestrial orchids i.e. Eulophia nuda (Lindl.) and Geodorum densiflorum (Lam.) Schltr. were carried out. Main focus was on the corms of both the orchids due to its huge medicinal properties like anticancerous, antioxidant, antidiabetic, antimicrobial, phytotoxic, etc. This is one of the prime reasons behind their threatened status along with endemic habitat. The parameters which were analysed during the study include macroand micro-morphology, preliminary phytochemistry, and antioxidant activity (DPPH activity). In macro-morphology of both the orchids, slight difference was reported and, thus sometimes Geodorum densiflorum is being used as an adulterant in place of Eulophia nuda. During micro-morphological analysis, the oxalate crystals, raphides, mucilage ducts, starch grains, and oil granules were observed from cortical region of corm and in the mesophyll tissue of leaves in both the orchids. Stomatal Index of leaf surface was calculated along with the type of stomata, i.e. Cyclic II type. Preliminary analysis of phytochemical constituents in both the orchids was performed with three different solvent systems. A comparative antioxidant activity between leaves extract of both the orchids was carried out.


Introduction
The two taxa under investigation belong to the family Orchidaceae, which is one of the largest and diversified families among angiosperms [1]. a). Eulophia nuda Lindl (= E. spectabilis (Dennst.) Suresh) a terrestrial orchid, which usually grows in shady places alongwith grasses and shrubs in forests. In ayurveda, it is known as Amarkand [2]. Its corms are medicinally important and are used for curing bronchitis, tumours, scrofulous glands, and also used as vermifuge. Anticancerous, anti-oxidant, and antibiotic properties are reported [3,4,5]. Due to overexploitation, it is under threatened status. Due to the similar morphology of their corms, Geodorum densiflorum corms are used as adulterant. b). Geodorum densiflorum (Lam.) Schltr. is mostly found in wet habitats like rainforests, moist deciduous forests, and grasslands with mostly terrestrial habit. In India, it is distributed in Kerala, Karnataka, Tamil Nadu and Maharashtra [6]. The corm extracts also shows antioxidant, cytotoxic, and antibacterial potentials [7,8].
In orchids, the flower is modified and diversified among the angiosperms such as zygomorphic flowers, which show variously modified labellum, epigyny, reduced stamens adhering with style and stigma to form a column (Gynandrium), pollens bound together to form a mass called as pollinia. Orchid seeds are very minute [9]. The underground fibrous corms contain important secondary metabolites like triterpenoids/steroids, saponins, proteins, anthraquinones, anthocyanins, etc [10,1]. Out of these phenanthrene derivatives are isolated from corms of Eulophia nuda i.e. Nudol possess anti-cancerous, antioxidant, and antimicrobial, anti-inflammatory, antiallergic, phytotoxic properties [4,11]. Phenanthrenes are tricyclic aromatic hydrocarbon (isomeric with anthracene) insoluble in water but soluble in most organic solvents like toluene, carbon tetrachloride, ether, chloroform, acetic acid, and benzene [11].

Collection of Plants
Primary step was scanning of probable regions for the availability of these two orchids-Eulophia nuda and Geodorum densiflorum. Eulophia was collected from Kolhapur and Melghat forest, Amravati; while Geodorum from Ghodazari forest (Chandrapur District) and Rawanwadi, (Bhandara District). The collected plants were identified with the help of standard literature like Flora of Maharashtra and Flora of Kolhapur [12].

Macroscopic characters
Macro-morphological characters of stem, leaf, and corm were analysed and compared between the two orchids manually. Observed differentiating characters were noted.

Microscopic characters
For micro-morphological characters, thin transverse sections of fresh leaves and corms were taken and dipped in 70% ethanol for 2 minutes, stained with safranin for 30 minutes and then dehydrated at 95% ethanol plus 0.5% picric acid for 30 seconds. Then sections were washed with 95% ethanol and 4 drops ammonium hydroxide per 100 mL for 30 seconds. For final dehydration step dipped the sections in absolute alcohol for 10 seconds. Later counterstained with Fast green and washed with absolute alcohol to remove excessive stain. At last kept the slides in xylene solution for 20 seconds and mounted in glycerine then observed under microscope (Johansen, 1940) [13].

Preliminary Phytochemistry
The leaves and corms of both the orchids were dried and crushed for preliminary phytochemical analysis. Three different solvent systems were used for comparative study as used by Maurya and Dongarwar, 2009 [14]. All the preliminary phytochemical tests were performed according to the methods given by Sadasivam and Manickam, 1996 [15]; and Harborne, 1973 [16].

DPPH Free Radical Scavenging Assay
In vitro antioxidant activity of the sample extracts analysed via DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical assay [17]. The sample stocks were diluted with methanol to prepare a concentration series of 25µL, 50µL, 75µL, 100µL, and 125µL. DPPH solution prepared by adding 1.3 mg/mL. To each concentration series 75 µL DPPH solution was added, shaken vigorously, and kept under dark for 30 minutes at room temperature. The decoloration of DPPH was determined by measuring the absorbance at 517nm using Shimudzu UV 1800 spectrophotometer. The percentage of scavenging is calculated by the following equation.

A. Macroscopic Characters
Stem of Eulophia is greenish yellow and that of Geodorum is green in color. Leaves are membranous; leaf surface is smooth in Geodorum whereas it is rough in Eulophia. Both the orchids have fibrous underground corms. In Eulophia, the corm shape is ovoid while, in Geodorum it is sub-globose. Size of tuber in Eulophia nuda is 3.5-4 cm ( Figure 2); in Geodorum densiflorum around 3-3.5 cm (Figure 1).

B. Microscopic Characters
For studying micro characters of Eulophia nuda and Geodorum densiflorum, thin sections of corm, roots, and leaf were taken.
Corm: The section of both the orchids shows epidermis with multilayered velamen tissue ( Figure 3A). In ground tissue, oxalate crystals, mucilage ducts, starch grains, oil granules, raphides, and scattered conjoint closed vascular bundles are present (Figure 3 A, B, C, D, F, G, H).
Leaf: It shows thin layer epidermis surrounding mesophyll tissue containing oil granules, raphides, and closed vascular bundles (Figure 3 E, I, J). Xylem and phloem are enclosed by sclerenchymatous bundle sheath cells (Figure 3 K).

Stomatal Index
Stomatal distributions on upper and lower leaf surface were calculated through stomatal index. The stomatal index is calculated by following formula: Stomatal Index = No. of stomata per mm 2 x 100 ÷ No. of stomata per mm 2 + No. of epidermal cells per mm 2 . According to the data presented in table 1 the lower leaf surface of Eulophia nuda shows 30.95% stomatal index and that of Geodorum densiflorum shows 29.26%. Whereas the upper leaf surface of both the orchids show zero stomatal index which indicates absence of stomata inspite of leaf being isobilateral.

Preliminary Phytochemical Analysis
The preliminary chemical tests of the leaf powder were carried out with three different solvents. Flavonoids, proteins, tannins, carbohydrates, phenols, quinones, terpenoids and steroids were present in all the three extracts of both the orchids. While saponins and anthocyanins were confirmed from methanolic and water extract of both the orchids; volatile oils from petroleum ether extract only of both the orchids. Few chemicals were completely absent in all the extracts like cardiac glycosides, anthrene glycosides, and coumarins ( Table 2).

DPPH Free Radical Scavenging Assay
Free radical scavenging activity of petroleum ether and methanolic extracts of Eulophia nuda and Geodorum densiflorum leaves were measured by 1, 1-diphenyl-2-picryl hydrazyl (DPPH). Ascorbic acid was used as the standard reference compound. Lower absorbance of the sample extract indicated higher free radical activity (Table 3, 4). Here control absorbance was of DPPH (0.632).

Conclusions
In the present investigation, a detailed macro-micromorphological characteristics were studied, which revealed the difference in appearance of corm, stem, leaves in both the orchids. Micro-morphology revealed the internal arrangement of cells and presence of various substances like oil granules, oxalate crystals, starch grains, mucilage ducts. Transverse section of corm shows the presence of scattered closed vascular bundles enclosed by sclerenchymatous bundle sheath cells. The preliminary phytochemical study of leaf powder using three different solvent systems revealed the presence of flavonoids, terpenoids/steroids, proteins, tannins, carbohydrates, phenols, and quinones in all the three solvent systems. While volatile oils were observed only in petroleum ether extract; anthocyanins and saponins were observed in methanol and distilled water extracts. The antioxidant activity was analysed by DPPH free radical scavenging assay with ascorbic acid as standard compound which showed better scavenging % in Eulophia nuda than Geodorum densiflorum in both petroleum ether and methanol extracts.