Desmodium oojeinense (Roxb.) H.Ohashi, (Syn. Ougeinia oojeinensis (Roxb.) Hochr.)is a tree belonging to family Fabaceae. It is native of India and also found in Nepal. Bark is astringent, acrid, cooling, sadorrific, depurative, stypic and rejuvenating. Bark is reported to posseses antiinflamatory, antispasmodic, hypoglycemic and hypolipidemic activity and useful in the management of diabetes[4-6]. It is used to treat diarrhoea, dysentery and used as fish poison and febrifuge[7-10]. It is also useful in anaemia, leucoderma, ulcer and biliousness. Methanolic extract of bark contains alkaloids, terpenoids, flavonoids, glycosides, tannins, saponins, proteins and carbohydrates.
Pharmacognostic and phyto chemical evaluation of bark has been reported but the detailed study was needed to standardize the bark drug as per the standards laid down by Ayurvedic Pharmacopoeaia of India and guidelines provided by WHO for medicinal plants.
2. Materials and methods
2.1 Plant material
Stem bark and flowering twigs of D. oojeinense were collected from the plant growing in the campus of Abasaheb Garware, Art and Science College, Pune, Maharashtra, India.
2.2 Identification and authentication of plant material
Plant material was identified and authenticated with the help of the Flora. Plant material was also compared with the herbarium speciemen available in the Botanical Survey of India, Western Circle, Pune.
2.3 Preparation of herbarium
Herbarium of the plant speciemen were prepared and deposited in the herbarium section of the RAIFR, Pune with voucher speciemen number 4526/2014.
2.4 Preservation of wet sample
Freshly collected and thoroughly washed bark pieces were kept in a glass bottle containing a mixture of formalin: glacial acetic acid: 70% ethyl alcohol [10:5:85].
2.5 Powder preparation
Shade dried, stem bark pieces were made in to powder using grinding mill; passed through #60 sieve and kept in airtight container for further analysis.
2.6 Macroscopic characterization
Macroscopic characters like fracture, shape, size,colour, taste, odour of bark and powder were determined by naked eyes.
2.7 Microscopic characterization
Free hand transverse sections (TS) of bark were taken and stained with Safranin and Phloroglucinol, followed by Hydrochloric acid. Micro photographs were snapped with the help of Deno Capture 2.0 version 1.4.2.D, the versetile digital microscope.
2.8 Histochemical and fluorescense analysis
Dried bark powder was used for the analysis of histochemical, physico-chemical aspects such as behaviour of powder, fluorescense analysis.
2.9 Determination of physicochemical parameters
Physicochemical parameters namely, loss on drying, ash value, acid insoluble ash, water soluble ash, water soluble extractive, alcohol soluble extractive were performed as per the standard procedures.
2.10 Preliminary qualitative analysis
Preliminary qualitative tests were performed by following standard methods.
2.11 Thin layer chromatography
Thin layer chromatography of aqueous and methanolic extract of D. oojeinense - stem bark was developed using solvent system toluene: ethyl acetate: acetic acid: methanol (5:3:1:1) which was saturated for 45 minute in CAMAG twin trough chamber. Both extractswere applied manually on TLC Silica gel 60 F254 Aluminum coated plate and run up to 8 cm. Plates were observed under day light, ultra violet light at 254 nm and 366nm and subsequently derivatized with Iodine vapour and anisaldehyde-sulphuric acid. Developed band colours and retention factor (Rf) were recorded. Photo documentation was done with the help of digital SLR Canon camera.
3. Results and discussion
3.1 Organoleptic Character of Powder
Stem bark powder is light brown in colour, coarse, fibrous, odourless, initially slightly sweet and then slightly astringent.
Mature pieces of bark are flat or slightly curved in shape, 5 to 6 cm in length, 2 to 2.5 cm in width and 4 to 8 mm in thickness. Outer surface of bark is powdery, slightly rough, and creamy to light brown in colour. There are small patches, oozing brown resin. Powdery layer alternating with rough surface transversely up to rhytidome; occasionally longitudinally cracked. Inner surface rough, yellowish brown, due to oozing of resin, splintery fractured (Figure 1).
Diagrammatically,transverse section of bark showed a rhytidome outside which occupies half area of entire bark with scattered groups of fibres, resin ducts and biseriate medullary rays. Rhytidome alternating with lignified irregular cells of powdery layer followed by cork. The innermost layer is phloem traverse with groups of fibres, resin duct, biseriate medullary rays and patches of compressed phloem (Figure 2.1).
Detailed T.S. of bark shows outer region of rhytidome composed of 6 to 7 rows of rectangular cells of cork, followed by 2 to 3 rows of groups of stone cells and sclerides. Pentagonal, hexagonal or polygonal cells of rhytidome traverse with groups of thin walled fibres, compressed phloem, groups of empty resin duct and biseriate medullary rays. Parenchymatous cells and medullary rays embedded with starch grains and prismatic crystals of calcium oxalate. Rhytidome alternating with 6 to 8 layers of irregular, lignified cells of powdery layer, followed by 6-7 layers of rectangular cells of cork. Below this group 2-5 layers of of stone cells were observed. Secondary phloem made up of somewhat polygonal to oval shaped cells traverse with groups of thin walled fibres, groups of resin duct, compressed cells of phloem and biseriate medullary rays. Phloem parenchyma and medullary rays embedded with simple, compound, oval starch grains and prismatic crystals of calcium oxalate (Figure 2.2 and 3).
3.4 Powder Microscopy
3.5 Histochemical and fluorescense analysis
Powder drug was analysed for histochemical tests for detection of lignin, aleurone grains, oils, mucilage and crystals. Analysis revealed that bark contains lignin, resins, oils, starch and calcium oxalate crystals which are the unique characters of bark (Table 1).
Powder drug was treated with different chemical reagents and observed in day light and ultra-violet light and inferences was recorded in Table 2. Drug colour changed after reacting with chemical and observed under 254 and 366 nm which is would be characteristics for particular drug and would be beneficial to identify the bark of D. oojeinense.
Behaviour of drug with different chemical reagent showed that drug immidiately move down in Glacial acetic acid, 5% FeCl3 solutions whereas powder colour changed in concentrated H2SO4, HNO3 and HCl solutions. Details of observations are exhibited in Table 3.
3.6 Determination of physicochemical parameters
Results obtained from physico- chemical parameters such as, loss on drying, total ash, acid insoluble ash, water soluble ash, water and alcohol soluble extracts are depicted in Table 4.
3.7 Preliminary qualitative analysis
Preliminary analysis revealed that bark contain, carbohydrates, saponins, anthroquinone, glycoside, tanins, phenol and organic acids. Details of the result given in Table 5.
3.8 Thin layer chromatography
TLC of methanolic extract developed 7 spots in 254 nm, 1 spot in 366 nm, 8 spot in anisaldehyde-sulphuric acid and 12 spots in Iodine. Whereas, single spot developed in aqueous extract at 0.72 Rf observed in 254 nm, and after derivatized with anisaldehyde-sulphuric acid and Iodine. Details are shown in Table 6 & 7 and Figure 5.
|Loss on drying||10.98|
|Acid insoluble ash||0.06|
|Water soluble ash||2.12|
|Water soluble extractive||26.43|
|Alcohol soluble extractive||10.13|
|Reducing sugar||a) Fehling’s test||+||+|
|b) Benedict’s test||+||+|
|Pentose sugar||Phloroglucinol Reag. test||+||+|
|Hexose sugar||a)Tollen’s Phloroglucinol test||+||+|
|b)Cobalt Chloride test||+||+|
|Protein||a) Biuret test||＿||＿|
|Amino acid||a)Ninhydrin test||＿||＿|
|b)Test for cysteine||＿||＿|
|Cardiac Glycosides||a) Legal test||＿||＿|
|b) Keller-Killiani test||＿||＿|
|Anthroquinone Glycoside||a)Borntrager’s test||＿||+|
|b) Modified Borntrager’s test||＿||+|
|b) Lead Acetate solution test||+||--|
|Coumarin Glycoside||a)Aromatic odour test||＿||＿|
|b) Fluorescence test||--||＿|
|Flavonoids||a) Shinoda test||--||＿|
|b) Lead acetate test||＿||--|
|Alkaloids||a) Dragendorff’s test||＿||＿|
|b) Mayer’s test||＿||＿|
|c) Wagner’s test||＿||＿|
|Tanins||a) Lead acetate solution test||+||--|
|b) Gelatine sol. test||＿||--|
|Phenol||a) Neutral Fecl3test||--||+|
|b) Indophenol reaction||＿||＿|
|Organic acids*||Oxalic acid (In NH4OH Extrat)||+|
|254 nm||366 nm||Anisaldehyde sulphuric acid||Iodine|
|Rf value||Band colour||Rf value||Band colour||Rf value||Band colour||Rf value||Band colour|
|0.72||Black||-||-||0.72||Navy blue||0.72||Brownish yellow|