Pharmacognostic standardization with various plant parts of Desmostachya bipinnata

Abstract

Context: Desmostachya bipinnata (L.) Stapf [Gramineae (Poaceae)] has been traditionally used to treat various disorders such as asthma, kidney stone, diarrhea, wound healing, etc.

Objective: The present study involves pharmacognostic, botanical, and preliminary phytochemical examination of various plant parts and powders of D. bipinnata.

Materials and methods: Leaves, stem, roots, underground and aerial part powders were microscopically examined. Pharmacognostic standardization parameters were determined as per the WHO guidelines. Parameters, including extractive value in different solvents, ash values, and loss on drying, were determined. Preliminary phytochemical studies, fluorescence analysis, and chromatographic profiling were performed for the correct identification of this crude drug and some of its phytoconstituents.

Results: Microscopical studies showed various characteristic features including, collateral vascular bundle, bundle sheath cells, and bulliform cells in leaf, conjoint, collateral and closed vascular bundles, and sclerenchymatous sheath in stem; and silica bodies in root. Phytochemical screening and chromatographic profile of aerial plant parts revealed the presence of alkaloids, tannins, flavonoids, steroids, glycosides, and coumarins. Underground plants parts indicated the presence of glycosides, steroids, flavonoids, coumarins, and alkaloids.

Discussion and conclusion: The results of the performed studies are helpful in correct identification, characterisation of D. bipinnata. Preliminary phytochemical studies and chromatographic profiling may be helpful in further isolation and purification of lead compounds from different extracts.

• Chromatography
• extraction
• microscopy
• phytoconstituent

Introduction

Desmostachya bipinnata (L.) Stapf (syn: Eragrostis cynosuroides) Gramineae (Poaceae), is commonly known as sacrificial grass, kusha (Khare, 2007), drabh (Qureshi et al., 2010), and dab (Praveen et al., 2007). D. bipinnata leaf paste is used to cure cuts and wounds (Katewa & Jain, 2006). D. bipinnata roots are used to treat rheumatism (Ahmad et al., 2010), carbuncles, piles, cholera, dysuria, (Qureshi et al., 2010) diuretic, galactagogue, astringent (Khare, 2007), dysentery, leucorrhea, and wounds (Praveen et al., 2007).

Flavonoids, viz., kaempferol, quercetin, quercetin-3-glucoside, trycin, and trycin-7-glucoside, have been isolated from D. bipinnata (Awaad et al., 2008). Trycin and trycin-7-glucoside showed promising antiulcerogenic activity (Awaad et al., 2008). D. bipinnata oil contains camphene (16.79%), isobornyl acetate (9.92%), tricyclene (4.30%), (±) trans-2,6-γ-irone (2.21%), caryophyllene diepoxide (12.29%), β-eudesmol (11.16%) eseroline (25.15%), and calarene (3.48%) (Kumar et al., 2010). Diphenyliodinium bromide, 1-limenone, 2-cyclohexene-1-one and 8-nitro-12-tridecanolide were also found in small amounts in D. bipinnata oil (Kumar et al., 2010). D. bipinnata oil showed good activity against different test microorganisms. Quercetin, isolated from D. bipinnata, is a useful chemoprotective agent in Helicobacter pylori infections (Ramadan & Safwat, 2009). Analgesic, antipyretic, and anti-inflammatory effects of various whole plant extracts in solvents, viz., petroleum ether, benzene chloroform, ethanol, and water, were investigated in albino rats (Panda et al., 2009). The alcoholic and aqueous root extracts of D. bipinnata were found effective in treating castor oil and charcoal meal–induced diarrhea (Hegde et al., 2010).

The present study deals with determination of several pharmacognostic parameters, preliminary phytochemical tests, and chromatographic profiles of the D. bipinnata.

Materials and methods

Plant material: collection, authentication

Whole plants of D. bipinnata were collected in September 2009 from Chirawa, district Jhunjhunu, Rajasthan, India. The collected plant was authenticated by Dr. R. P. Pandey from Botanical Survey of India, Jodhpur, India. A voucher specimen, JNU/PH/2010/D_b D_2, was deposited in the herbarium of Jodhpur National University, Jodhpur, India.

Microscopy, powder analysis and standardization parameters

Hand sections of fresh root, stem and leaves were used for microscopical characterization. Sections were treated with chloral hydrate to remove chlorophyll. Saffranin, phloroglucinol HCl, iodine, ruthenium, sudan red, sulphuric acid, etc., were used to stain different sections (Khandelwal, 2005).

Different plant parts, viz., roots, stems and leaves, were dried in shade for 1 month. Dried plant parts were crushed and ground using an electric mixer-grinder and screened using BSS standard sieve no. 22 of average aperture size 710 μm. Shade-dried, coarsely powdered drug was used for powder analysis, fluorescence studies (Mukherjee, 2003), and preparation of various extracts. Total ash value, acid- insoluble ash value, sulphated ash value, water soluble ash value, loss on drying, and extractable matter in petroleum ether, chloroform, ethyl acetate, ethanol, hydro alcoholic (1:1) and water were estimated, as per standard procedures given in World Health Organization (WHO) guidelines (WHO, 1998).

Preparation of plant extracts

The powdered crude drug, aerial and underground parts (10 g) were extracted in a Soxhlet extractor with petroleum ether, chloroform, ethanol, ethanol (50%) and water separately to extract non-polar and polar compounds. The obtained extracts were filtered through Whatman filter paper, concentrated by evaporation on water bath, and residual moisture was removed in dessiccator.

Preliminary phytochemical studies

Phytochemical analysis, from aerial and underground parts, was performed for various phytoconstituents, like alkaloids, glycosides, terpenes, coumarins, flavonoids, carbohydrates, proteins, volatile oils, saponins, etc., with the help of standard reagents and solutions (Khandelwal, 2005). Chromatographic profiles were generated with several solvent systems for different phytoconstituents (Egon, 2007; Wagner & Blat, 2007).

Results and discussion

Microscopy

Leaf

Microscopic characters of leaves are presented in Figure 1. Mid-rib collateral vascular bundle consists of two metaxylem and a phloem in between them (Figure 1c). This type of vascular bundle is also present in Cynodon dactylon L. Pers. (Poaceae) leaves. Vascular bundle is covered with continuous layer of sclerenchymatous cells (Figure 1c). Thick wall, small cells are present on lower portion of the leaf, while cells on upper portion are relatively larger with thin wall (Figure 1e).

Figure 1. Microscopical characters of Desmostachya bipinnata leaf. (a) T.S. of leaf lamina, (b) T.S. of leaf margin, bundle sheath cells, (c) vascular bundles, phloem, xylem, sclerenchymatous cells, (d) cuticle and bulliform cells and (e) T.S. of mid rib with lamina.

Leaf lamina section and marginal section

Lower epidermis of leaf lamina is smooth and comprises circular cells with thick cuticle, while upper epidermis is not so smooth and comprises circular cells with papillate walls (Figure 1a). In marginal section of leaf, both epidermal layers are smooth and cells are square shaped with thickening (Figure 1a). Vascular bundles are somewhat smaller than mid rib vascular bundles (Figure 1e). Leaf has furrows, where epidermal cells and mesophyll cells forms bulliform cells (Figure 1d). Xylem and pholem are less distinct and covered with 9 to 10 chloroplast bearing bundle sheath cells (Figure 1b) but vascular bundle is covered with 5 to 6 chloroplast bearing bundle sheath cells in Cynodon dactylon leaves.

Stem

A continuous layer of epidermis is covered with cuticle. Ground tissue, present on the inner side, consists of parenchymatous cells. Epidermis and parenchymatous cells (2–3 layers) are lignified followed by parenchymatous cells, which shows the presence of starch grains and oil glands (Figure 2a). Vascular bundles are conjoint, collateral, and closed (Figure 2e). Different size vascular bundles are scattered in ground tissue (Figure 2c), covered with sclerenchymatous sheath (Figure 2f), and shows lignin deposition (Figure 2b). Same type of vascular bundle scattered in ground disuse and lignin depositon is also observed in Saccharum spontaneum Linn (Poaceae) stem. Roots have been found emerging from stem node (Figure 2d). Saccharum spontaneum and D. bipinnata belongs to the family Poaceae (Suresh Kumar et al., 2009).

Figure 2. Microscopical characters of Desmostachya bipinnata stem. (a) Starch with oil glands, (b) cortical region, sclerenchymatous sheath followed by vascular bundles, (c) scattered vascular bundles, (d) emerging root from node, (e) meta xylem and pholeum and (f) sclerenchymatous sheath.

Root

A continuous layer of epidermis was observed with thin wall, semicircular shape epidermal cells, which have projections on outer walls (Figure 3a). Outer cortex consists of three to four layers of sclerenchymatous cells (Figure 3b). Inner cortex comprises of aerenchymatous cells, consisting of radially elongated wide air chambers with thin partition filaments (Figure 3d). These aerenchymatous cells with elongated wide air chambers are also present in Imperata cylindrica Linn (Poaceae). Spherical cells have been observed in these partition filaments (Figure 3d). The stele is covered by endodermis and deposition of silica bodies at inner side (Figure 3c). Circular, thin wall meta xylem varies in number from 9 to 10 (Figure 3c). These thin walled meta xylem present in Imperata cylindrica varies in number from 5 to 7. In between, two meta xylem, 2 to 3 phloem are present. Pith tissue consists of sclerenchyamatous cells also present in Imperata cylindrica.

Figure 3. Microscopical characters of Desmostachya bipinnata root. (a) T.S. of root, (b) T.S. showing steler region having sclerenchymatous cells, (c) endodermis, metaxylem and phloem and (d) epidermis, trichomes and aerenchymatous cells.

Microchemical tests, applied to leaves, rhizomes, and roots, for examining the presence of starch, lignin, calcium oxalate, etc. are given in Table 1. Starch and lignin were present in leaf and stem, while root contained only lignin. Additionally, oil glands were observed in stem.

Table 1. Microchemical tests performed with various plant parts of Desmostachya bipinnata (L.) Stapf.

Part of plant	Reagents used	Name of phyto-constituent	Observation/Inference	Histological zone
Leaf	Safranin	–	Red	Whole section
	Iodine solution	Starch	Blue	Bundle sheath cells
	Phloroglucinol and dilute  hydrochloric acid	Lignin	Pink	Vascular bundles
	Ruthenium red solution	Mucilage	No red color	–
	Sudan red solution	Oil globules	No red color	–
	Concentrated hydrochloric acid	Crystals	No change	–
Root	Safranin	–	Red	Whole section
	Iodine solution	Starch	No color	–
	Phloroglucinol and dilute  Hydrochloric acid	Lignin	Pink	Vascular bundles
	Ruthenium red solution	Mucilage	No red color	–
	Sudan red solution	Oil globules	No red color	–
	Concentrated hydrochloric acid	Crystals	No change	–
Stem	Safranin	–	Red	Whole section
	Iodine solution	Starch	Blue	Outer parenchymatous cells
	Phloroglucinol and dilute  hydrochloric acid	Lignin	Pink	Sclerenchymatous sheath,  oil glands and epidermis
	Ruthenium red solution	Mucilage	No red color	–
	Sudan red solution	Oil globules	Red color	Oil glands
	Concentrated hydrochloric acid	Crystals	No change	–

Powder analysis

Aerial plant parts powder showed the presence of parenchymatous cells (58.8 µm length and 14.7 µm width) (Figure 4c and d), vessels (223.65 µm length and 50.4 µm width) both simple and lignified (Figure 4a and d), trichomes (186.69 µm length and 14.7 µm width), starch grains (27.93 µm length and 22.86 µm width), phloem (Figure 4b), and fibers (306.02 µm length and 14.7 µm width) (Figure 4c and d). Underground plant part powder analysis revealed the presence of trichomes (235.08 µm length and 17.35 µm width) (Figure 5a), vessels (241.68 µm length and 35.35 µm width) (Figure 5b), fibers (339.96 µm length and 17.27 µm width) (Figure 5c), starch grains (37.57 µm length and 32.46 µm width) (Figure 5d), sclerenchymatous cells (32.19 µm length and 15.15 µm width) (Figure 5b), and endodermal cells (Figure 5b). Measument of these powder characteristics are summarized in Table 2.

Figure 4. Powder characteristics of aerial parts of Desmostachya bipinnata. (a) Lignified vessel, (b) phloem, (c) fiber, parenchymatous cells and (d) fibers, epidermal cells, parenchymatous cells and vessels.

Figure 5. Powder characteristics of underground parts of Desmostachya bipinnata. (a) Endodermal cells, trichomes, (b) sclerenchymatous cells, (c) fiber and (d) starch grains, vessels.

Table 2. Microscopic measurements for several powder characteristics in Desmostachya bipinnata (L.) Stapf.

		Average size (µm)
Plant part	Parameters	Length	Width
Aerial parts	Fiber	306.02	14.7
	Trichome	186.69	14.7
	Vessel	223.65	50.4
	Starch grains	27.93	22.86
	Cell	58.8	14.7
Underground parts	Fibre	339.96	17.27
	Trichome	235.08	17.35
	Vessel	241.68	35.35
	Starch grains	37.57	32.46
	Cell	32.19	15.15

Fluorescence studies with different solvents and powder (aerial and underground parts) were conducted (Table 3). In aerial parts, at long UV (365 nm), greenish white fluorescence was observed with water, sulphuric acid, concentrated nitric acid, acetic acid, and ammonia; whitish fluorescence was observed with methanol, ethanol, ethanolic sodium hydroxide, methanolic sodium hydroxide, concentrated nitric acid, chloroform, petroleum ether, sodium hydroxide, and potassium hydroxide. At short UV (254 nm), greenish fluorescence was observed with ethanolic potassium hydroxide, methanolic potassium hydroxide, acetic acid, and ammonia. In underground plant parts, at long UV (365 nm), greenish fluorescence was observed with water and concentrated hydrochloric acid; whitish fluorescence was observed with methanol, ethanol, acetic acid, ammonia, chloroform, petroleum ether, sodium hydroxide, and potassium hydroxide. At short UV (254 nm), greenish fluorescence was observed with ethanolic sodium hydroxide, ethanolic potassium hydroxide, methanolic potassium hydroxide, ethanolic potassium hydroxide, concentrated hydrochloric acid, petroleum ether, sodium hydroxide, potassium hydroxide, acetic acid, and ammonia.

Table 3. Fluorescence studies with different plant parts of Desmostachya bipinnata (L.) Stapf.

Drug + solvent	Plant part	Visible	Short UV (254 nm)	Long UV (365 nm)
Drug	Aerial Parts	Green	Green	White Fluorescence
	Underground Parts	Brown	Brown	Brownish White Fluorescence
Drug + Water	Aerial Parts	Green	Greenish Brown	Greenish White Fluorescence
	Underground Parts	Light Brown	Light Brown	Greenish Fluorescence
Drug + Methanol	Aerial Parts	Greenish Brown	Dark Green	Whitish Flourescence
	Underground Parts	Brown	Brown	Whitish Flourescence
Drug + Ethanol	Aerial Parts	Greenish Brown	Dark Green	Whitish Flourescence
	Underground Parts	Brown	Brown	Whitish Flourescence
Drug + Ethanolic sodium hydroxide solution	Aerial Parts	Greenish Brown	Greenish Brown	Whitish Flourescence
	Underground Parts	Brown	Greenish Flourescence	Greenish White Fluorescence
Drug + Methanolic sodium hydroxide solution	Aerial Parts	Greenish Brown	Greenish Brown	Whitish Flourescence
	Underground Parts	Brown	Greenish Flourescence	Greenish White Fluorescence
Drug + Ethanolic potassium hydroxide solution	Aerial Parts	Greenish Brown	Greenish Flourescence	Green
	Underground Parts	Brown	Greenish Flourescence	Greenish White Fluorescence
Drug + Methanolic potassium hydroxide solution	Aerial Parts	Greenish Brown	Greenish Flourescence	Green
	Underground Parts	Brown	Greenish Flourescence	Greenish White Fluorescence
Drug + Conc. hydrochloric acid	Aerial Parts	Brownish Green	Brownish Green	Greenish White Fluorescence
	Underground Parts	Light Brown	Greenish Flourescence	Greenish Fluorescence
Drug + Conc. nitric acid	Aerial Parts	Greenish Brown	Greenish Brown	Whitish Flourescence
	Underground Parts	Light Brown	Light Brown	Brownish White Fluorescence
Drug + Conc. sulphuric acid	Aerial Parts	Dark Brown	Black	Greenish White Fluorescence
	Underground Parts	Dark Brown	Navy Blue	Black
Drug + Petroleum ether	Aerial Parts	Brown	Dark Green	Whitish Flourescence
	Underground Parts	Brown	Greenish Flourescence	Whitish Flourescence
Drug + Chloroform	Aerial Parts	Brown	Dark Green	Whitish Flourescence
	Underground Parts	Brown	Brown	Whitish Flourescence
Drug + Sodium hydroxide solution	Aerial Parts	Greenish Brown	Dark Green	Whitish Flourescence
	Underground Parts	Brown	Greenish Flourescence	Whitish Flourescence
Drug + Potassium hydroxide solution	Aerial Parts	Greenish Brown	Dark Green	Whitish Flourescence
	Underground Parts	Brown	Greenish Flourescence	Whitish Flourescence
Drug + Acetic acid	Aerial Parts	Greenish Brown	Greenish Flourescence	Whitish Green Flourescence
	Underground Parts	Brown	Greenish Flourescence	Whitish Flourescence
Drug + Ammonia	Aerial Parts	Greenish Brown	Greenish Flourescence	Whitish Green Flourescence
	Underground Parts	Brown	Greenish Flourescence	Whitish Flourescence

W.H.O standardization parameters

Different ash values, viz., total ash value, an acid-insoluble ash value, a water-soluble ash value, and sulphated ash value were determined to find the inorganic content in each sample and results are presented in Table 4. Crude fiber content, foreign organic matter, swelling index, foaming index, etc., were also estimated for aerial and underground parts (Table 4). Solvents of different polarity ranges were used to determine the extractive value and extract yield in the petroleum ether, ethyl acetate, chloroform, ethanol, ethanol (50%), and water for aerial and underground parts (Tables 4 and 5). Further phytochemical screening (Table 6) and chromatographic profile (Table 7) of aerial plant parts revealed the presence of alkaloids, tannins, flavonoids, steroids, glycosides, and coumarins in hydro-alcoholic and alcoholic extracts; flavonoids and glycosides in water extract; alkaloids, flavonoids, and steroids in chloroform extract; while, steroids were found in ethyl acetate extract. Phytochemical screening of underground plants parts indicated the presence of glycosides and alkaloids in water extract; glycosides, flavonoids, coumarins, and alkaloids in hydroalcoholic extract; glycosides, flavonoids, steroids, coumarins, and alkaloids in alcoholic extract; coumarins and alkaloids in chloroform extract; flavonoids, coumarins, and alkaloids in ethyl acetate extract.

Table 4. Estimated standardization parameters of Desmostachya bipinnata (L.) Stapf as per WHO guidelines.

Parameter	Aerial parts	Underground parts
LOD	7.25 ± 0.25%	7.00 ± 0.66%
Total ash values	6.91 ± 0.14%	9.25 ± 0.86%
Acid insoluble ash	4.60 ± 0.10%	3.83 ± 0.52%
Water soluble ash	0.91 ± 0.14%	3.06 ± 0.60%
Sulphated ash	4.90 ± 0.17%	1.58 ± 0.14%
Crude fiber content	40.66 ± 1.6%	27.9 ± 1.35%
Foreign organic matter	Not found	Not found
Swelling index	8 ml	6 ml
Foaming index	<100	<100
Extractive value in water	8.53 ± 0.61%	10.06 ± 1.02%
Extractive value in  hydroalcholic solvent	10.00 ± 0.80%	10.40 ± 0.40%
Extractive value in alcohol	14.06 ± 1.02%	3.73 ± 0.61%
Extractive value in chloroform	1.67 ± 0.11%	1.66 ± 0.11%
Extractive value in petroleum ether	1.60 ± 0.40%	0.60 ± 0.20%
Extractive value in ethyl acetate	1.73 ± 0.23%	2.26 ± 0.23%

Table 5. Extractive yields estimated for various plant parts of Desmostachya bipinnata (L.) Stapf.

Extractive yield in solvent	Aerial parts	Underground parts
Water	18.36 ± 1.5	8.33 ± 0.92
Hydroalcoholic	16.06 ± 0.32	10.45 ± 0.19
Alcohol	11.01 ± 0.79	5.21 ± 0.07
Chloroform	2.83 ± 0.11	1.54 ± 0.06
Ethyl acetate	3.73 ± 0.20	2.38 ± 0.16

Table 6. Phytochemical tests performed with various plant parts of Desmostachya bipinnata (L.) Stapf.

			Observation
Phyto-chemicals	Chemical test	Plant Part	Water extract	Hydro-alcoholic extract	Alcoholic extract	Chloroform extract	Petroleum ether extract	Ethyl acetate extract
Carbohydrates	Molisch’s test	Aerial Parts	+	+	+	+	+	−
		Underground Parts	+	+	+	−	−	+
	Benedict’s test	Aerial Parts	+	+	+	+	+	−
		Underground Parts	+	+	−	−	−	−
	Starch	Aerial Parts	−	−	−	−	−	−
		Underground Parts	−	−	−	−	−	−
Alkaloids	Dragendroff’s test	Aerial Parts	−	+	+	+	+	−
		Underground Parts	+	+	+	+	+	+
	Mayer’s test	Aerial Parts	−	+	+	+	+	−
		Underground Parts	−	−	−	+	+	−
	Marqui’s test	Aerial Parts	−	+	+	+	−	−
		Underground Parts	−	−	+	+	+	−
Steroids	Salkowski test	Aerial Parts	−	+	+	+	+	−
		Underground Parts	−	+	+	+	+	−
Proteins	Biuret test	Aerial Parts	−	−	−	−	−	−
		Underground Parts	−	−	−	−	−	−
	Millon’s test	Aerial Parts	−	−	−	−	−	−
		Underground Parts	−	−	−	−	−	−
Glycosides	Borntager	Aerial Parts	−	−	−	−	−	−
		Underground Parts	−	−	−	−	−	−
	Mod. Borntager	Aerial Parts	−	−	−	−	−	−
		Underground Parts	+	+	+	−	−	−
	Killer killani	Aerial Parts	+	+	+	−	−	−
		Underground Parts	+	+	+	−	−	+
	Legal	Aerial Parts	+	+	+	−	−	−
		Underground Parts	−	−	−	−	−	−
Fats and oils	Filter paper spot test	Aerial Parts	−	−	−	−	−	−
		Underground Parts	−	−	−	−	−	−
Saponins	Foam test	Aerial Parts	+	+	−	−	−	−
		Underground Parts	+	+	−	−	−	−
Tannins and phenolic compounds	5% FeCl3	Aerial Parts	+	+	+	−	−	−
		Underground Parts	−	−	−	−	−	−
	Lead acetate	Aerial Parts	+	+	+	−	+	−
		Underground Parts	−	−	−	−	−	−
	Gelatin	Aerial Parts	+	+	+	−	+	−
		Underground Parts	−	−	−	−	−	−
Flavanoids	Shinoda	Aerial Parts	+	+	+	+	+	−
		Underground Parts	−	+	+	−	+	+
Coumarins	FeCl3+ HNO3	Aerial Parts	−	+	+	−	−	−
		Underground Parts	−	+	+	+	+	+
	NaOH + UV	Aerial Parts	−	+	+	−	−	−
		Underground Parts	−	+	+	+	+	+
Volatile oils	Sudan III	Aerial Parts	+	+	+	+	+	+
		Underground Parts	−	+	+	−	+	−

Table 7. Chromatographic profile of various plant parts of Desmostachya bipinnata.

Phytoconstituents	Detecting reagent	Solvent system	Water	Hydroalcoholic	Alcohol	Chloroform	Ethyl acetate
(a) Desmostachya bipinnata aerial plant parts
Alkaloids	Dragendroff’s reagent	Ethanol:Formic acid (8:2)	–	0.6	0.63	0.62	–
Tannins	Ferric chloride	Toluene:Chloroform:   Acetone (40:25:35)	–	0.7	0.7	–	–
Flavonoids	Iodine and short UV 254	Chloroform:Methanol (9:1)	0.3	0.32	0.10, 0.30	0.10, 0.30, 0.36	–
Steroids	Iodine and long UV 364	Chloroform:Methanol:   Water (65:50:10)	–	0.49	0.49	0.47	0.44
Glycosides	Iodine and long UV 364	Ethylacetate:Formic acid:   Glacial acetic acid:   Water (100:11:11:26)	0.23	0.23	0.23	–	–
Coumarins	Iodine and UV	Butanol:Acetic acid:Water (4:1:5)	–	0.6	0.63	–	–
(b) Desmostachya bipinnata underground plant parts
Alkaloids	Dragendroff’s reagent	Ethanol:Formic acid (8:2)	0.66	0.67	0.64	0.7	0.6
Tannins	Ferric chloride	Toluene:Chloroform:   Acetone (40:25:35)	–	–	–	–	–
Flavonoids	Iodine and short UV 254	Chloroform:Methanol (9:1)	–	0.85	0.12, 0.41, 0.46,  0.54, 0.85	–	0.37, 0.53, 0.85
Steroids	Iodine and long UV 364	Chloroform:Methanol:   Water (65:50:10)	–	–	0.48, 0.15	–	–
Glycosides	Iodine and long UV 364	Ethylacetate:Formic acid:   Glacial acetic acid:   Water (100:11:11:26)	0.36, 0.40	0.27, 0.32	0.24, 0.31	–	–
Coumarins	Iodine and UV	Butanol:Acetic acid:Water (4:1:5)	–	0.79	0.79	0.79	0.79

Conclusion

Pharmacognostical and preliminary phytochemical investigations were performed on various plant parts of D. bipinnata. The results of these investigations are expected to help in correct identification, characterization and other relevant findings. Standard profile of D. bipinnata may be prepared from identified characters and characterized parameters. The outcomes of preliminary phytochemical investigations may guide isolation and purification of lead compounds responsible for traditional therapeutic claims.

Declaration of interest

The authors declare no conflict of interests. The authors alone are responsible for the content and writing of this article.

Acknowledgements

The authors express their thanks to Dr. R. P. Pandey from Botanical Survey of India, Jodhpur, India, for authentication of plant material.