Microscopic and Phytochemical analysis of Desmodium velutinum (Willd) DC and Desmodium gangeticum (L.) DC. Panchanga powder

Jigyasa Pathak¹, Aswathi M. P.², B. R. Patel³, Harisha C. R.⁴, Shukla Vinay J.⁵

^1,2Ph. D. Scholar, Dept. of Dravyaguna, IPGT & RA, Jamnagar.

³Associate Professor, Dept. of Dravyaguna, IPGT & RA, Gujarat Ayurved University, Jamnagar.

⁴Head, Pharmacognosy Laboratory, IPGT & RA, Jamnagar.

⁵Head, Pharmaceutical Chemistry Laboratory, IPGT & RA, Jamnagar.

*Corresponding Author E-mail: jigyasapathak30@gmail.com

ABSTRACT:

Background: Desmodium velutinum (Fabaceae) is one of the wild edible plants used traditionally to treat diarrhoea, haematuria, infertility and also externally used to treat scorpion bites. Though various parts of the plant have been used for therapeutic purpose, panchanga of the plant has not been studied for its Pharmacognostical and phytochemical characteristics in detail, following scientific parameters. Hence in the present study Desmodium velutinum whole plant has been studied for its pharmacognostical and phytochemical characters and compared with commonly used species Desmodium gangeticum. Methods: Whole plant of Desmodim velutinum was collected from Paikmal, Odisha and Desmodium gangeticum was collected from Junagarh, Gujarat in the month of November 2019 following good collection practices and shade dried. Pharmacognostical study and analytical study were performed following standard pharmacopoeial procedures. Results: Whole plant (Panchanga) of former is sweetish and latter is bitter. Powder microscopy of Desmodium gangeticum showed presence of rhomboidal crystals and lignified annular and spiral vessels. Desmodium velutinum showed presence of prismatic crystals and lignified pitted vessels. Physico-chemical analysis showed higher loss on drying in Desmodium gangeticum, and higher water soluble extractive values of Desmodium velutinum. Phytochemical estimation showed the presence of Proteins, tannins, and carbohydrates in both samples. HPTLC study showed similar Rf value 0.82 and 0.94 at 254 and 0.03 and 0.41 at 366nm. Conclusion: Basing upon the analytical and microscopic similarities Desmodium velutinum may be considered as substitute of Desmodium gangeticum

KEYWORDS: Desmodium gangeticum, Desmodium velutinum, Microscopic, Phytochemical, Shalaparni.

INTRODUCTION:

Desmodium velutinum (Willd.) D.C. of family Fabaceae [1] is a popular plant which has been used as traditional medicine in India for various ailments. [2] It is a perennial plant local to tropical Africa and subtropical Asia (China, Taiwan, India, Indonesia, Laos, Malaysia, Myanmar, Sri Lanka, Thailand and Vietnam). [2] It is an erect under shrub, 4-6ft. high. Leaves are one-foliate. Leaflet two to five by one to three inches, broad-ovate, truncate or cordate and 3 nerved at the base, hispid above, densely soft-pubescent beneath; tip rounded. Petiole about four to seven inches long.

Flowers bright purple, clustered on axillary and terminal racemes. Calyx minute with linear teeth. Pod jointed, much constricted between joints. Flowering occurs in August, September and fruiting from November. [3] The plant is well known for its ethno medicinal claims and the leaves of the drug are widely used in diarrhoea, vomiting, dysentery and indigestion. [4] The decoction of leaves and roots is used for general body vitality and treating infertility [4]. This plant known as velvet leaf Desmodium has common names in India like Jagru or Lagawang (Hindi), Chitkibhoota or Latkan (Madhya pradesh), Chithamalli (Tamil), Orila (Malayalam), Lepati or Chirmi (Maratti), and Latkana (Orissa) [1] [2].

Desmodium gangeticum is a sub-erect under shrub, stem reaching one meter in height, woody slightly angular, clothed with short grey hair. Leaves alternate, petiolate, long, unifoliate, leaflets membranous or subcoriaceous. Flowers white, bracteates, pods falcate, jointed, glabrescent or clothed with minute hooked hair. [5] Different part wise uses of Desmodium gangeticum is found in practice in various tribal communities of India. Specific uses of leaf and roots are found, whereas uses of whole plant are also observed. Root is advised in whooping cough by tribal communities of Rewa district of M.P. and also used by chenchu tribal communities of A.P. Roots and Panchang (Whole plant) are used as antidote for snake bite by tribes of Bihar, A.P, various tribes of U.P., W.B., and in Rajasthan, where also used as antidote for scorpion sting by tribal communities of santhal pargana of Bihar. Tribes of W.B. claimed that victim of snake bites are cured by oral use of root paste. Leaves, roots and whole plant are used in treating fever. [6]

Though various parts of these plants are used by tribals, the Panchanga (whole plant) is not yet evaluated in a scientific way for its pharmacognostical characters and phytochemical constituent. Hence this article brings insight on pharmacognostical characters, preliminary phytochemical constituents including HPTLC and GCMS profile.

MATERIAL AND METHODS:

Plant Collection and authentication:

Whole plant of Desmodium velutinum was collected with the help of traditional practitioner from its natural habitat Gandhamardan hills, Paikmal, Odisha (Fig. 1a) and Desmodium gangeticum was collected from its natural habitat, in and around forest areas in Junagarh, Gujarat (Fig.1b) during the month of November 2019.

Figure 1: Desmodium velutinum and Desmodium gangeticum in natural habitat

Preparation of the sample:

Both the drugs were processed at the pharmacy, I.P.G.T.R.A, and panchanga powder was obtained.

Organoleptic characters:

Macroscopic observations were made with naked eyes and centimeter scale was used for measurement. The stem was washed and transverse section was taken cleared with chloral hydrate to observe the anatomy of stem with help of Quasmo binocular compound microscope.

For powder microscopy, to obtain a powder shaded dried pieces of panchanga (whole plant) stem were grounded by the mechanical grinder and sieved through 60# [7] [8]

Analytical study:

Physico-chemical and preliminary phytochemical screening:

The loss on drying, moisture, ash, extractive value and other physico-chemical constant were determined by using the association of Official Analytical Chemist [9] [10]. Preliminary phytochemical investigations such as Molisch’s test, Salkowski test, Keller-Killiani test, Flavonoid test, Dragendorff’s test and test for tannins were performed according to Harbone following standard protocol [11].

HPTLC study:

Chromatography is a powerful analytical method suitable for the separation and quantitative determination of a considerable number of compounds even from complicated matrix. [12] HPTLC was carried out with methanolic extract of the samples spotted on pre-coated Silica Gel GF254 plates by means of Camag Linomat V sample applicator. The mobile method consisted of Toluene: ethyl acetate 7.5:2.5V/V. After development, the densitometry scan was performed with a Camag T. L. C. scanner III in reflectance absorbance mode at 299nm and the control of Win cats software [12] [13]

GCMS study:

Preparation of extract for GC-MS:

The powdered sample of Desmodium velutinum panchanga (100g) were extracted with ethanol (750ml, 52h) a temperature between 50-600C by using Soxhlet extractor. The solvent was evaporated by rotary-evaporator in vacuum to obtain a viscous semi-solid mass. The crude extract was then filtered through Whatman No. 41 filter paper to obtain dust free plant crude extract and used for GC-MS analysis. [13] [14] [15]

RESULT AND DISCUSSION:

Pharmacognostical study:

The organoleptic characters of panchanga powder of Desmodium gangeticum and Desmodium velutinum are described in table 1

Table 1: Organoleptic characters of panchanga powder of Desmodium gangeticum and Desmodium velutinum

Character	*D.gangeticum*	*D.velutinum*
Colour	Dark green	Green
Odour	Characteristic	Characteristic
Taste	Bitter	Sweetish , mucilage
Touch	Coarse	Coarse

Powder microscopy:

Desmodium gangeticum:

Diagnostic powder characters of Panchanga of Desmodium gangeticum showed the presence of annular spiral vessels, simple trichome, simple and compound starch grains, rhomboidal crystals, spongy parenchyma, group of fibers, warty trichomes, group of lignified fibers, fragment of lignified spiral vessels, pitted and annular vessels (figure 2a-k)

Desmodium velutinum:

Diagnostic powder characters of Desmodium velutinum shows presence of trichomes, oil content, starch grain, Fibers, cork cells, bordered pitted vessels, group of pitted stone cells, lignified pitted vessels, lignified stone cells, lignified group of fibers, prismatic crystals, epidermal cells, spongy parenchyma, wavy parenchyma, rhomboidal crystals (Figure 3a-o)

Physicochemical and phytochemical study:

Physico-chemical analysis:

The result of the physicochemical properties of the samples are presented in table 2

Table 2: Physicochemical parameters of Desmodium gangeticum and Desmodium velutinum

Sr. No	Parameters	*Desmodium gangeticum*	*Desmodium velutinum*
1.	Loss on Drying % w/w	7.42	4.014
2.	Total Ash Content %w/w	4.65	2.71
3.	Water Soluble Extractive Value %w/w	6.08	8.6
4.	Alcohol Soluble Extractive Value %w/w	4.88	4.64
5.	pH in 5 % aqueous solution	5.3	5.7

LOD is designed to measure the amount of water and volatile matters in a sample. Loss on drying is higher in Desmodium gangeticum indicating that it has higher moisture content. Ash value is also high in Desmodium gangeticum, indicate the presence of inorganic and salt materials. [16] Water soluble extractives were higher in Desmodium velutinum which indicates that the drug can be used as Kashaya, hima, phanṭa preparations. [17] Alcohol soluble extractive value is almost similar for both compounds and indicates the presence of polar constituents. pH value of the both the powder showed it is a weak acid, so it may be comparatively rich in organic acids.

Phytochemical analysis:

The results of phytochemical analysis of the two samples have been presented in table 3.

Table 3: Preliminary qualitative chemical test for Desmodium gangeticum and Desmodium velutinum

Sr. No	Active Constituent	Test (waterand methanolic extract)	*Desmodium gangeticum*	*Desmodium velutinum*
1.	Carbohydrate	Molish’s test	+	+
2.	Protein	Biuret test	+	+
3.	Amino acids	Ninhydrin test	-	-
4.	Steroids	Salkowski test	-	-
5.	Glycosides	Kellar Killiani test	-	-
6.	Saponins	Foam test	+	+
7.	Alkaloids	Dragendorff’s test	-	-
8.	Tannins	Lead acetate test	+	+
9.	Flavonoids	Lead acetate test	-	-

+: Present, –: Absent

Preliminary Phytochemical screening showed carbohydrates, proteins and tannins present in water and alcohol soluble extracts. These phytochemicals are medicinally active as well as exhibit physiological activity.

HPTLC:

Chromatographic investigation was carried out as per the standard materials and methods. The solvent system which was designed for HPTLC studies was in the ratio Toluene: Ethyl acetate: Acetic acid (7: 2: 1 v/v). Results are depicted in Table 4 [Figure 4, 5].

· Sample loading: Methanolic extract of panchanga (1 mg/ml)

Table 4: High Performance Thin Layer Chromatography studies of methanolic extracts at 254- 366 nm

Sample	No. of spots	Rf value at 254nm	No. of spots	Rf value at 366 nm
Desmodium gangeticum	10	0.03,0.33,0.36,0.42,0.59,0.69, 0.82,0.83,0.89,0.94	7	0.03,0.17,0.35,0.41,0.59,0.62,0.82,0.83,0.89
Desmodium velutinum	11	0.04,0.12,0.21,0.24,0.38, 0.40, 0.44, 0.50, 0.73, 0.82, 0.94	7	0.03, 0.12, 0.41, 0.50, 0.55, 0.61,0.73

HPTLC profile showed ten spots of Desmodium gangeticum and eleven spots for Desmodium velutinum at 254 nm in which two Rf value (0.82 and 0.94) are common in both. Similarly seven spots are found at 366nm for both Desmodium gangeticum and Desmodium velutinum, in which two Rf value (0.03, 0.41) are common in both.

Figure 4. a: Peak display of Desmodium gangeticum at 254nm, b: Peak display of Desmodium velutinum at 254nm, c: 3D graph of Desmodium gangeticum and Desmodium velutinum at 254nm, d: Peak display of Desmodium gangeticum at 366nm, e: Peak display of Desmodium velutinum at 366nm, f: 3D graph of Desmodium gangeticum and Desmodium velutinum at 366nm

After spectral overlap from 200-800nm similar spectra was found as shown in figure 5(a-h)

Figure 5 a: 3D graph Multiwavelength, b: Spectral comparison depicting similar spectra at 0.02 Rf, c: Spectral comparison depicting similar spectra at 0.36 Rf, d: Spectral comparison depicting similar spectra at 0.42 Rf, e: Spectral comparison depicting similar spectra at 0.59 Rf, f: Spectral comparison depicting similar spectra at 0.69 Rf, g: Spectral comparison depicting similar spectra at 0.82 Rf, h: Spectral comparison depicting similar spectra at 0.94 Rf

GCMS:

Gas Chromatography- Mass Spectrometry (GC-MS) is a hyphenated analytical technique that combines the separation properties of gas-liquid chromatography with the detection feature of mass spectrometry to identify different substances within a test sample. GC is used to separate the volatile and thermally stable substitutes in a sample whereas GC-MS fragments the analyte to be identified on the basis of its mass. The further addition of mass spectrometer in it leads to GC-MS/MS [18] the study was carried out for Desmodium velutinum and since in this procedure no peaks were obtained during the procedure we may infer that methanolic extract contains thermo labile constituents which could not be detected.

On the other hand 18 compounds have been detected and reported from GCMS study of Desmodium gangeticum [13]

A clinical study is going at the Institute to assess the efficacy of panchanga of Desmodium velutinum as substitute of Shalaparni Desmodium gangeticum in the management of Kasa. [19]

The analytical and microscopic study done above reveals that both the species show almost similar characters. However it can be differentiated by few other characters.

CONCLUSION:

The physicochemical parameter, HPTLC profile of the panchanga can be used for ensuring quality and purity of the raw drug. The information obtained from this research may be useful to establish certain botanical standards. From the study conducted for both plants, it can be concluded that both the species show almost similar morphological and microscopical characters. Therefore we may conclude that Desmodium velutinum may be used as substitute of Desmodium gangeticum

ACKNOWLEDGEMENT:

The authors express sincere gratitude to Head-Pharmacognostical Laboratory, Head-Pharmaceutical Laboratory and to Director of Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University-Jamnagar

CONFLICT OF INTEREST:

None.

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Received on 21.04.2020 Modified on 29.05.2020

Research J. Pharm. and Tech. 2021; 14(6):2950-2956.

DOI: 10.52711/0974-360X.2021.00517