Comparative phytochemical study of root versus small branches of Desmodium gangeticum using High Performance Thin Layer Chromatographic UV detection Method

 

S. C. Verma¹*, S. Subhani¹, E. Vashishth¹, R.K. Tiwari², R. Singh¹, P. Pant¹, M. M. Padhi¹,

K.S Dhiman¹

¹Central Council for Research in Ayurvedic Sciences,

61-65, Institutional Area, Opp.-D-Block, Janakpuri, New Delhi-110058, India.

²NVRI and H, Sector -25, Indra Nagar, Lucknow.

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

Desmodium gangeticum belongs to family Leguminoceae. It is a versatile plant with many features and considerable prospective. Whole plant is employed as traditional medicine for the treatment of several disorders. D. gangeticum is a traditional Ayurvedic plant used for centuries as an anthelminthic, anti-catarrahal, diuretic, expectorant, astrin-gent, febrifuge, nervine tonic, anti diarrheal, bronchodilator, vasopressor, analgesic, antipyretic, cardio tonic, stimulant, antioxidant and anti-inflammatory agent. Chromatographic fingerprint and  similarity measurement could efficiently identify and distinguish between root and small branches of  D. gangeticum. The phytochemical fingerprint profiling of root and small branches of D. gangeticum were found similar as an official part of D. gangeticum plant i.e. root, therefore small branches may be used in place of root and vice-versa after comparison and confirmation of same pharmacological activities.

 

 

 

INTRODUCTION:  

Desmodium gangeticum is known as Shalparni. It is an endangered ethnomedicinal plant belonging to family Leguminoceae sub family Fabaceae^1,2. According to Ayurveda it contains (i) Gunna (properties) – guru (heavy) and snigdh (slimy), (ii) Rasa (taste) – madhur (sweet) and tickta (bitter), (iii)Vitya (potency) – ushan (hot)³. This plant is one among the Dashamoola (ten roots) of Ayurveda and is an important ingredient of many famous Ayurvedic drugs like Dashamoolarishta, Chyavanaprasha, Dhanwantharam Kuzhambu, Rasnadhi decoction, Agusthya Rasayanam, Sukumara gritham, Dasamula Katuthiayadi kashayam, Dasamula thailam, Danvantra thailam, Mahamasah thailam, Anu thailam, Vidaryadi gritham and Brahma Rasayan ^4-8. It is found all over areas that are up to the height of 5000 feet. It is a small plant that has a height of 2 to 4 feet. The stem is angular.

 

 

Leaves are ovate in shape that is 3 to 6 inch in length. The lower surface of the leaf is of light green in color. Flowers are purple or white in color. These are small and are placed in a pattern. Pod is thin, flat, curved having 6 to 8 nodes. It also bears hairs like structures on them. The plant shows flowers and fruits especially in early summers.³

 

PHYTOCHEMISTRY:

Phytochemical screening of D. gangeticum revealed the presence of tryptamines, phenethylamines like alkaloids and their N-oxides, pterocarpnoid, desmocarpin, isoflavanoid glycoside, cardiac glycoside, steroids, saponins, salicylic acid, rutin, chlorogenic acid, caffeic acid, phospholipids, glycosphingolipids, uridine triacetate, trans – 5 – hexadecanoic acid, 5-O-methylgenistein-7-O-β-d-glucopyranoside,1– Tritriacontanol, 1- heptadecanol, β – amyrone and flavanoid glycosides like 4,5,7- trihydroxy-8-prenylflavone-4’Ox-L-rhamnopyranosyl-(1-6)-D-glucopyranoside and 8-C-prenyl-5,7,5-trimethoxy-3,4- methylenedioxy flavones3,4- dihydroxy benzoic acid, kaempferol- 7-O-β-d-glucopyranoside, and uridine triacetate^9,10. Aerial parts contain β-sitosterol, α-amyrone, lupeol and its acetate, stigmastrol, five tryptamine derivatives, Nb-Me-tetrahydroharman and 6-OMe-2-Me- β-carbolinium cation 5-methoxy N, N-dimethyl tryptamine and indole-3-alkyl-amines. Flavones like 4/,5,7-Trihydroxy-8-prenylflavone, 4-O-α-L-rhamnopyranosyl-(1→6)-β-d-glucopyranoside, 8-C-prenyl-5,7,5-trimethoxy- 3,4-methylenedioxyflavone, rutin and quercetin-7-O-β-d-glucopyranoside were also reported from the aerial parts. From the stem bark, N-methylserotonin Bufotenine N-oxide, Hypaphorine, 6-Methoxy-2-methyl-β-Carbolinum, Nb-methyltetra hydroharman, hordenine are isolated. Pterocarpans such as gangetin, gangetinin, desmodin, and desmocarpin were reported to be present in roots¹¹. A new Pterocarpans - gangetial, had been isolated from the chloroform extract of the roots¹². Seeds contain β-carboline alkaloid, indole-3-alkylamine, carbolines, aminoglucosyl glycerolipid¹³ and five phospholipids. Seeds also showed presence of sugars, fatty oil and alkaloids. From the methanolic extract of leaves of D. gangeticum simple indole base namely, N, N-Dimethyltryptamine was isolated^{14, 15}.

 

MEDICINAL USES:

D. gangeticum is of great therapeutic value in treating various diseases. Owing to various flavonoids and alkaloids present in this plant, it is known for various biological activities such as anti-inflammatory, anti-nociceptive activity, anti-ulcer, cytoprotective, anti-secretary activity, hypocholesterolemic, anti-diabetic activity and anti-leishmanial activity. The aqueous extract of D. gangeticum has been reported to have strong anti-writhing and central nervous system (CNS) depressant activity, in improving memory and in healing different types of wounds. It has also been found to be a promising candidate for the management of dementia and Alzheimer disease. The plant of D. gangeticum is bitter, sweet, thermogenic, nervine tonic, aphrodisiac, demulcent, anthelmintic, cardic tonic, febrifuge, anti-inflammatory, diuretic, haemostatic, rejuvenating and useful in neuromuscular, ophthalmic disorders, loss of appetite, flatulence, diarrhea, dysentery, nausea, piles and helminthiasis. It is used in angina pain, cardic disorders, tuberculosis, cough, seminal weakness, urinary disorders, fever, debility and gout. ¹⁶ The root of D. gangaticum is one of the constituent of famous Ayurvedic preparation Dasmoola kvatha which is antipyretic and bitter tonic. It is reported to be beneficial in treatment of typhoid fever, biliousness and also diuretic and aphrodisiac¹². The whole plant decoction is given to treat digestive disorders, edema, diarrhea, intermittent fevers, malaria, and urinary tract infections¹⁷. In folklore medicine, decoction from D. gangeticum leaves used for stones of the gall bladder and kidneys. The flavone and isoflavonoid glycosides present in this plant form the ingredient of many Ayurvedic formulations for diabetics. ^18.The root is nervine tonic, diuretic, cardiotonic, and expectorant¹⁹. The root decoction is used for the treatment of heart diseases, especially in angina pectoris and myocardial infarction. It strengthens heart muscles and reduces cholesterol. Roots are chewed daily for the cure of typhoid and pneumonia.²⁰

 

Figure 1: D. gangaticum

 

Figure2: Root

 

Figure 3: Small branches

Taxonomic / Scientific Classification¹³

Kingdom	Plantae
Subkingdom	Tracheobionta
Super division	Spermatophyta
Division	Magnoliophyta
Class	Magnoliopsida
Subclass	Rosidae
Order	Fabales
Family	Fabaceae
Genus	Desmodium
Species	gangeticum (L.) DC
Binomial name	Desmodium gangeticum (L.) DC

 

MATERIALS AND METHODS:

Plant Materials and Chemicals:

Plant materials i.e Root (Fig.2) and small branches of stem (Fig.3) of D. gangeticum were collected in December 2013 and authenticated by Dr. R. K. Tiwari, Research Officer, Pharmacognosy, National Veterinary Research Institute and Hospital, Lucknow. All chemicals (AR grade) and TLC plates were purchased from E. Merck Pvt. Ltd. (Mumbai, India).

 

Sample preparation:

The plant parts were dried under a gentle stream of air in the laboratory till no loss in weight (temperature 30+ 2°C and relative humidity 50 + 5%) and powdered in an electric grinder.

 

Conventional extraction of Root and small branches of stem of D. gangeticum were performed at room temperature (28^o ± 3^oC) with a variety of solvents ranging from non-polar to polar ones, i.e. n-hexane, ethyl acetate and ethanol. Dried and powdered parts of D. gangeticum (10 g each) were extracted three times (3 × 50 mL) for 18 h of each extraction with each of the above-mentioned solvents separately. Each extract was filtered by using Whatman filter paper no. 1 and the solvents were removed under vacuum at 50°C, separately and concentrated up to 10 mL to get the sample solution of 100 mg mL^-1.  5 µL of each sample was applied separately to TLC plate for the development of fingerprints.

 

HPTLC-UV detection Method:

High Performance Thin Layer Chromatography was performed on 10 cm × 10 cm TLC plates pre-coated with 0.25 μm thin layers of silica gel 60 F₂₅₄ (E. Merck). Both samples (Root and small branches) were applied on the plates as bands 10 mm wide by use of a Linomat-IV applicator (CAMAG, Switzerland) fitted with a 100 μL syringe (Hamilton, Switzerland). The application positions X and Y were both 10 mm, to avoid edge effects. Linear ascending development to a distance of 80 mm with Toluene: Ethyl acetate 9:1 (v/v) and as mobile phase for both n-hexane extract was performed in a twin-trough glass chamber (20 cm × 10 cm) previously saturated with vapors of mobile phase for 20 min. The plates were dried in air and visualized under λ 254 nm and λ 366 nm for ultra violet detection and taken the fingerprints as evident in Figures 4 – 5. Further, the same TLC plate was derivatized with anisaldehyde-sulphuric acid reagent and visualized in white light obtained fingerprints were as evident in Figures 6 using CAMAG Reprostar and WinCATs software (V 1.4.2; CAMAG). HPTLC of ethyl acetate extract and alcoholic extract of both drugs were performed with same procedure in the mobile phase of Toluene: Ethyl acetate 8: 2 (v/v) and Toluene: Ethyl acetate 6:4(v/v)  and then visualized in λ 254 nm, λ 366 nm and white light using CAMAG Reprostar and WinCATs software as shown in Figure 7-12.

 

                             1                   2                                               1                      2                                            1                         2 

                                   254 nm                                                       366 nm                                                                After derivatization

                                  Figure 4                                                        Figure 5                                                             Figure 6    

Figure 4-6: TLC fingerprint of n- hexane extract of D. gangeticum (1= Rt.; 2= Sm. Br.)

                           1                    2                                                           1                  2                                                    1                     2

                                254 nm                                                             366 nm                                                            After derivatization

                                Figure 7                                                                Figure 8                                                       Figures 9

Figure 7-9: TLC fingerprint of ethyl acetate extract of D. gangeticum (1= Rt.; 2= Sm. Br.)

 

 

Table 1: R_f value of phytochemicals present in n-hexane, ethyl acetate and ethanol extract of D. gangeticum (Rt. and Sm. Br.) at different wave-lengths.

 

 

RESULTS AND DISCUSSION:

No such study was found in literature for comparative phytochemical study of Root versus small branches of D. gangeticum Linn by using High Performance Thin Layer Chromatographic-Ultra Violet detection Method. Comparative study of TLC fingerprints of Root and small branches of D. gangeticum revealed that many similarities in phytochemical fingerprints were found and evident in Table-1 and Fig. 4-12

 

Phytochemical fingerprints of n-hexane extract of root and small branches under 254 nm showed no band in both parts. Under 366 nm UV detection, root and small branches showed three and six band respectively, out of which, three bands at R_f 0.33 (blue), 0.54( light blue) and 0.75 (blue) were found similar. After TLC plate derivatized with Anisaldehyde sulphuric acid reagent and visualized under white light, root and small branches both were showed six and five bands respectively, out of which, five bands at R_f 0.34 (blue), 0.42 (blue), 0.49 (blue), 0.54 (violet), 0.87(blue) were found similar as represented in Table 1 and Fig. 4-6.

 

Ethyl acetate extract of Root and small branches (stem) under 254 nm, both extract showed no band in both parts. Under 366 nm UV detection, Root and small branches showed two and five bands respectively, out of which, two bands at R_f 0.67(blue) and 0.80 (red) were found similar. After derivatized with Anisaldehyde sulphuric acid reagent and visualized under white light,both  Root and small branches both showed seven  bands and all seven  bands at R_f 0.04(blue), 0.20(blue), 0.24(blue), 0.44(blue), 0.53(blue), 0.63(violet) and 0.86(violet)were found similar as showed in Table 1 and Fig. 7-9.

 

                          1                     2                                                          1                2                                                     1                    2

                              254 nm                                                                 366 nm                                                            After derivatization

                            Figure 10                                                                  Figure 11                                                      Figures 12

Figure 10-12: TLC fingerprint of ethanol extract of D. gangeticum (1= Rt.; 2= Sm. Br.)

 

 

Phytochemical fingerprints of ethanol extract of Root and small branches under UV detection at 254 nm showed no band in both parts.  While under 366 nm UV detection, Root and small branches showed five and seven bands respectively, out of which four bands at R_f 0.08 (red), 0.64 (blue), 0.71 (blue) and 0.77(red) were found similar. After TLC plate derivatized with Anisaldehyde sulphuric acid reagent and visualized under white light, Root and small branches both showed four bands and all four bands at R_f  0.42, 0.51, 0.60 and 0.86 (all are blue) were similar in both parts (Rt. and Sm. Br.) as evident in Table 1 and Fig.10-12.

 

CONCLUSION:

The phytochemical fingerprint profiling of Root and small branches of D. gangeticum were found similar as an official part of D. gangeticum plant i.e. Root , therefore small branches may be used in place of Root and vice-versa after comparison and confirmation of same pharmacological activities.TLC phytochemical fingerprint profiling of n-hexane, ethyl acetate, ethanolic extracts of Root and small branches of D. gangeticum have been given an idea about the presence of various phytochemicals in their reported parts. The TLC spots provided valuable clue regarding presence or absence of various phytochemicals or metabolites of the plants.

 

ACKNOWLEDGMENTS:

Authors are thankful to Director General, Central Council for Research in Ayurvedic Sciences, New Delhi to provide the financial support under 2^nd IMR scheme for this research work.

 

CONFLICT OF INTEREST:

Authors have no conflict of interest.

 

REFERENCES:

1.        Oommen S, Ved DK and Krishnan R. Tropical Indian medicinal plants: propagation methods. Foundation for Revitalization of Local Health Traditions. 2000; 128–132.

2.        Cheruvathur KM, Abraham J and Thomas TD. Plant Regeneration through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC. Applied Biochemistry and Biotechnology. 169; 2013:1799–1810.

3.        www.ayushveda.com/herbs/Desmodium-gangeticum.html.site accessed on 27/02/2015.

4.        Kirtikarand KR and Basu BD. Indian Medicinal Plants. International Book Distributors. 3; 1987: 2057–2059.

5.        Niranjan A, and Tewari SK. Phytochemical composition and antioxidant potential of Desmodium gangeticum (Linn.)DC. Natural Product Radiance.7 (1); 2008:35-36.

6.        S T , Kirubha V, Jegadeesan M and Kavimani S .Studies on Desmodium gangeticum: A review. Journal of Chemical and Pharmaceutical Research. 3(6); 2011: 850-855.

7.        Rao ML and Savithramma N. Antimicrobial activity of Dasamoola – An Ayurvedic Drug. World Journal of Pharmaceutical Research. 1(3); 2012: 803 – 812.

 

 

8.        Rathi A, Rao Ch V, Ravishankar B , Deb S and  Mehrotra S. Anti-inflammatory and anti-nociceptive activity of the water decoction Desmodium gangeticum. Journal of Ethnopharmacology. 2004; 95: 259 – 263.

9.        Anurag S and Singh PK. An ethnobotanical study of medicinal plants in Chandauli District of Uttar Pradesh, India. Journal of Ethnopharmacology.  121; 2009: 324–29.

10.     Deshpande HA and Bhalsing SR. A Review of Phytochemical Profile of Desmodium gangeticum (L.) DC: A Valued Endangered Medicinal Plant. International Journal of Pharma Research and Health Sciences. 1(4); 2014: 36-48.

11.     Subha R, Madan MP and Ajay KSR. An ethnomedicinal, phytochemical and pharmacological profile of Desmodium gangeticum (L.) DC. and Desmodium adscendens (Sw.) DC. Journal of Ethnopharmacology .136; 2011:283-96.

12.     Varaprasad MV, Balakrishna K, Sukumar E and Patra A. Gangetial, a new pterocarpan from the roots of Desmodium gangeticum. Journal of Indian Chemical Society .86; 2009: 654–56.

13.     Mishra PK, Singh N, Ahmad G, Dube A and Maurya R. Glycolipids and other constituents from Desmodium gangeticum with antileishmanial and immunomodulatory activities. Bioorganic and Medicinal Chemistry Letters.2005; 15: 4543-46.

14.     Shrivastava R, Sinha BN, A HPTLC report on Desmodium gangeticum. International Journal of Pharmaceutical Sciences. (3); 2011: 379 – 381.

15.     Bhattacharjee A, Shashidhara CS and Saha S Phytochemical and ethno-pharmacological profile of Desmodium gangeticum (L.) DC. A review. International Journal of Biomedical Research. 4 (10); 2013:507-515.

16.     Vaghela B, Buddhadev S and Shukla L. Pharmacological Activities of Desmodium Gangeticum: An Overview. International Journal of Pharmacy and Pharmaceutical Sciences. 4(4); 2013: 264-278.

17.     Chopra RN, Nayer SL and Chopra IC. Glossary of Indian Medicinal Plants, CSIR, New Delhi. 12(5); 1956: 157.

18.     Retnam RK and Martin P. Ethnomedicinal plants. Jodhpur. Agrobios. 2006: 69.

19.     Govindarajan R, Rastogi S, Vijayakumar M, Rawat AKS, Shirwaikar A, Mehrotra S and Pushpangadan P, Studies on antioxidant activities of Desmodium gangeticum. Biological and Pharmaceutical Bulletin 26; 2003: 1424 – 1427.

20.     Masoumeh Rashidi and S. S. Deokule. Studies on biodeterioration of somechemical constituents in fresh and market roots of drug Desmodium gangeticum DC. under storage. European Journal of Experimental Biology. 3(1); 2013: 161-165.

 

 

 

Received on 27.03.2015         Modified on 08.04.2015

Accepted on 15.04.2015         © AJRC All right reserved