International Journal of Drug Development and Research

Key words

HPTLC, Chemical profile, Flavonoids, Euphorbia neriifolia

INTRODUCTION

Plants generally contain primary and secondary metabolites and these phytoconstituents impart the specific characteristics and properties of plants. Therefore, it is obligatory to resolve all of the phytochemical constituents present in the plants in order to ensure the consistency and repeatability of pharmacological, antimicrobial and clinical research, to understand their bioactivities, to identify the active principles (components) and possible side effects of active compounds and to enhance product quality control[1]. These phytoconstituents are estimated quantitatively and qualitatively by a variety of techniques such as spectroscopy and chromatography. Chromatography techniques are the most useful and popular tools used for the qualitative and separation studies. Now-a-days, HPTLC is gaining increasing importance for the analysis of plant extracts. The qualitative analysis which produces a “fingerprint” chromatogram obtained under standard conditions can be very useful for quality control of phytochemicals[2-4]. Euphorbia neriifolia (Euphorbiaceae) Linn. popularly known as Sehund in Ayurveda and is widely distributed in India [5]. Traditionally, the leaves of this plant are reported as aphrodisiac and diuretic and are also used in the treatment of cough, cold and bronchitis. Hydro-ethanolic extract of leaves possesses antioxidant free radical scavenging[6,7], analgesic[8] and anticancer properties[8-12]. The plant extract is reported to have anti-inflammatory, antimicrobial activity and antidepressant activities[13,14]. With this knowledge the present study was aimed to develop a screening programme covering the most important class of phytochemicals (i.e. flavonoids) using HPTLC as technique and to perform screening of different extracts using selected mobile phases in order to detect possible phytochemical flavonoids present in the medicinally important plant E. neriifolia.

MATERIALS AND METHODS

Chemicals and reagents

All chemicals and reagents used in the study were of analytical grade and were purchased from reliable firms and institutes (CDH, MERCK, and SUYOG). Silica gel (G) 60F and 0.25 readymade aluminium sheets (Merck KGaA, Germany). Silica gel 60 F254, HPTLC aluminium sheets 20 x 20 cm, Merck KGaA, Germany Ord. No. 1.05554.

Procurement and authentication of experimental plant

Euphorbia neriifolia leaves were collected from medicinal garden of Banasthali University, Banasthali and near by areas of Banasthali, Rajasthan, India (Latitude N-26°24’14.8414”; Longitude E-73°52’9.7194”), in the month of October, 2009 and was taxonomically identified by Botanist of Krishi Vigyan Kendra, Banasthali University, Banasthali, Rajasthan, India.

Preparation of extracts

Shade dried leaves were powdered (250 g), soxhlet extracted with 70% (v/v) ethanol and vacuum concentrated to dryness under reduced pressure at 60º±1ºC. After drying in hot air oven (40-45°C), it was stored in an air tight container in refrigerator at 5°C. The residue was designated as hydro-ethanolic extract of E. neriifolia (HEEN). Dried leaves of Euphorbia neriifolia (250 g) were also extracted successively with pet-ether, benzene, chloroform, ethyl acetate, and ethanol and finally macerated with distilled water (non-polar to polar) to get respective extracts. Flavonoids presence were determined by the standard methods[15-17], using quercetin and rutin as standard. Figure 1 represents the schematic diagram of secondary metabolite profiling extraction method.

HPTLC method for the estimation of flavonoids

For the present study complete CAMAG HPTLC equipment, supported by the CURIE, DST has been used. The equipment consists of a fully automatic sample Linomat V sample applicator, developing chamber, TLC Scanner III for densitometric evaluation of chromatograms and CATS 4 software for interpretation of data. In the present study Sony Cyber shot 16 mega pixel camera for photo documentation was used. Silica gel 60 F254, HPTLC aluminium sheets were used as adsorbent (stationary phase). The 10 μl extracts were applied point-wise on HPTLC aluminium sheets (20 x 10 cm with 250 μm thickness) as different tracks in the form of 6 mm wide bands by using Camag semi-automatic Linomat 5 spotter at a distance of 12 mm. Nitrogen gas was also supplied for simultaneous drying of bands and then drier was used for complete drying of bands. HPTLC of different extracts were performed by using altered mobile phase summarized as: 1) n-butanol: acetic acid: water (4:1:5 & 5:1:6), and 2) Ethylacetate: methanol: water (7.5: 2: 4). To saturate the chamber, 10 ml mobile phase was placed in each flat-bottomed Camag twin trough TLC chamber, 30 minutes before the development of the HPTLC plate. The chamber was sealed with parafilm and covered with a steel lid. The sample loaded plates were kept in this chamber with respective mobile phase. The developed plates were then dried and scanned using TLC scanner 3 with Wincats software under 364 nm[18]. The HPTLC plates were air-dried at room-temperature for minimum 1 hour and by hot air to evaporate solvents from the plate before derivatization. After drying the plates, they were exposed to ammonia and iodine vapours by placing for 1 min in a chamber that was saturated with iodine and ammonia vapours. All plates were visualized directly after drying and a fingerprint profile was photo documented using Camag Reproster – 3 under 254 nm and 366 nm in UV and white light. Finally the digital images of the chromatograms were evaluated and scanning was done at 254 nm (CAMAG video Scanner). The captured image was subjected to a visual inspection on the computer screen. The results of the screening are presented individually for each extract. The differences found in the Rf values of a compound, are specified by the HPTLC system.

RESULTS

Different compositions of the mobile phase for HPTLC analysis were tested in order to obtain high resolution and reproducible peaks. Authentic markers of flavonol (quercetin) and flavonoid glycoside (rutin) obtained commercially were cochromatographed. Blue brown colour zone was detected in UV derivatisation in the chromatogram which (Figure 2 to 4) confirms the presence of flavonoids. The extracts were run along with the standard polyphenols compound. HPTLC of different extracts of E. neriifolia were found to be similar as those obtained with rutin and quercetin standards on different mobile phases. Rf values of the all the extracts were found to be comparable with Rf values of standards quercetin and rutin as depicted in table 1 to 3 and figure 2 to 4. HPTLC chromatogram of standards quercetin and rutin, and all extracts with varying mobile phase is depicted in figure 5 to 7. The benzene extract of E. neriifolia leaves illustrated the presence of 5 different types of flavonoids with 5 different Rf values ranging from 0.06 to 0.96 by using ethyl-acetate: methanol: water (7.5: 2: 4) as mobile phase. The chloroform extract of E. neriifolia demonstrated the presence of 3, 7 and 9 different types of flavonoids with different Rf values ranging from 0.03 to 0.96 by using varying mobile phase i.e. n-butanol: acetic acid: water (5: 1: 6 & 4: 1: 5) and ethyl-acetate: methanol: water (7.5: 2: 4). The ethylacetate extract of E. neriifolia demonstrated the presence of 6, 9 and 12 different types of flavonoids with different Rf values ranging from 0.02 to 0.97 by using varying mobile phase i.e. n-butanol: acetic acid: water (5: 1: 6 & 4: 1: 5) and ethyl-acetate: methanol: water (7.5: 2: 4). The ethanol extract of EN demonstrated the presence of 5 different types of flavonoids with different Rf values ranging from 0.24 to 0.82 by using mobile phase n-butanol: acetic acid: water (5: 1: 6). Whereas the aqueous extract of plant depicted the presence of 12 different spots with varying Rf values ranging from 0.03 to 0.95 by using specific mobile phase n-butanol: acetic acid: water (5: 1: 6). Along with these the hydro-ethanol extract showed the presence of 6, 7 and 9 altered flavonoids with different Rf values ranging from 0.03 to 0.93 on varying mobile phases as depicted in table 1 to 3. Maximum number of flavonoids has been observed in ethyl-acetate and aqueous extracts (12) followed by petroleum ether (10) and chloroform (9) extracts. Whereas minimum numbers of flavonoids were observed in ethanol (5) and benzene extract (5). The n-butanol: acetic acid: water with volume proportion 5: 1: 6 showed good separation of the phytoconstituents for ethyl-acetate and aqueous extracts (Figure 4 and 7). HPTLC chromatogram showed that maximum numbers of components were observed under UV and florescence absorbance mode. The characteristics pattern of all extracts showed well separated pattern of peaks and Rf values.

DISCUSSION

Secondary metabolites are produced by a large variety of organisms, including bacteria, fungi, plants and animals. Especially higher plants use secondary metabolites for their defensive mechanisms against the biotic and abiotic factors. Of these secondary metabolites flavonoids are a group of about 4000 naturally polyphenolic compounds, found universally in plant origin [19]. Flavonoids, a broad class of polyphenolic compounds ubiquitously widespread among photosynthesizing cells, possesses an impressive array of pharmacological activity[20,21], including free radical scavenging, inhibition of a vast spectrum of enzymes, and estrogenic activity, because of their spasmolytic, antiphlogistic, and diuretic properties[22]. It was also indicated in epidemiological studies that their consumption always assured a reduced risk of cancer and cardiovascular disease[23-29]. In the last two decades HPTLC method has emerged as an important tool for the qualitative and quantitative phytochemical analysis of herbal drugs and formulations [30] and gives better choice of analysis as it can handle several samples of divergent nature and composition by several analysts at the same time. Chromatographic HPTLC method for detection of flavonoids from Euphorbia neriifolia has not been so far reported in literature. Densitometric HPTLC has been used in the present work for the quantification of quercetin and rutin and other unknown flavonoids from sequential extracts of E. neriifolia. In fact this is the first attempt in which the different flavonoid profiles in the leaves extracts of E. neriifolia were elucidated. In present study the chromatographic screening of all extracts depicted that E. neriifolia contained wide range of flavonoids. All mobile phases used for the chromatography were good and suitable for the extraction of flavonoids from plant extracts[16]. Chloroform: Methanol solvent system was also used by Gohar et al.[29] for isolation of acylated flavonol glycoside from Ceratonia siliqua L. seeds. Meena & Patni,[31] adopted same procedure for the isolation & identification of flavonoid “quercetin” from Citrullus colocynthis. Benzene and methanol with volume proportion 9.5: 0.5 showed good separation of the phytoconstituents for petroleum ether extract of E. neriifolia[30]. Our results are also supported by various other previous reports[32,33]. Quercetin has been reported to have interesting biological activities. In present study the ethanolic extract of Euphorbia neriifolia acts as a better source of quercetin. We have already reported the higher antioxidant activity of HEEN could be attributed to the presence of flavonoids and other active ingredients [6,7]. Using this analytical method quercetin and rutin could be determined simultaneously and the validity of the method was also verified. Putative therapeutic effects of many traditional medicines might be ascribed to the presence of flavonoids[34]. The results of the present study authenticated and confirmed the folkloric usage, traditional practices, ethno-botanical, anti-microbial and pharmacological values of the medicinally important plant E. neriifolia and suggested that the leaves extracts of E. neriifolia possess compounds with bioactivity properties that can be used as active principles or agents in new drugs for the therapy of infectious and degenerative diseases.

Conclusion

For the first time HPTLC profile for the benzene, chloroform, ethyl acetate, ethanol, aqueous and hydro-ethanol extracts of E. neriifolia leaves was developed. A broad screening programme, covering the most important phytochemical group of compounds (e.g. Flavonoids) was developed on the basis of HPTLC. Chromatographic screening studies gave 5 to 12 spots in petroleum ether, benzene, chloroform, ethyl acetate, ethanol and aqueous extracts at various mobile phases respectively. Thus, it could be concluded that HPTLC can be used as a pharmacognostical tool in the pharmaceutical industry to identify medicinally important plant. In addition it can be adopted as a chemo-taxonomical tool in the plant systematic. Present study also supported the view that the leaves of E. neriifolia could be a potential source of natural antioxidant and anti-carcinogenic drugs. Further in near future the separation and characterization of the bioactive compound from the plants is also to be evaluated.

ACKNOWLEDGEMENTS

The authors are grateful to University Grants Commission for providing financial assistance and to authorities of Banasthali University, Rajasthan for providing necessary facilities to carry out the present study. We also acknowledge Dr. Sonika Jain, Assistant Professor, Department of Chemistry, Banasthali University, for her valuable help and guidance to conduct this study.

CONFLICT OF INTERESTS

The authors have declared that no conflict of interest exists.

Conflict of Interest: NIL

Source of Support: NONE

Tables at a glance

Table 1	Table 2	Table 3

Figures at a glance

Figure 1	Figure 2	Figure 3	Figure 4
Figure 5	Figure 6	Figure 7

References

1. Amani M, El-Mousallamy D. Leaf flavonoids of Albizialebbeck. Phytochem. 1998; 48(4): 759 -761.
2. Dash S, Das C, Sahoo DC. Phytochemical and anthelmintic screening of crude bark extract of Adenantherapavonina Linn. Int J Comp Pharm. 2010; 2(10): 1-4.
3. Yamunadevi M, Wesely EG, Johnson M. Phytochemical studies on the terpenoids of medicinally important plant Aervalanata L. using HPTLC. Asian Pac J Trop Biomed. 2011; 1(2): S220–S225.
4. Bobby MDN, Wesely EG, Johnson M. High performance thin layer chromatography profile studies on the alkaloids of Albizialebbeck. Asian Pac J Trop Biomed. 2012; 2(1): S1–S6.
5. Sharma V, Janmeda P, Singh L. A Review on Euphorbia neriifolia (Sehund).Spatulla DD. 2011; 1: 107-111.
6. Pracheta, Sharma V, Paliwal R, Sharma S. In vitro free radical scavenging and antioxidant potential of ethanolic extract of Euphorbia neriifolia Linn. Int J Pharm Pharm Sci. 2011; 3: 238-242.
7. Pracheta, Sharma V, Paliwal R, Sharma S. Preliminary phytochemical screening and in vitro antioxidant potential of hydro-ethanolic extract of Euphorbia neriifolia Linn. Int J PharmTech Res. 2011; 3(1): 124-132.
8. Sharma V, Janmeda P, Paliwal R, Sharma C. Elucidation of analgesic activity of hydro-ethanolic extract of Euphorbia neriifolia leaves in Swiss albino mice. J Plant Dev Sci. 2012; 4: 183-189.
9. Pracheta, Sharma V, Paliwal R, Sharma S, Singh L, Janmeda BS, Savita, Yadav S, Sharma SH, Chemoprotective activity of hydro-ethanolic extract of Euphorbia neriifolia Linn leaves against DENAinduced liver carcinogenesis in mice. Biol Med. 2011; 3(2): 36-44.
10. Janmeda P, Sharma V, Singh L, Paliwal R, Sharma S, Yadav S, Sharma SH. Chemopreventive effect of hydro-ethanolic extract of Euphorbia neriifolia leaves against DENA-induced renal carcinogenesis in mice. Asian Pac J Cancer Prev. 2011; 12: 677- 683.
11. Sharma V, Pracheta, Paliwal R, Singh L, Sharma V, Sharma SH. Anticarcinogenic potential of Euphorbia neriifolia leaves against NNitrosodiethylamine- induced Nephrotoxicity in mice. J Biochem Cell Archives.2011; 11: 393-398.
12. Sharma V, Janmeda P, Paliwal R, Sharma SH. Anti hepatotoxic activity of Euphorbia neriifolia extract against N-nitrosodiethylamine-induced hepatocarcinogenesis in mice. J Chin Integr Med. 2012; 10: 1303-1309.
13. Bigoniya P, Rana AC. Immunomodulatory activity of Euphorbia neriifolia leaf hydro-alcoholic extract in rats. Indian Drugs. 2008; 45(2): 90-97.
14. Gaur K, Rana AC, Chauhan LS, Sharma CS, Nema RK, Kori ML, Yashwant. Investigation of Immunomodulatory Potential of Euphorbia neriifolia Linn.against Betamethasone Induced Immunosuppression. Int J Pharm Phytochem Res. 2009; 1(1): 8-11.
15. Wagner H, Bladt. Plant Drug Analysis: A thin layer chromatography Atlas. 2nd Ed, Springer-Verlag Berlin Heidelberg London, New York, 1996; 349- 354.
16. Harborne JB. Phytochemical Methods, A Guide to Modern Techniques of Plant Analysis, Chapman & Hall, London, UK, 3rd edition, 1998; 1-7.
17. Beknal AK, Korwar PG, Halkai MA, Kulkarni U, Patil BS, Soodam SR. Phytochemical investigation and antioxidant activity study of Drynariaquercifolia Linn rhizome. Int J Current Pharm Res. 2010; 2(4): 36-39
18. Harborne JB. Nature, distribution and function of plant flavonoids, in plant flavonoids in biology and medicine. In: Biochemical, Pharmacological and Structure- Activity Relationships (Eds) Cody V, Middleton E, Jr, Harborne JB, Alan R. Liss, Inc. New York, 1986; 15-24.
19. Hausten B. Flavonoids: a class of natural products of high pharmacological potency. BiochemPharmacol. 1983; 32(7): 1141–1148.
20. Hollman PCH, Hertog MGL, Katan MB. Analysis and health effects of flavonoids.Food Chem. 1996; 57: 43-46.
21. Cao G, Sofic E, Prior RL. Antioxidant and prooxidant behavior of flavonoids: structureactivity relationships. Free RadicBiol Med. 1997; 22(5): 749-76022) Wattenburg LW. Chemoprevention of cancer. Cancer Res. 1985; 45: 1-8.
22. Middleton E, Kandaswami C. In The Flavonoids; Harborne JB, Ed; Chapman & Hall: London, 1986; Chapter 15.
23. Wei H, Tye L, Bresnick E, Birt DF. Inhibitory effect of apigenin, a plant flavonoid, on epidermal ornithine decarboxylase and skin tumor promotion in mice.Cancer Res. 1990; 50: 499-502.
24. Bohm BA. Introduction to Flavonoids; Harwood Academic Publishers: Singapore, 1998; Chapter 2.
25. Kaur C, Kapoor HC. Antioxidants in fruits and vegetables – the millennium’s health.Int J Food Sci Technol. 2001; 36: 703-725.
26. Springob K, Saito K. Metabolic engineering of plant secondary metabolism: Promising approach to the production of pharmaceuticals. SciCul. 2002; 68: 76-85.
27. Sharma DK. Pharmacological properties of flavonoids including flavonoligans- Integration of petrocrops with drug development from plants.J Scientific Industrial Res. 2006; 65: 477-484.
28. Gohar A, Gedara SR, Baraka HN. New acylatedflavonol glycoside from Ceratoniasiliqua L. seeds. J Med Plants Res. 2009; 3(5): 424-428.
29. Sharma V, Pracheta. Microscopic studies and preliminary pharmacognostical evaluation of Euphorbia neriifolia (Linn.) leaves. Indian J Nat Prod Resour. 2013. In Press
30. Meena MC, Patni V. Isolation and identification of Flavonoid “Ouercetin” from Citrulluscolocynthis (Linn.) Schrad.Asian J Exp Sci. 2008; 22: 137-142.
31. Lakshmi T, Rajendran R, Madhusudhanan N. Chromatographic fingerprint analysis of Acacia Catechu Ethanolic leaf extract by HPTLC Technique. Int J Drug Dev Res. 2012; 4(1): 180-185.
32. Schultz SC, Bahraminejad S, Asenstorfer RE, Riley IT. Analysis of the antimicrobial activity of flavonoids and saponins isolated from the shoots of Oats (Avena sativa L.). J Phytopathol.2008; 156: 1–7.
33. Nagai N, Nakai A, Nagata K. Quercetin suppresses heat shock response by down regulation of HSF1. BiochemBiophys Res Commun.1995; 208: 1099- 1105.