Preliminary phytochemical screening of Quercus infectoria Oliv . for treatment of skin diseases

In present work, fruits of Quercus infectoria are selected on the basis of traditional claim. Fingerprinting analysis for the said extract along with the biomarker, gallic acid was performed using high performance thin layer chromatography (HPTLC). The extract and biomarker were standardized with various instrumental techniques like Newton magnetic resonance (NMR), Fourier transform infrared (FTIR) and ultraviolet (UV) spectroscopies. Preliminary phytochemical screening was performed for establishing the profile of extract for its nature of chemical composition. The extracts showed the presence of tannins, mucilage and saponins. HPTLC fingerprinting showed better separation of the components. Planer chromatogram generated was used to determine existence of present phytoconstituents. The Rf value was found to be 0.19. The extract exhibited good correlation with selected marker in different analytical techniques like NMR, FTIR and UV spectroscopies. UVSpectroscopic analysis showed good correlation between plant extract and standard (gallic acid). The extract and biomarker exhibited 0.074, 0.0511 and 0.003, 0.0429 intercept and slope values, respectively.


INTRODUCTION
The ancient literature of Ayurveda has served the wellbeing of mankind because of empirical knowledge of the observations and the experience of Ayurveda practitioner.Though traditional medicines offer a safe and inexpensive approach to treat many skin diseases, it has been neglected due to some reasons like shortcoming in treating chronic condition and unavailability of pathological data (Govindarajan, 2007;Al-Quran, 2008).
In this work, fruits of Quercus infectoria Oliv.(Fagaceae) which is also traditionally known as Majuphal was selected on the basis of traditional claim (Khouzami, 2009;Kaur, 2008).The reported constituents of plant are tannins, saponins and mucilage.This study has been focused on phytochemical investigation of Q. infectoria which has been reported as potent candidate for treating skin diseases and inflammatory conditions (Umachigia, 2008;Leela, 2011).The existence of the aforementioned phytoconstituents is determined using proximate analysis, fingerprinting analysis of extract and biomarker using high performance thin layer chromatography (HPTLC) and other instrumental techniques like Newton magnetic resonance (NMR), Fourier transform infrared (FTIR) and ultraviolet (UV) spectroscopies.

Plant collection and extraction
The fresh fruits of Q. infectoria were selected for this study.Selected plant was collected from Pune region and authenticated *Corresponding author.E-mail: v.vaidya123@gmail.com.Tel: +91-9423324249.from Department of Botany, University of Pune, Maharashtra, India (Voucher No. Bot/34/2010) (Figure 1).Proximate analysis of powder was carried out for different physicochemical standards such as ash values, extractive values and loss on drying (Mukherjee, 2002).
The hydroalcoholic extracts in the proportion 60:40 for the selected herbal plant was prepared by simple maceration for about 72 h and concentrated and stored in air tight container (Chusri, 2009;Vermani, 2010).

Phytochemical analysis
The crude extract was subjected to preliminary phytochemical screening for the detection of various phytoconstituents (Ghafour, 2010;Wallis, 1985).

Histology
The plant specimen for the study was cut and fixed in formalin acetic acid (FAA) and was dehydrated (Sass, 1940).Infiltration was carried out by addition of paraffin wax and this paraffin embedded specimens were sectioned with the help of rotary microtone.The sections were stained with Toluidine blue and observed under microscope (O'Brien et al., 1964).Photographs of different magnifications were taken with Nikon Laboratory Photo 2 Microscopic Unit with the help of plane polarized light for the study of various organs (Esau, 1964).

Fingerprinting and spectroscopic analysis
The crude extract of selected plant was analyzed for HPTLC fingerprinting using Camag HPTLC system equipped with an automatic TLC sampler and TLC scanner with a UV cabinet.Planer chromatograms of samples prepared using general solvent system Toulene:Chloroform:Ethanol (8:8:2) and solution prepared as 100 mg in 5 ml of methanol.UV-Spectroscopic analysis of extract and biomarker was performed using Shimadzu 1700 UV spectrophotometer.NMR as well as FTIR investigation was performed on extract and biomarker (Jamil, 2012;Rodriguez, 2008;Carmen, 2008).

Pharmacognostic study
The various physicochemical standards such as ash values, extractive values and loss on drying was performed.Preliminary phytochemical screening was performed for establishing the profile of extract for its nature of chemical composition (Soon et al., 2007;Aroonrerk, 2009).

Phytochemical analysis
Preliminary phytochemical screening was performed for establishing the profile of extract for its nature of chemical composition.The extracts showed the presence of tannins, mucilage and saponins.The results are listed in Table 1.

Histology
It shows the pericarp of around 250 µm thick and consist of thin walled parechymatous cells somewhat radialy oblong and possess dense accumulation of tannins.Mesocarp shows thin layers of parenchyma with the vascular strands including masses of fibers, xylem and phloem.The endocarp shows columnar sclerides.The cotyledon is seen in parallel longitudinal sections.It is bilobed and embryo is placed in wide longitudinal cylindrical chamber (Figures 2 to 4).

Fingerprinting and spectroscopic analysis
HPTLC fingerprinting showed better separation of the components.Planer chromatogram generated was used to determine existence of present phytoconstituents.The R f value was found to be 0.19 (Graph 1 and Figure 5).UV calibration curve was plotted for plant extract as well as biomarker, gallic acid at 275 and 260 nm, respectively.UV-Spectroscopic analysis showed good correlation between plant extract and standard (gallic acid).The extract and biomarker exhibited 0.074, 0.0511 and 0.003, 0.0429 intercept and slope values, respectively (Tables 2 and 3; Graphs 2 and 3).UV-Spectroscopic analysis showed good correlation between plant extract and standard (gallic acid).FTIR and NMR spectra were run for extract and biomarker.The extract and marker showed good correlation in FTIR.

DISCUSSION
Thus in recent years there is spurt in the interest regarding survival of Ayurvedic forms of medication due to shorcomings of modern medicines.Tannins serves as natural defense mechanism against microbial infections can also be used in some inflammatory conditions (Abdul, 2005;Adel, 2010).Phytochemical analysis of hydroalcoholic extract of fruits of Q. infectoria showed the presence of tannins (Basri, 2004) which is confirmed by fingerprinting and spectroscopic analysis.The study can be further extended in formulation of this potent candidate for treatment of skin diseases as tannins act as free radical scavengers as well as antimicrobial agents.

Figure 3 .
Figure 3. Entire section of fruit.

Table 3 .
Calibration curve of gallic acid at 260 nm.