Therapeutic phytoelemental profile of Trichosanthes dioica

In India, Trichosanthes dioica is known as pointed gourd (in English), parwal (in Hindi) and potol (in Oriya) it is distributed in equatorial and subtropical regions and widely found in northern India. The present study is a unique example of interdisciplinary research as it deals not only with the phytochemical investigation of Trichosanthes dioica, using natural product technology but also correlates the presence of certain trace elements with its biological activities using Laser induced break down Spectroscopy (LIBS). The study reveals the isolation of one known and two unknown flavonoids and the presence of certain glycemic elements, responsible for the observed antidiabetic activity of Trichosanthes dioica. Hence, the presence of isolated flavonoids and trace elements can be very well correlated with its medicinal value. Copyright © 2010 VBRI press.

Dr. Watal has five Patents for her inventions and more than eighty publications in journals of high repute to her credit. Recently she has published two book chapters on Advanced Protocols in Oxidative Stress in an edited book of International publisher, "Methods in Molecular Biology Series". Dr. Watal is currently involved in a screening program of herbal extracts from India, China and Nigeria, for their bioactivities. She has designed the laboratory control and test procedures, to assure the potency and purity of activity testing, which can be ratified from any independent lab in the world. She has invented highly effective oral medication for diabetic patients. She believes that natural resources can play a big role in Diabetes management. Her motto is "Integration of natural medicine into conventional treatments".

Introduction
Trichosanthes dioica, (green potato) is cultivated widely in eastern India. It is a rich source of carbohydrates, vitamin A, and vitamin C and major nutrients and certain essential trace elements such as magnesium, potassium, copper, sulfur, and chlorine which that seems an important role in human physiology [1][2][3][4]. The phytochemical investigation carried out in past with other species of Trichosanthes reports a number of different classes of compounds viz. flavonoids [5], alkaloids, glycosides [6], terpenes and sterols [7], from fruits and seeds of the plant could be responsible for their medicinal value.
Flavonoids are secondary metabolites [8] helps in regulation of normal plant development [9] and are responsible for colors of plant organs. These compounds are not only present in plants as constitutive agents but are also accumulated in plant tissues in response to microbial attack [10]. Flavonoids form a large group of natural Copyright © 2010 VBRI press. phenolic compounds, considered as safe and natural antioxidant [11].
Traditional use of Trichosanthes dioica in folk medicines for treating diabetes mellitus [1] and presence of flavonoids [5] in addition to trace elements viz. Cu, K and Mg etc. [1] inspired us for conducting this study. It has been reported that the presence of flavonoids and trace elements viz. Cu, K and Mg are responsible for antidiabetic activity of plants [4]. Laser-induced breakdown spectroscopy (LIBS) is a useful technique for varied applications. The most important is material analysis. The interaction of a high power-pulsed laser beam with the surface of the target material leads to the formation of a laser-induced plasma. Optical emission from the plasma consists of atomic and ionic spectral lines, characteristic of the constituent species, superimposed on a broad band continuum that is due to electron-ion recombination and free-free interactions. Identification of spectral lines and measurement of their intensities provides qualitative and quantitative information about the constituents of the target material [12,13]. In the present study the LIBS has been used for identifying the complete phytoelemental profile of Trichosanthes dioica responsible for its bioactivity. Based on natural product technology one known and two unknown flavonoids have also been isolated from the seeds of Trichosanthes dioica in this study.

Preparation of material
Fresh ripe fruits of Trichosanthes dioica were purchased from the local market of Allahabad, India and authenticated by Prof. Satya Narayan, Taxonomist, Department of Botany, University of Allahabad, India. A voucher specimen (AA/512/06) has been submitted. The fruits were cut into pieces and the seeds were collected and shade dried. The dried seeds (2 kg) were mechanically crushed and extracted with distilled water using Soxhlet up to 48 h. The extract was filtered and concentrated in rotatory evaporator at 35 5 o C under reduced pressure, to obtain semisolid material, which was then lyophilized to get a powder (yield about 10.5% w/w).

Extraction and isolation of compounds
The whole seed was mechanically crushed and extracted with different solvents in order of increasing polarity viz. hexane, benzene, ethyl acetate and ethanol. The ethanol fraction was loaded in a silica gel column and was run in gradient of increasing polarity using different solvents (hexane → ethyl acetate → chloroform → ethanol → methanol → 30% of aqueous methanol). Three pure compounds 1-3 were isolated from the last fraction of 30% aqueous methanol and characterized. TLC reveals the Rf of compounds 1, 2 and 3 as 0.45, 0.52 and 0.65 respectively (DCM: Methanol, 3:7).

Characterization of compounds
Melting points were determined by open glass capillary tubes and are uncorrected. IR and UV spectra were recorded on Perkin-Elmer 599 B and Shimadzu UV-250 spectrometers respectively. 1 H NMR, 13 C NMR, spectra were recorded in DMSO-d 6 on Bruker 300 DPX, FT NMR ( 1 H: 400 MHz, 13 C: 100 MHz) spectrometers. Mass spectra were measured on a JEOL AX 500 spectrometer. Silica gel G (60-120 mesh) was used for TLC and column chromatography, respectively. The collected fractions of the column were subjected to check homogeneity. Chromatographically identical fractions were combined and concentrated. The TLC spots were visualized under UV light and I 2 exposure as well. Spectral data for compound 1 and 2 was as follows:

Detection of trace elements through LIBS
A schematic diagram of the experimental setup for recording the LIBS spectra is shown in Fig. 1. The LIB spectra of T. dioica seeds extract powder, dissolved in distilled water, was recorded for identifying the presence of best set of elements responsible for its antidiabetic efficacy. The solution was prepared by dissolving 1.0 g of lyophilized material in 10 ml of distilled water. The spectra presented in Fig. 2 and 3, are the average of 100 scans (100 shots). The 4-channel spectrometer equipped with Change Coupled Device (CCD) (Ocean optics LIBS 2000+) comprising the 4-grating was used to get the dispersed light from the plasma. The initial three gratings had the resolution of 0.1 nm covering the wavelength range from 200-310 nm, 310-400 nm and 400-510 nm, respectively, while the fourth grating, termed a broad band grating, covered the wavelength range from 200-1100 nm and had a resolution of 0.75 nm. All four gratings were used simultaneously to record the LIBS spectra. A pulsed laser beam from a Q-switched Nd: YAG laser (Continuum Surellite III-10) was focused on the sample using a converging lens Quartz) of 30 cm focal length, the temperature of the locally heated region rose rapidly and resulted in plasma formation on sample surface. The emitted light from micro-plasma was collected using an optical fiber tip placed in the vertical plane at 45 o with respect to the laser beam and finally fed into an entrance slit of the multichannel spectrometer (Ocean Optics Copyright © 2010 VBRI press. LIBS2000+) equipped with CCD and 4 gratings. In case of aqueous extract of T. dioica the LIBS spectra were recorded at a repetition rate of 2 Hz and 175-mJ-laser energy.

Preliminary phytochemical investigation
Initial phytochemical analysis of the seeds extract powder was carried out and flavonoids were identified according to Kokte and Harborne [14,15]. The plant extract powder (200 mg) was dissolved in 100 ml ethanol and filtered. 2 ml of this filtrate was mixed with equal volume of concentrated HCL followed by addition of the Magnesium ribbon. The appearance of tomato red color indicated the presence of flavonoids in the extract of Trichosanthes dioica seeds. The similar test was performed for each solvent fraction and ethanol fraction was found to be positive.

LIBS analysis of Trichosanthes dioica extract
LIBS spectra of T. dioica seeds extract, shown in Fig. 2  and 3, were taken at optimized experimental conditions. It clearly revealed the presence of Mg, Fe, Na, K, Zn, Ca, H, O, C and N elements in the spectral range from 200nm to 900 nm. According to the Boltzmann's distribution law, intensity is directly related to concentration [20].
Therefore, the intensity of the observed spectral lines corresponding to major and minor elements present in the extract is not only an indication of their concentration but it also assists in defining their role in diabetes as well as in diabetes-induced oxidative stress management. The ratio of intensities of the detected elements (Mg, Fe, Na, K, Ca, C, H, O and N) to the intensities of reference lines (C and O that are the essential elements of plant materials), was estimated to evaluate their proportional concentration. Since gratings with different resolutions were used, the whole spectra was divided in two sections: the first (resolution 0.1 nm) covered the wavelength range from 200-510 nm and the second (0.75 nm resolution) spanning the wavelength range between 510 and 1100 nm. To find the intensity ratios of spectral lines, the C line at 247.88 nm (as a reference line for the spectral range of λ 200-510 nm) and the O line at 844.10 nm (as the reference line for spectral wavelength range of 500-1100) nm have been selected. Calculated intensity ratios of Fe/C, Mg/C, Ca/C, K/O, Na/O, H/O and N/O are given in Table 1 and 2.
Demand of interdisciplinary research is increasing in the new millennium. The present study is an excellent example of unique combination of Physics, Chemistry and Biological techniques used for unveiling the scientific facts. LIBS can scientifically unfold the mystery enclosed in medicinal plants based on their inorganic constituents. The study is based on systematic, scientific and critical evaluation of Indian medicinal herbs in treating diabetes mellitus. Since, there are no reports till date on glycemic elemental assessment of such herbs based on LIBS, therefore, the present study is the first reporting of its type. It deals with the monitoring of the role of the trace mineral elements, present in seeds extracts in management of diabetes and its complications. Our results and discussion clearly show that the specific concentration of these elements play an important role in diabetes management. Function of most of these elements has already been reported in different literature. Most essential trace mineral elements act preliminary as catalyst or as co-factors in enzyme systems [21]. The role of some inorganic elements like Ca, K, Mg, Al, and traces of Fe etc. in the improvement of impaired glucose tolerance and their indirect role in the management of diabetes mellitus are being increasingly recognized. Ca and other trace elements play an important role in the release of insulin from β cells of islets of Langerhans by activating gene expression via CREP (calcium responsive element binding protein), which is responsible for exocytosis of stored insulin [22]. According to recent reports, specific concentration of K takes part in carbohydrate metabolism; it is active in glycogen and glucose metabolism, converting glucose to glycogen that can be stored in the liver for energy [23]. Mg helps in maintaining this specific amount of K in the cell thus, Na and K balance is finely tuned. Mg is a cofactor in various enzyme pathways involved in glucose oxidation and it also modulates glucose transport Copyright © 2010 VBRI press. across cell membrane. Hypomagnesaemia is common in patients with diabetes due to the excess of urinary Mg losses. Deficiency of Mg can potentially cause state of insulin resistance. Hence, low levels of Mg are commonly seen in the people with diabetes [24]. There is evidence that Mg supplementation may be helpful in improving insulin resistance [4]. Since, its role in glycemic control was unknown therefore the present study is a forwarding step in this direction.

Conclusion
In the present study, we were inferring isolated flavonoids and trace elements which can be very well correlated with medicinal value of T. dioica. Also, the results clearly demonstrate that LIBS technique can be used as a basic yet powerful tool for determining the presence of glycemic elements.