Phytochemical, Fluorescence and GC-MS Analysis of Methanolic Extract of Sterculia foetida L. Seeds

— The present work phytoconstituents of the seed powder of Sterculia foetidaL. extracted with 98% methanol. The extracted phytochemical compound subjected to qualitative analysis, quantitative analysis, fluorescence analysis and GC-MS analysis. The results of qualitative phytochemical screening confirm that the presence of tannins, phenols, steroids, cardiac glycoside and coumarin. The significant amount of carbohydrate, protein, lipid, tannin and total phenol estimated through quantitative analysis of phytochemicals. The seed powder with the picric acid exhibited fluorescent yellow during fluorescence analysis undertaken with short ultra violet light at 254 nm. There are 13 bioactive compounds were identified through GC-MS analysis of seed powder of S. foetida L. These various bioactive compounds possess a wide range of activities such as disease control, pest control and microbicidal effect.


INTRODUCTION
The medicinal plants are widely used in traditional medicine to prevent and treat various diseases. The phytoconstituents present in the various part of the plant can be exhibit anti-cancer, anti-tumour, anti-diabetic, antispasmodic, anti-inflammatory, anti-oxidant and antibacterial actvities 23 . Sterculia foetida is commonly called as Wild Indian almond belonging to the family Sterculiaceae. The fruits, seeds and leaves of S. foetida have been conventionally known for its many therapeutic purpose 5 . A different variety of pharmacologically active compounds have been isolated from the leaves of S. foetida and these compounds from the leaves used as astringent, laxative, antifungal, anti-inflammatory and antiulcer medicines 26 . Nanadagopalan et al., (2015) 23 isolated twenty seven bioactive compounds from the methanolic extract of S. urens leaves. The important compounds present in the leaves extract were 3, 7, 11, 15-Tetramethyl-2-hexadecen-1-ol, sucrose, 2, 4-Dihydroxy2, 5-dimethyl-3(2H)-furan-3-one, 5(2H)-Oxazolone, 4-(phenylmethyl)-, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-, Megastigmatrienone and 2-Methoxy-4-vinylphenol etc and these compounds could contribute the medicinal quality of the S. foetida leaves. S. foetida seeds are not toxic and edible to humans and animals 5 . The bioactive compounds sterculinine-I, sterculinine-II, and soyacerebroside-I were isolated from the seeds of S. lychnophora 34 . In view of that the present work aimed to identify the pharmacologically active biomolecules responsible for anti-cancer and antioxidant from the methanolic extract of S. foetida seed. were collected from dried fruits were grinded and sieved to make fine powder for further study.

Preparation of extracts
Two fifty grams (250 g) from seed powder was extracted with 250 mL of 98% methanol in Soxhlet apparatus for 24 hours. After the extraction the crude extract was filtered through a Whatman number 1 filter paper. Later, the crude extract was subjected to evaporation in a rotary vacuum evaporator for dryness. After the evaporation process completed the concentrated extract was collected and stored at 4°C for further analysis 9,31 .

Quantitative analysis of phytochemicals
The methanolic seed extract was subjected to quantitative analysis by spectrophotometer method. The extract was analyzed for Carbohydrate by Anthrone method 12 , Protein by Lowry's method 20 , Total Lipids 35 , Total Phenol 2 and Total Tannin 2 .

Fluorescence analysis
Fluorescence analysis is the most important parameter of pharmacognostical evaluation. This analysis was carried out as per the standard protocol 16,18,29 . In the present work, the seed powder was treated with different solvents and chemicals. The seed powder was subjected to fluorescence analysis in visible/daylight and UV light (254 nm).

GC-MS analysis
Aim of this analysis is to identify the pharmacologically active biomolecule (anticancer & antioxidant) present in the seed extract. The methanolic extract of S. foetida seed powder was subjected to GC-MS analysis on the instrument GC and MS JEOL GC mate equipped with secondary electron multiplier (Agilent Technologies 6890N Network GC system for gas chromatography). The column (HP5) was fused silica 50 m×0.25 mm I.D. The study conditions were 20 min. at 100°C, 235°C for column temperature at 3 minutes and 240°C for injector temperature, carrier gas was helium, and split ratio was 5:4. The 1 μl of the sample was evaporated in a split-less injector at 300°C and the run time was 22 min. The phytoconstituents of the extract was identified by Gas Chromatography coupled with Mass Spectrometry. The GC-MS spectrum was analyzed using the NIST08 library which has more than 62,000 patterns 4,25 .

Qualitative analysis of phytochemicals
The essential information regarding the phytochemical constituents is generally determined through qualitative phytochemical analysis of plant extracts. The qualitative analysis of the methanolic seed extract showed the presence of secondary metabolites such as tannins, phenols, steroids, cardiac glycoside and coumarin and this analysis also confirmed that the absence of the phytochemical phlobatanin (

Quantitative analysis of phytochemicals
The amount of Carbohydrate, Protein, Lipid, Phenol, and Tannin present in the seed extract was obtained by plotting the Optical Density value for the standard test tubes. The optical density value for sample tube is compared with one of the same optical density value of the standard tube. The optical density reading of the sample tube is plotted on the graph and the concentration of the component is determined. The amount of carbohydrate present in the 2ml of seed sample is estimated to be 100µg as shown in the Fig. 1a. The amount of protein present in the 2ml of seed sample is estimated to be 20µg as shown in the Fig.  1b. The amount of lipid present in the 2ml of seed sample is estimated to be 50µg as shown in the Fig. 1c. The amount of phenol present in the 2ml of seed sample is estimated to be 60µg as shown in the Fig. 1d. The amount of tannin present in the 2ml of seed sample is estimated to be 60µg as shown in the Fig. 1e. The secondary metabolites like carbohydrate, protein and lipid have antioxidant, antimicrobial and antiviral properties 21 .
According to the literatures, phenolic compounds are known to exhibit antioxidants, anticancer, antiinflammatory, antimicrobial, anti-allergic and antifertility activity 14,30 . Tannin is one among the major active secondary metabolite found in wide variety of plants 11 . Tannin has been reported to possessing antiviral, antioxidant, antibacterial and antitumor activity 6,17 . In the present work confirm that there is a significant quantities of pharmacologically active secondary metabolites such as carbohydrate, protein, lipid, phenol, and tannin are present.

Fluorescence analysis
The results of fluorescent analysis of dried seed powder of S. foetida with different chemical reagents are given in  32 reported that the secondary metabolites like coumarin, sapogenin and terpenoids show yellowish green fluorescence under short UV light. In the present work, the major bioactive metabolites present in the crude powder of S. foetida seed was found to be coumarin, phenols, tannins and sterols and this results could confirm that the pharmacognostic properties of S. foetida seed.

GC-MS analysis
The GC-MS analysis of methanolic extract of S. foetida seed powder divulge the presence of thirteen phytochemical compounds that could possess the pharmacological and microbicidal activity. The identification of the biomolecule was confirmed based on the retention time and molecular formula. The biologically active compounds with their Retention time (RT), Molecular formula, Molecular weight, Molecular structure and their Biological activity are presented in Table 3

IV. CONCLUSION
On the basis of above findings, it can be concluded that the phytochemical screening and fluorescence analysis confirm that the methanolic extract of Sterculia foetida seed contains various pharmacologically active secondary metabolites. Furthermore, there are thirteen pharmacologically active biomolecules were identified through GC-MS study. Further studies are required to find out the efficacy of methanolic extract of Sterculia foetida seed as it may offer an effective alternative source against many diseases with less to no side effects.