Identification of Active Compounds

Some plants have more than one chemical as an active principle responsible for their biological properties. Phytochemical tests was done to find the presence of the active chemical constituents such as alkaloid, glycosides, flavonoids, reducing sugars, triterpenes, phenolic acid and tannis. Detailed Phytochemical screening was carried out as per standard methods. (Kokate, 2000; Habone, 1999; Tiwari et al., 2011). Gas Chromatography Mass Spectrometry Analysis: To find the bioactive compound in the extracts and their quantification, it was further analyzed by GC/Ms. The various crude extracts of the plants were analyzed by using a Shimadzu, 2010 equipped with a splitless mode injector system,a flame photometric detector and a TRB 5 MS capillary column with 30m length,0.25 mm internal diameter and 0.25 um stationary film thickness prepared from Phenomenex was used for determination of biopesticide. The oven temperature was hold at 50? for 1 min then programmed at 25 ?/min to 125? ,from 125? to 300? at the rate of 10oC/min and finally holds for 15 min. The minimum detection limit of GC-MS for the determination of pesticide was 0.1ppb.The total time for one GC run was 30 min.Other operating conditions were as follows:ultra pure helium,passed through a molecular sieve trap and trap oxygen was used as the carrier gas at constant linear velocity of 40.7 cm/sec. The injection port temperature was 250?and used in splitless mode at ratio of 10:00.The detector temperature was hold at 280?.A hydrogen generator instrument was applied to supply hydrogen gas for flame photometric detector (FPD)at a flow of 1.22ml/min . The percentage composition of the crude extract constituents was expressed as a percentage by peak area. Total GC running time was. Interpretation on mass spectrum GC-MS was conducted using the database of national institute of standard and technology (NIST). The mass spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST Library. Results:There are several factors that can interfere qualitatively and quantitatively in the results, in the metabolites presence, one of them can be seasonal factor, which may be considered as the period of seed collection. The present study made an effort to screen for its photochemicals the photochemical screening of methanolic extract of piper nigrum showed maximum number of metabolites like alkaloid, flavanoids, carbohydrates, proteins, phenols, phytosterols, tannins in table 1. Table No1: Photochemical screening of Piper NigrumS. No Phytochemicals Test Performed Result1. Alkaloids Hager's testMayer's Reagent Positive2. Carbohydrates Molish test Positive3. Saponins Froth test Negative4. Glycosides Borntager,s test Negative5. Protien&Amino acids Biuret's Test Positive6. Phytosterol Libermann –Burchard'test Negative7. Phenolic compounds Ferric chloride test compounds Positive8. Flavonoids Alkaline Reagent testLead Acetate test Positive9. Terpenoids Salkowski's test Positive10. Tannins Neutral FeCl3 PositiveFg: GC-MS Chromatogram of Piper Nigrum L. Table 2: Quantitaive analysis of P.Nigrum: Name Conc(%) Ret.Time m/z Area1. 3-Carene 7.846 3.574 93.00 9772. Delta-3-carene 18.671 3.655 93.00 23253. Alpha –Limonene 1.233 3.828 93.00 1554. Cinene 15.492 3.903 93.00 23125. Beta-Linalool 1.328 5.049 71.00 1656. Delta-Elemene 11.678 10.100 121.00 14547. Ylangene 0.418 10.420 121.00 528. Copaene 6.270 11.048 105.00 7819. Levo-beta-Elemene 1.222 11.390 93.00 15210. Caryophyllene 32.261 12.117 69.00 401911. Alpha Caryophyllene 7.053 12.919 93.00 87812. Alpha-Curcumene 0.557 13.473 73.00 8313. Eudesma-4(14),11-diene 0.749 13.684 93.00 9314. Alpha-Selinene 0.561 13.879 189.00 6915. Beta-Bisabolene 0.426 14.104 69.00 5316. Delta-Cadinene 2.335 14.441 161.00 29017. Caryophylleneoxide 1.110 15.953 43.00 13818. Myristyl chloride 0.614 16.049 43.00 7619. Ledol 0.097 16.558 43.00 1220. (-)-Spathulenol 3.700 17044 43.00 46021. Ar-tumerone 3.657 17.740 83.00 455In the present study ,the chemical profile of black pepper was identified using GC-MS.GC-MS investigation revealed that 21 compounds are identified in Black pepper extract . The major components of black pepper were caryophyllene (32.261),delta.3-carene (18.671) as well as Cinene(15.492) as indicated in Table 2.DiscussionStudies have reported that the spicy tang of pepper is due to the presence of piperamides which are the pungent bioactive alkaloids accumulate in the skin and seeds of fruit.(Nahak and Sahu, 2011) reported the presence of alkaloids, glycosides, tannins, phenol, essential oil and protein in Piper nigrum.The result of the study corroborates with findings of (Zahira et al.,2016) which shows alkaloid,flavanoid,carbohydrates,tannins,proteins,phenols,phytosterols,while terpeniods and saponins were absent.(Priya and Sarvana 2016) Revealed the presence of 15 components of blackpepper out of that highest integrated percentage of Aspartic acid is present and moderate amout of Limonene,Betasteosterol,and Asarinin.Previous study on GC-MS analysis were carried by (Morshed et al.,2017 )they reported that the Carophyllene ,Limonene and camphene were major components in oil of blacl pepper which were close to our findings. The differences are probably due to various geographic and ecological conditions.ConclusionThe phytochemical screening of methanolic extract of black pepper decleared the presence of many phytochemical components.The GC-MS analysis showed a number of components.The present study may be useful for further study in future to explore the biosynthetic activity of black pepper.