Identification of novel anticancer terpenoids from Prosopis juliflora (Sw) DC (Leguminosae) pods

Purpose: To identify a novel source of terpenoid anticancer compounds from P. juliflora (Sw.) DC. (Leguminosae) pods as a medicinal substitute for cancer medicines. Methods: The pods were collected, dried and pulverized. The ethanol extract was prepared by maceration. Various phyto-constituents were detected in the extract by UV-VIS spectroscopy at a wavelength ranging from 200 800 nm. The molecular formula, chemical structure, and percent peak area of these phyto-constituents were determined by gas chromatography-mass spectrometry (GC-MS). attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used for functional group determination of bioactive compounds. Results: P. juliflora pods were rich in tannins, carotenoid and terpenoids. Nineteen bioactive compounds were detected. Out of these, thirteen are here reported for the first time with four of them exhibiting anticancer activities, while two belong chemically to terpenoids. Furthermore, FTIR established characteristic peaks for the various biologically-active functional groups. Conclusion: The results show that P. juliflora pods is a valuable source of anticancer, antitumor and chemoprotective compounds, especially terpenoids, that can potentially be developed as alternatives to current painful and costly cancer therapies.


Medicinal plants have been used orally and as
Plant terpenoids are extensively used for their aromatic qualities and have played a role in traditional herbal remedies in India and China [10,11].
Since the discovery of terpenes, more than 150 years ago, scientists have identified and isolated more than 50,000 terpenoids.But the source of medicinal terpenoids, was mainly fungi.In the current study, a plant source was used for the first time, to identify anticancer terpenoids.Identification and confirmation of natural compounds is a crucial and vital step for anticancer drug discovery.GC-MS is a very powerful technique towards the specific detection and potential identification of compounds.For detailed screening of terpenoids, ultravioletvisible spectroscopy [12], attenuated total internal Reflectance-Fourier transform infrared spectroscopy for functional group detection and GC-MS were used [13].

EXPERIMENTAL Plant material
The pods of P. juliflora were picked from Jinnah Garden, Lahore, Pakistan during summer season, 2017 and identified by a taxonomist, Dr Zubaida Yousaf (Department of Botany, Lahore College for Women University).A voucher specimen (no.LCWU-15-128) was deposited in Prem Madam Herbarium, Department of Botany, Lahore College for Women University, Lahore, Pakistan.

P. juliflora pods extraction
P. juliflora pods were cut, shade dried, and powdered for further use.Two grams of powdered pods were extracted with 50 mL of ethanol with gentle stirring for 72 h, filtered and crude extract was collected.

P. juliflora extract analysis
Freshly prepared extract was exposed to standard methods of phytochemical analysis to detect the constituents, viz.terpenoids, flavonoids, alkaloids, phenolics and glycosides [14].In our previous experiment, we have also investigated the qualitative and quantitative terpenoid contents with high anti-oxidative potential [9,15].
For UV-VIS analysis, the extract was centrifuged at 2000 rpm (15 min) and processed through micro filters.The sample was diluted to 1:4 with ethanol.The extract was scanned using wavelength ranging from 200 -900 nm through Perkin Elmer Spectrophotometer and the characteristic peaks were noticed.
ATR-FTIR analyzed the phyto-constituent in extract.Infrared spectra were obtained with a Harrick Split-pea ATR microscope interfaced to a Perkin Elmer 2000 Fourier transform infrared spectrometer with a single bounce diamond ATR cell.Spectra over the 4000 -1000 cm -1 range were recorded in % transmittance mode to fetch the characteristic peaks of the functional groups.The collected data are processed by optical user software.
The phytochemical constituents were determined by GC-MS analysis system (Clarus 500, Perkin Elmer, CT, USA) containing Agilent DB-5 (Thomas no.2713R99) with specification (5 % diphenyl dimethyl polysiloxane, 30 m × 0.25 mm × 0.25 µm, 5 cage).Constant flow of Helium gas was maintained at 1 mL/min.The oven temperature was maintained at 110 °C for 2 min and then increased to 280 °C in 9 min.Aliquots (2 µL) of ethanol extract were injected into the instrument, injector temperature was 250 °C.The MS detection took 36 min to screen the predominant constituents with computer-driven algorithm and using mass spectrum library (NIST version 2.0, 2005) [17].Turbomass 5.2 software program was used in the analyzer.

Phytochemical profile
Qualitative phytochemical analysis of pods extract indicated the presence of terpenoids, falvonoids, phenols, quinine, saponins, glycosides and alkaloids.

DISCUSSION
Pakistan being an agricultural land is a precious source of medicinal as well as ornamental plants.
A huge population of Pakistan cannot afford costly imported anticancer synthetic medicines.Terpenoids will not only reduce economic burden but also provide local medication with low side effects.
Spectroscopic methods are becoming a powerful analytical tool for secondary metabolite profiling as well as the qualitative and quantitative fingerprinting of bioactive compounds.The qualitative UV-Vis spectroscopy of ethanol extract of P. juliflora displayed characteristic peaks of major bioactive compounds like terpenoid, carotenoids and tannins.The results obtained were confirmed by comparing the absorption values and wavelength with already published research data [18].These results showed that the pods are rich source of terpenoids [15].Previously, (-) mesquitol was isolated from P. juliflora, heartwood [19].The antibacterial properties of alkaloid from P. juliflora were also determined [6].There seems a connection between terpenoids and antimicrobial activity, hence, we used pods for the first time to identify anticancer terpenoids and fortunately, we are successful.It will bring a revolution in cancer treatment with less side effects and low cost.Many scientists proposed that terpenoids are natural products for cancer therapy [20].
Previously, we revealed that P. juliflora pods had 25 % of total terpenoid contents [15], and these may be major contributing factors for the highest antioxidant potential of pods by DPPH free radical scavenging activity (92 %) with IC 50 (0.045 ± 0.005) [9].
For further confirmation of bioactive compounds, these findings must be supplemented with other analytical techniques like FTIR and GC-MS, for proper extract characterization and finger-printing of metabolites.The FTIR spectrum identifies the functional group of the active components on the basis of peak value in infrared radiation region.The functional groups were identified by comparing the wavelength of the phytochemicals with infrared spectroscopy absorption Table published online by UC Davis ChemWiki and OChemonline.The results confirmed the presence of phenol, cyclohexane, alkene and aromatic compounds [18].
Functional groups play in the activity of secondary metabolites, their kinetics, and overall role in chemical activity and therapeutic functioning of compounds [21].
GC-MS analysis identified 19 bioactive compounds comprising 5 anticancer compounds with 2 novel anticancer compounds and 2 anticancer terpenoids from the ethanol extract of pods of P. juliflora (Figure,3).Furthermore, for the first time, in the pods a total of 13 novel compounds, 2 anticancer compounds have been identified i.e. dibutyl phthalate and hydroxyurea.Dibutyl phthalate (Genoplast B), magnificent potential compound against breast and bladder cancer.Similarly, hydroxyurea (0.15 %) has been used in in vitro anticancer drug screening.Although alkaloids were identified from P.
juliflora, but they are used under different sources like heartwood and leaves [6,19].
Previously, these terpenoids have been synthetically produced or obtained from microbes like fungi, bacteria and marine organisms [22].

CONCLUSION
The findings of this study indicate that P. juliflora pods are a novel source of anticancer, chemopreventive and medicinally important bioactive compounds, especially terpenoid.Thus, further investigations, including preclinical studies are recommended on cancer animal model.

Figure 2 :
Figure 2: FTIR spectra of ethanol extract of P. juliflora pods with peak values and functional groups

GC-MS data
Interpretation of GC-MS was conducted by comparing spectrum, SI (similarity index), RT (retention time) and RI (retention indices) of the unknown component with database of NIST with 62,000 patterns and Scifinder having more than 250,000 patterns of compounds.The percentage relative amounts of all components were calculated by comparing it average peak areas to the total areas.The biological activity of the compound was based on Duke's Phytochemical and ethnobotanical Databases by Jim Duke (Agricultural Research Service/USDA) [17].The name, molecular weight and structure of chemical motifs of the test materials were presented in Tables (3,4,5) and Figure 3.

Table 1 :
UV-VIS peak values of ethanol extract of P.

Table 2 :
FTIR spectral peak values and functional groups obtained from ethanol extract of P. juliflora pods

Table 5 :
Identified bioactive compounds of ethanol extract of P. juliflora by GC-MS (contd)