Phyto-chemicals and Spectral Characterization of Novel Glaucarubinone Quassinoid from Simarouba glauca Leaves: In vitro Anti-proliferative Potential against Breast Adenocarcinoma Cell line

Breast cancer is the second most prevalent type of cancer in women.¹ The compromised immune system brought in by radiation and surgery, together with its high expense, contributes to the growing cancer burden worldwide.² One strategy to decrease the incidence of cancer globally is to find a greater availability of innovative anticancer phytomolecules that can be combined with chemotherapy. Because evidence-based, safe and effective are thought to be useful in treating and preventing a wide range of illnesses.^3,4

The Simarouba glauca leaf is a member of the Simaroubaceae family, also referred to as “Laxmitaru,” “Paradise tree,” or dysentery bark.⁵ The leaf decoction of SG has been claimed to anticancer, antimalarial, antiviral, antibacterial and anthelmintic properties in traditional medicine.⁶ Quassinoids of SG include dehydroglaucarubinone, glaucarubine, glaucarubolone, glaucarubinone,benzoquinone, holacanthone, melianone, canthin, simaroubidin, simarolide, simarubin, simarubolide, sitosterol and tirucalla.⁷ Quassinoids are triterpene lactones used in folk medicine for their pharmacological properties and seem to be well documented.⁸ Anticancer studies on the crude leaf decoction of SG have been recorded but have not been proven scientifically. Furthermore, in folk practice, the aqueous leaf decoction of SG has to be prepared daily, which leads to non-compliance.

The current research has been focused on the isolation of a novel lead phytochemical, ‘Quassinoid’ in the extract of Simarouba glauca leaf and evaluating the anticancer potential of the isolated extract using an in vitro assay against the MDA-MB 231 breast adenocarcinoma cell line.

All chemicals used for this research were of analytical grade from Pure Chemicals, Loba Chemicals and Merck; Standard Quassinoid-Glaucarubinone was from (NCI) Frederick, 21702, United States. MTT (Himedia), all cell culture reagents and disposable dishes were purchased from Thermo Scientific, Olympus and Tarson’s India Limited respectively. MDA MB-231 cell lines were purchased from NCCS in Pune. FTIR, UV-Spectrophotometer (1650 PC Shimadzu, Japan), UPLC (Waters, USA), Acetonitrile and CDCl₃ (Sigma Aldrich), NMR spectrometer (BRUKER 400MHz), Superfit Vacuum Rotary Evaporator, HPTLC (Camag, Muttenz, Switzerland), Digital Ultra Bath Sonicator (Labman Scientific).

Fresh leaves of Simarouba glauca were collected from the herbal garden maintained and voucher specimens were stored by PSG College of Pharmacy, PSGIMSR and Hospital Campus, Coimbatore, India. Plant were identified and authenticated (BSI/ SRC/5/23/2021/Tech./3L8) by plant taxonomist Dr. M. U. Sharief, Botanical Survey of India (BSI), TNAU Campus, Coimbatore, Collected SG leaves were washed with tap water to remove the dust and adhering debris before being shade dried and pulverized into coarse powder for the extraction process.

Simarouba glauca leaf extracts (chloroform, methanol and aqueous) were produced using a Digital ultra-bath sonicator and temperature controller (Frequency 40 kHz) at 30°C with a power of 60-100W. To maximize the extraction efficiency, the defatted SG leaf marc was extracted using 250 mL of solvents employing fine ultra-wave contact duration of about 40 min. The prepared SG leaf extracts were concentrated using a rotary evaporator and calculate the percentage yield of the SG leaf extracts.⁹

The acquired SG leaf extracts performed phytochemical analysis to identify the following phytocompounds such as proteins, alkaloids, glycosides, sterols, terpenoids, carbohydrates, flavonoids, saponins, tannins, volatile oils and mucilage.¹⁰

The MDA MB-231 cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM) high glucose medium. The culture was carried out in a CO2 incubator at 37°C with a 5% CO₂ atmosphere and 18-20% O2. SG leaf extracts were assessed for their in vitro antiproliferative potentials using the MTT assay.¹¹ 20,000 cells per well were seeded into 96-well plates. Different doses of SG leaf extracts were added (12.5, 25, 50, 100, 200 μg/mL) and 1 μM/mL of Doxorubicin was used as standard. The plate was incubated for 24 hr at 37°C. After the incubation period, the plates were filled with 0.5 mg/mL MTT reagent. To protect the plate from light, they are covered with aluminium foil and incubated for 3 hr. Then, 100 μL DMSO solution was added to enhance solubility and read the absorbance at 570 nm. Table 1 and Figure 1 provide % cell viability data analysis along with IC₅₀ value of the respective SG leaf extracts.

Figure 1:
% Cell Viability of SG leaf extracts treated with MDA MB 231 cells after 24 hr.

The IC₅₀ value was calculated using a linear regression equation.

Where,

Y=50, M and C values are taken from the viability.

The prepared SG leaf extracts were analyzed using TLC.¹² Preparative TLC analysis was done to isolate the quassinoid. The TLC plates were made manually and activated. Then, the bioactive extract of SG leaf was dissolved in methanol and applied to the activated plates for separation of quassinoid. A chromatogram was developed using n-hexane: ethyl acetate (2:8) as a mobile phase. Then the separated chromatograms were marked with 10 distinct colour bands under daylight, at 254 nm and 366 nm using a UV lamp.¹³ It was scraped off dissolved in methanol and filtered. After the eluent was evaporated, the calculated yield was assigned the following names as Fr1 (220 mg), Fr2 (140 mg), Fr3 (90 mg), Fr4 (190 mg), Fr5 (110 mg), Fr6 (14 mg), Fr7 (19 mg), Fr8 (43 mg), Fr9 (20 mg) and Fr10 (32 mg). The fractions were then stored in eppendorf tubes. Using the HPTLC fingerprint technique, the amount of quassinoid present in bioactive fractions of SG leaf was estimated and displayed in Figure 2.

Figure 2:
Quantification of Quassinoid from bioactive fractions of SG leaf extract by HPTLC.

SG leaf fractions were subjected to quantification using the HPTLC method. The mobile phase used was n-hexane: ethyl acetate (2:8)¹⁴ and standard quassinoid concentrations of 2, 3, 4, 5 and 6 μL of (Glaucarubinone). Fingerprint profiles were obtained by scanning the tracks at a wavelength of 254 nm. Table 2 and Figure 2 display 3D overlay, standard and fraction 4 chromatograms with R_f values. The amount of quassinoid in the bioactive fraction (Fr4) of SG leaf, or the QEAF (Quassinoid Enriched Active Fraction), was calculated using a calibration curve with standard quassinoid (GB).

The values are mean± SD (n=3).

The isolated quassinoid in QEAF of SG leaf was measured for UV absorption spectra at 250 nm. FT-IR analysis was performed and significant functional groups were characterized as illustrated in Table 3. LC-MS phytochemical profiling of isolated quassinoids in QEAF of SG leaf was performed. The compound was identified using the BEH C18 column. The gradient elution was done with solvent A: 0.1% formic acid in water and solvent B: acetonitrile. The solvents ratio (A: B) was varied from 0-1 min (A: 98% and B: 2%), 1-6 min (A: 50% and B: 50%), 12-16 min (A: 5% and B: 95%) and 17-20 min (A: 98% and B: 2%). The sample injection volume (QEAF) was set to 5 μL, with a flow rate of 0.2 mL/min. The m/z was measured at 496 g/mol [M+H],^15,16 and shown in Figure 3 and Table 4.¹³C and ¹H NMR analysis of isolated quassinoid in QEAF of SG leaf was predicted by BRUKER 400MHz using CDCl₃ as solvent.^17,18 The ¹³C and ¹H NMR peaks of the isolated quassinoid in the bioactive fraction of SG leaf are shown in Figure 4.

Figure 3:
LC-MS analysis of Quassinoid in QEAF of Simarouba glauca leaf.

Figure 4:
¹³C & ¹H NMR spectra of Isolated Quassinoid in QEAF-SG leaf (Solvent: CDCl₃).

Simarouba glauca is an evergreen flowering tree belongs to the Simaroubaceae family. The current investigations used with SG leaves were collected from the PSGCP Herbal Garden, India. It was acknowledged and authenticated (No.BSI/SRC/5/23/2021/Tech./328) from plant taxonomist Dr. M. U. SHARIEF, Botanical Survey of India (BSI), TNAU Campus. The percentage yields of Simarouba glauca leaf extracts (chloroform, methanol and aqueous) obtained from UAE (Ultrasonic assisted extraction) method. In this study highest percentage (%) w/w yield was obtained from aqueous SG leaf (23.08%) when compared to other two extracts chloroform (9.18%) and methanol (15.84%) respectively.

The prepared SG leaf extracts were subjected to phytochemical analysis and they showed the presence of flavonoids, alkaloids, glycosides, saponins, terpenoids, tannins, steroids and proteins. However, alkaloids, terpenoids and steroids were absent from the aqueous SG leaf extract. Saponins and carbohydrates were absent from the chloroform and methanol SG leaf extracts.

In vitro cytotoxicity assay of Simarouba glauca leaf extracts was done using MTT assay and the results are shown in Table 1 and Figure 1. All were effectively cytotoxic with significant IC₅₀ values of 123.05 μg/mL, 117.81 μg/mL and 155.06 μg/mL on MDA-MB 231 respectively. The results suggested that methanol SG leaf extract proved to possess effective cytotoxic potentials on MDA MB 231 cells with an IC₅₀ value of 117.81 ug/mL. Chloroform and aqueous SG leaf extracts were observed to have moderate cytotoxic potentials. Hence, the valuable bioactive extract was found to be methanol SG leaf extract. Hence, it was purified and isolated through the preparative TLC method.

The quantification of quassinoid content in 10 distinct fractions of the bioactive SG leaf extract was achieved via the HPTLC fingerprint technique. The mobile phase, hexane-ethyl acetate (2:8), was chosen based on the TLC study. Afterward, the quassinoid chemical compound found in the bioactive SG leaf extract was isolated using a preparative TLC study. The calibration curve was generated in HPTLC using different concentrations of standard quassinoid (GB) and to determine the amount of quassinoid (12.573 μg) present in the SG leaf fraction (Fr4). The results of this technique demonstrate that the separated quassinoids in fraction 4 of the bioactive extract’s SG leaf had an R_f value of 0.22, which is comparable to the Rf value of 0.24 found for standard quassinoids. The standard and the bioactive fraction 4 chromatograms with Rf values and 3D overlay are shown in Table 2 and Figure 2.

The UV absorption spectrum (λ_max) of isolated Quassinoid in Enriched Active Fraction 4 (QEAF) of SG leaf was seen at 250 nm. FT-IR analysis was employed to analyze the functional groups of the isolated quassinoid in QEAF. Table 3 shown the outcome of the IR functional groups interpretations of bioactive SG leaf fraction 4.

The Mass spectra of the isolated quassinoid peak were observed at m/z 496.33 g/mol [M+H], as well as other fragmented peaks (m/z) were also measured with a retention period of 12.113 min and their m/z values were annotated in positive mode and shown in Figure 3 and Table 4. This study compares the measured mass value m/z, 496 of the isolated quassinoid found in the QEAF of SG leaf to the precise mass value of a known quassinoid (Glaucarubinone) 495 g/mol.

¹³C and ¹H NMR spectra of isolated quassinoid in QEAF of Simarouba glauca leaf was predicated by BRUKER 400MHz using CDCl₃ as solvent. The respective peaks associated with quassinoids (Glaucarubinone) present in the QEAF of SG leaf are recorded in Figure 4. Chemical shift (ppm) values of ₁₃C and ₁H NMR are as follows:¹³C NMR (δ ppm): 7.5 (1C, s),10.0 (1C,s), 14.7 (1C,s), 23.0 (1C, s), 24.8 (1C, s), 25.46 (1C, s),31.3 (1C,s), 32.9 (1C,s), 41.6 (1C, s), 44.4 (1C, s), 45.2 (1C, s),45.5 (1C,s), 47.4 (1C,s), 70.0 (1C, s), 71.1 (1C, s), 75.0 (1C, s),76.7 (1C,s), 79.1 (1C, s), 82.9 (1C, s), 108.8 (1C, s), 124.3 (1C, s), 164.5 (1C, s), 166.7 (1C, s), 176.3 (1C, s), 195.9 (1C, s).¹H NMR (δ ppm): 0.96 (3H, t), 1.11 (3H, d), 1.20 (3H, s), 1.44 (3H,s), 2.39 (3H, s), 2.02 (4H, m), 2.30 (1H, m), 2.75 (1H, s), 2.97 (2H, m), 3.57 (1H, d), 3.70 (1H, q), 3.96 (1H, q), 4.09 (1H, s), 4.64 (1H, t), 5.23-5.63 (1H, d), 6.15 (1H, m), 7.87 (1H, s).

Current research has shown that a powerful phytochemical-rich fraction derived from natural sources is effective against cancer.¹⁹ Plant-based anticancer drug discovery stands as a golden mark and gained popularity in the present scenario.²⁰ In addition to this new finding, other studies have shown that treating many tumours with single therapy leads to the development of drug resistance and the failure of the treatments.²¹ Therefore, there is a growing need for phytomedicine made from natural materials because these phytomolecules are thought to be safer, more cost-effective, effective and supported by evidence for the prevention and treatment of a variety of illnesses.^22,23Simarouba glauca is known as the “tree of the solace of cancer,” as the traditional medicinal practitioners employed the leaf decoction to treat the first and second stages of cancer, while the quality of life significantly seems to improve in patients with third and fourth stages of many forms of cancer. It also reduces the negative effects of chemotherapy, minimizes appetite loss and guarantees a speedy recovery without causing adverse effects.²⁴

Early cancer screening performed by the National Cancer Institute (NCI) in 1976 indicated that an alcohol extract of Simarouba root has toxic effects against leukemic cancer cells at very low concentration (less than 20 μg/mL).²⁵ The presence of quassinoids in the Simaroubaceae family is a class of bitter compounds with several biological properties including anticancer activity.²⁶ More research on crude Simarouba glauca leaf decoction has been done and the literature review reveals that the crude methanolic SG leaf extract possesses significant anticancer potentials.^27–30 The overall observation of this study, the initial process starts with the collection and authentication of Simarouba glauca (SG) leaves. Then, Chloroform, methanol and aqueous extracts of Simarouba glauca leaves were prepared by the eco-friendly ultrasonic extraction method. In this study, the highest percentage (%) w/w yield was obtained from aqueous SG leaf (23.08%) when compared to the other two extracts, chloroform (9.18%) and methanol (15.84%) respectively. The prepared SG leaf extracts were subjected to phytochemical analysis and they showed the presence of flavonoids, alkaloids, glycosides, saponins, terpenoids, tannins, steroids and proteins.

In vitro, an anti-proliferative assay of Simarouba glauca leaf extracts (chloroform, methanol and aqueous) was done using the MTT method. All three SG leaf extracts were effectively cytotoxic with significant IC₅₀ values of 123.05 μg/mL, 117.81 μg/mL and 155.06 μg/mL on Human breast cancer cell lines (MDA-MB 231) respectively. The results suggested that methanol SG leaf extract proved to possess effective anti-breast cancer potentials on MDA MB 231 cells with an IC₅₀ value of 117.81 ug/mL compared to other SG leaf extracts (chloroform and aqueous). Furthermore, a qualitative TLC study was carried out using different solvent systems such as n-hexane: ethyl acetate (2:8), chloroform: methanol (3:7), chloroform: methanol (5:5) and chloroform: methanol (7:3). The best solvent was chosen for further chromatography studies both preparative TLC and HPTLC. In these techniques, hexane-ethyl acetate (2:8) was used as a mobile phase to isolate and estimate the quantity of quassinoid from 10 different fractions of the bioactive SG leaf extract. The outcome of this approach proves that the amount of quassinoid in fraction 4 of the Simarouba glauca leaf of the bioactive extract was determined and the R_f value of bioactive Fr4 was found to be 0.22 which is close to the 0.24 R_f value of the standard quassinoid. The UV absorption spectrum (λ_max) of isolated Quassinoid Enriched Active Fraction 4 (QEAF) of SG leaf was seen at 250 nm. The FT-IR spectra, ¹³C and ¹H NMR spectral characterization were done for structure elucidation. In LC-MS, the spectrum of the isolated quassinoid peak was observed at m/z 496.33 g/mol [M+H]. This study compares the measured mass value m/z, 496 g/mol of the isolated quassinoid found in the QEAF of SG leaf to the precise mass value of a known quassinoid (Glaucarubinone) which is m/z 495 g/mol.

At the outset, this particular research was undertaken based on the earlier reports and the inspiration to overcome hurdles in folk practice and scientific evidence to prove the standardized systematic study has not been done till date. Hence, the extraction process needed a change over from conventional to an eco-friendly greener manner using the modern ultrasonic assisted approach of extract preparation by avoiding excess usage of solvents in a much lesser time and for increased percentage yield was followed as the prime process. The current research has proved scientifically that the novel quassinoid, glaucarubinone from Simarouba glauca leaf has been identified through the HPTLC fingerprint technique also to evaluate the anti-proliferative potentials of novel glaucarubinone quassinoid; present in the active fraction of Simarouba glauca leaf is very much complementing. Futuristic studies will be concentrated on proving the underlying mechanism so that the process may be fruitful for the management and treatment of breast cancer.

The authors are thankful to the Principal and Management of PSG College of Pharmacy, Coimbatore, Tamil Nadu for providing the facilities and other research assistance towards the completion of this research work. The authors are thankful to Dr. John A. Beutler, National Cancer Institute (NCI), Chemotherapeutics Repository, Molecular Targets Laboratory, Frederick, USA, for providing Glaucarubinone. The authors are also thankful to The Tamil Nadu Dr. MGR Medical University for the support given during the PhD research process.