Isolation, Characterization, and HPTLC-Quantiﬁcation of Compounds with Anticancer Potential from Loranthus Acaciae Zucc.

: The cytotoxic e ﬀ ects of the crude extract of Loranthus acaciae Zucc. and its n -hexane, chloroform, and n -butanol fractions were assessed against three cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell apoptosis was determined using an annexin V-phycoerythrin / 7-aminoactinomycin kit. We observed that the L. acaciae n -hexane extract (LAHE) could inhibit cancer cell growth, particularly of MCF7 and A549 cells. Chromatographic puriﬁcation of LAHE and nuclear magnetic resonance analysis led to the identiﬁcation of two compounds from this plant species, namely, betulinic acid and β -sitosterol, for the ﬁrst time. Flow cytometry study suggested that betulinic acid induced cell death via apoptosis, as a distinguished marked enhancement in the early and late apoptosis of human lung (A549) and breast (MCF-7) cancer cell lines. The isolated compounds were further estimated concurrently in LAHE using a validated high-performance thin-layer chromatographic (HPTLC) method on a 10 × 10 cm 2 HPTLC plate with chloroform, methanol, and glacial acetic acid (97:2:1, v / v / v ) as the mobile phase and a λ max of 540 nm. The amounts of betulinic acid and β -sitosterol in LAHE were 69.46 and 135.53 µ g / mg of dried weight of extract, respectively. The excellent cytotoxic e ﬀ ect of LAHE could be attributed to the presence of ample amounts of betulinic acid. in the with 5 µ L of annexin V-phycoerythrin (PE) and 5 µ L of 7-aminoactinomycin (7-AAD) for 15 min at room temperature. After the incubation period, 400 µ L of annexin-binding bu ﬀ er was and using a ﬂow cytometer with three replicates.


Introduction
Cancer is the second reason of death around the world. Approximately, 17 million individuals were diagnosed with cancer, and 9.6 million cases of deaths were reported worldwide in 2018; the most common types include lung cancer, breast cancer, prostate cancer, colorectal, and skin cancer, they account for around 40% mortality [1]. Current conventional cancer treatments put the patients

Preparation of the Extracts and Fractions
The aerial parts were dried in artificial heat at 45 • C and coarsely powdered (500 g) before extraction with ethanol (3000 mL) using a Soxhlet apparatus. The liquid extract was concentrated under vacuum using a rotavap to obtain 61 g of the crude semi-solid ethanol extract. This crude extract was further suspended in distilled water and successively partitioned with solvents of different polarities, i.e., n-hexane, chloroform, and n-butanol, to produce 6.5, 11, and 20 g of dried fractions, respectively.

Compound Isolation and Identification
LAHE (5 g) was separated via silica gel column chromatography (72 g, 80 × 3 cm). Elution began with 3% hexane:ethyl acetate, and the polarity was increased with ethyl acetate. After checking the thin-layer chromatography (TLC) behavior, homogenous fractions were pooled to generate seven fractions (A-G). Since the anticancer activity remained in fraction B, fraction B (50 mg) was further eluted with 5% hexane:ethyl acetate and rechromatographed on a normal-phase column (7.2 g, 60 × 1 cm) to obtain compounds 1 and 2.

Determination of Cell Viability
Cell viability was determined via the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay (MTT) assay against the breast (MCF-7), lung (A549), and HepG2 (liver) cancer cell lines, which were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and against human umbilical vein endothelial cells (HUVEC cells) 16549, which were purchased (Cat no. PCS-100-010; ATTCC, MD, USA). The assay was performed as described previously [25]. Briefly, culture plates (24-well) were seeded with 5 × 104 HepG2 (liver), MCF-7 (breast), or A549 (lung) cancer cells or normal human umbilical vein endothelial cells (HUVECs) in 1 mL of DMEM medium and allowed to attach overnight at 37 • C in a 5% CO 2 incubator. After 24 h, the cells were treated with different concentrations of LAHE for 48 h. Then, 100 µL of MTT (5 mg/mL) was added to each well, and the plate was incubated at 37 • C in 5% CO 2 for 4 h. A solubilization solution (0.01 N HCl/isopropanol) was added to the wells to solubilize the formazan, and the plate was placed on a shaker for 10 min. The absorbance at λ = 570 nm was measured on a microplate reader (BioTek, Winooski, VT, USA). The percentage viability of treated cells normalized to that of negative-control was assessed, and the IC 50 (tested sample concentration required to inhibit cell growth by 50 percent) values of each tested extract or compound were calculated from the dose-response curves using the following formula: Cell viability (%) = [optical density (OD) of the treated sample)/(OD of the untreated sample] × 100%

Flow Cytometric Analysis of Cell Apoptosis
A PE Annexin V Apoptosis Detection Kit with 7-Aminoactinomycin D (BioLegend, San Diego, CA, USA). was used to detect the induction of apoptosis after treatment with the isolated betulinic acid following the manufacturer's instructions. Briefly, both MCF-7 and A549 cells were seeded into 12-well tissue culture plates at a density of 5 × 10 4 cells/well for 24 h at 37 • C for adherence, followed by treatment with betulinic acid at its IC 50 value (17 µg/mL) for 24 h. Cells were collected, washed twice in cold PBS, and resuspended in kit-specific binding buffer. They were then incubated in the dark with 5 µL of annexin V-phycoerythrin (PE) and 5 µL of 7-aminoactinomycin (7-AAD) for 15 min at room temperature. After the incubation period, 400 µL of annexin-binding buffer was added, and the samples were immediately analyzed using a flow cytometer (Beckman Coulter, Brea, CA, USA) with three replicates.

Development of the HPTLC Procedure to Determine Betulinic Acid and β-Sitosterol in LAHE
Betulinic acid and β-sitosterol procured from Sigma Aldrich (St Louis, MO, USA) were used for the quantitative analysis in LAHE using validated HPTLC method and carried out on a 10 × 10 cm 2 NP-HPTLC plate (Merck, Darmstadt, Germany). Stock solutions of betulinic acid and β-sitosterol standards (1 mg/mL) were prepared in chloroform and diluted further with chloroform to obtain seven different dilutions (20-140 µg/mL). The prepared dilutions of betulinic acid and β-sitosterol, as well as LAHE (5 µL), were applied to the HPTLC plate using a microliter syringe attached to an Automatic TLC Sampler-4 (CAMAG, Switzerland) with 6-mm band size at a speed of 160 nL/s to obtain a linearity range of 200-1400 ng/band. The loaded TLC plate was then developed in a 20 × 10 cm 2 pre-saturated glass chamber under controlled temperature (25 • C ± 2 • C) and humidity (60% ± 5%). The developed TLC plate was visualized using a spray reagent (vanillin/HCl, Noida, India) to visualize the spots of the standards and phytoconstituents present in the samples (extract fractions). Quantitative analysis was conducted at an optimized UV wavelength of 540 nm in the absorbance mode. The proposed chromatographic method was validated by considering the following parameters suggested in the ICH guidelines [26]: limit of detection (LOD), limit of quantification (LOQ), precision, recovery (as accuracy), and robustness.

Isolated Compounds from LAHE
Separations 2020, 7, x FOR PEER REVIEW 5 of 12

Cytotoxic Activity
The potential anticancer effect of L. acaciae was tested against selected human malignant cell lines, namely, A549 (lung), HepG2 (liver), and MCF-7 cells (breast), as well as against HUVECs. Both the crude extract and its fractions exerted selective dose-dependent cytotoxic effects on both tumor  Compound 2 Compound 2 ( Figure 1) was also isolated as a white powder. Its mass spectral data suggested a molecular formula of C 29 H 50 O. The 1 H NMR spectrum of 2 revealed the presence of six methyl signals that appeared as two methyl singlets at δ 0.69 and 1.01; three methyl doublets at δ 0.81, 0.83, and 0.93; and a methyl triplet at δ 0.85. The 1 H NMR spectrum of 2 also displayed one olefinic proton at δ 5.36 and a proton corresponding to the proton connected to the C-3 hydroxyl group, which appeared as a triplet of doublets at δ 3.53. The 13 C NMR together with COSY, HMQC, and HMBC revealed 29 carbon signals, including 6 methyl, 11 methylene, 10 methane, and 3 quaternary carbons. A comparison of the 1 H-NMR and 13 C-NMR results of 2 with published data indicated that the compound was β-sitosterol. This represents the isolation of this compound from L. acaciae for the first time. However, it was previously isolated from other plant species, including Plicosepalus curviflorus [27].

Cytotoxic Activity
The potential anticancer effect of L. acaciae was tested against selected human malignant cell lines, namely, A549 (lung), HepG2 (liver), and MCF-7 cells (breast), as well as against HUVECs. Both the crude extract and its fractions exerted selective dose-dependent cytotoxic effects on both tumor cells Separations 2020, 7, 43 6 of 12 and HUVECs. According to the IC 50 values, these extracts exhibited considerable cytotoxic activity against MCF-7 and A549 cells (Table 2). Compared with other fractions, LAHE exhibited highly selective anticancer activity against all tested cancer cells ( Figure 2A); therefore, it was selected for further studies. Betulinic acid, which was isolated from LAHE, displayed strong cytotoxic activity ( Figure 2B), whereas β-sitosterol exhibited weak activity (data not shown).
Separations 2020, 7, x FOR PEER REVIEW 6 of 12 cells and HUVECs. According to the IC50 values, these extracts exhibited considerable cytotoxic activity against MCF-7 and A549 cells (Table 2). Compared with other fractions, LAHE exhibited highly selective anticancer activity against all tested cancer cells ( Figure 2A); therefore, it was selected for further studies. Betulinic acid, which was isolated from LAHE, displayed strong cytotoxic activity ( Figure 2B), whereas β-sitosterol exhibited weak activity (data not shown).  In chemoprevention studies, bioactive compounds that can induce apoptosis rather than necrosis in cancer cells are more desirable. Therefore, the induction of apoptosis was investigated  In chemoprevention studies, bioactive compounds that can induce apoptosis rather than necrosis in cancer cells are more desirable. Therefore, the induction of apoptosis was investigated after treatment with betulinic acid. Phosphatidylserine exposure in the cell surface is a classic feature of cells undergoing apoptosis [28]. Using flow cytometry, this method can identify apoptosis at an earlier stage as compared with assays based on nuclear changes, such as DNA fragmentation [29].
In this study, a PE annexin V apoptosis detection kit with 7-AAD was used. In the kit, annexin V, which is conjugated to the fluorochrome PE, has a high affinity for phosphatidylserine, which is redistributed from the inner to the outer leaflet of the plasma membrane during early apoptotic events. Conversely, 7-AAD detects dead cells by binding to cellular DNA inside the cell. As shown in Figure 3, following the treatment of A549 and MCF-7 with isolated betulinic acid at its IC 50 value (17 µg/mL), a noticeable increase in the percentage of apoptotic cells was reported. The percentages of surviving, early apoptotic, late apoptotic, and necrotic cells in A549 cells were 58.87% ± 0.6%, 9. 31% ± 0.4%, 15.21% ± 0.3%, and 15.16% ± 0.6% and those in MCF-7 cells were 48.29% ± 0.4%, 11.87% ± 0.3%, 23.4% ± 0.56%, and 15.3% ± 0.4%, respectively, compared to in the untreated cells ( Figure 3B). after treatment with betulinic acid. Phosphatidylserine exposure in the cell surface is a classic feature of cells undergoing apoptosis [28]. Using flow cytometry, this method can identify apoptosis at an earlier stage as compared with assays based on nuclear changes, such as DNA fragmentation [29].
In this study, a PE annexin V apoptosis detection kit with 7-AAD was used. In the kit, annexin V, which is conjugated to the fluorochrome PE, has a high affinity for phosphatidylserine, which is redistributed from the inner to the outer leaflet of the plasma membrane during early apoptotic events. Conversely, 7-AAD detects dead cells by binding to cellular DNA inside the cell. As shown in Figure 3, following the treatment of A549 and MCF-7 with isolated betulinic acid at its IC50 value (17 µg/mL), a noticeable increase in the percentage of apoptotic cells was reported. The percentages of surviving, early apoptotic, late apoptotic, and necrotic cells in A549 cells were 58.87 ± 0.6 %, 9. 31 % ± 0.4 %, 15.21 % ± 0.3 %, and 15.16 % ± 0.6 % and those in MCF-7 cells were 48.29 % ± 0.4 %, 11.87 % ± 0.3 %, 23.4 % ± 0.56 %, and 15.3 % ± 0.4 %, respectively, compared to in the untreated cells ( Figure  3B).
Betulinic acid is a pentacyclic triterpenoid that is widely distributed in the plant kingdom. It possesses various biological activities, including anticancer activity against different cancer cell lines [30]. After exploring its role as an anticancer compound, the focus has shifted toward elucidating its mechanism of action. Various studies have shown that it induces apoptosis in several cell lines, including human neuroblastoma [31], leukemia [32], and different adenocarcinoma cell lines (i.e., breast, lung, and colon cancer cells) [33]. We reported the isolation of betulinic acid from L. acaciae for the first time and demonstrated its excellent anticancer activity via the inhibition of cell viability and induction of apoptosis.  Betulinic acid is a pentacyclic triterpenoid that is widely distributed in the plant kingdom. It possesses various biological activities, including anticancer activity against different cancer cell lines [30]. After exploring its role as an anticancer compound, the focus has shifted toward elucidating Separations 2020, 7, 43 8 of 12 its mechanism of action. Various studies have shown that it induces apoptosis in several cell lines, including human neuroblastoma [31], leukemia [32], and different adenocarcinoma cell lines (i.e., breast, lung, and colon cancer cells) [33]. We reported the isolation of betulinic acid from L. acaciae for the first time and demonstrated its excellent anticancer activity via the inhibition of cell viability and induction of apoptosis.

Concurrent Analysis of Betulinic Acid and β-Sitosterol in LAHE by a Validated HPTLC Method
By testing various compositions of different solvents, the most suitable mobile phase for betulinic acid and β-sitosterol analysis in LAHE was a mixture of chloroform, methanol, and glacial acetic acid [97:2:1 (v/v/v)]. Intense and sharp peaks of betulinic acid and β-sitosterol were observed at Rfs (retention factors) of 0.31 ± 0.001 and 0.41 ± 0.001, respectively ( Figure 4A). This approach was found to clearly separate the standards of betulinic acid and β-sitosterol and isolated compounds of LAHE ( Figure 4B). The authentication of bands was conducted by matching (overlaying) the spectra of the extract with those of betulinic acid and β-sitosterol ( Figure 4C).

Concurrent Analysis of Betulinic Acid and β-Sitosterol in LAHE by a Validated HPTLC Method
By testing various compositions of different solvents, the most suitable mobile phase for betulinic acid and β-sitosterol analysis in LAHE was a mixture of chloroform, methanol, and glacial acetic acid [97:2:1 (v/v/v)]. Intense and sharp peaks of betulinic acid and β-sitosterol were observed at Rfs (retention factors) of 0.31 ± 0.001 and 0.41 ± 0.001, respectively ( Figure 4A). This approach was found to clearly separate the standards of betulinic acid and β-sitosterol and isolated compounds of LAHE ( Figure 4B). The authentication of bands was conducted by matching (overlaying) the spectra of the extract with those of betulinic acid and β-sitosterol ( Figure 4C). The developed procedure was approved by the ICH guideline of 2005 to evaluate LOD, LOQ, precision, accuracy, and robustness. It was discovered that the created technique was completely selective with good baseline resolution. In the linearity range of 200-1400 ng/spot, the regression equations and correlation coefficients were Y = 5.206X + 117.77 and Y = 4.514X + 1039.7 and 0.996 and 0.9979 for betulinic acid and β-sitosterol, respectively. LOD and LOQ were 34.51 and 104.57 ng for betulinic acid and 40.95 and 124.11 ng for β-sitosterol, respectively ( Table 3). The results of the recovery analysis of the developed procedure are presented in Table 4.
The recoveries and relative standard deviations (RSDs, %) were 98.85-99.71% and 0.932-1.29% for betulinic acid and 98.8-99.40% and 0.95-1.06% for β-sitosterol, respectively. The intra-and interday precisions for the developed technique are displayed in Table 5. The developed procedure was approved by the ICH guideline of 2005 to evaluate LOD, LOQ, precision, accuracy, and robustness. It was discovered that the created technique was completely selective with good baseline resolution. In the linearity range of 200-1400 ng/spot, the regression equations and correlation coefficients were Y = 5.206X + 117.77 and Y = 4.514X + 1039.7 and 0.996 and 0.9979 for betulinic acid and β-sitosterol, respectively. LOD and LOQ were 34.51 and 104.57 ng for betulinic acid and 40.95 and 124.11 ng for β-sitosterol, respectively ( Table 3). The results of the recovery analysis of the developed procedure are presented in Table 4. Table 3. Rf, linear regression data for the calibration curve of betulinic acid and β-sitosterol (n = 6).
The recoveries and relative standard deviations (RSDs, %) were 98.85-99.71% and 0.932-1.29% for betulinic acid and 98.8-99.40% and 0.95-1.06% for β-sitosterol, respectively. The intra-and inter-day precisions for the developed technique are displayed in Table 5. The RSDs for intra-and inter-day precisions (n = 6) were 1.09-1.30% and 1.08-1.29% for betulinic acid and 1.01-1.18% and 0.99-1.16% for β-sitosterol, respectively, demonstrating the high accuracy of the developed technique. To check the validity of the developed procedure, small but intentional changes (in mobile phase composition, saturation time, and mobile phase volume) were applied; the gathered data are presented in Table 6. The low values of SD and RSD illustrate that the developed HPTLC procedure is durable. The validated HPTLC method was applied to analyze betulinic acid and β-sitosterol concurrently in LAHE ( Figure 4D). The amounts of betulinic acid and β-sitosterol in LAHE were estimated to be 69.46 and 135.53 µg/mg of dried weight of extract, respectively. The presence of betulinic acid at high levels (as evaluated by the HPTLC method) in LAHE validated its excellent anticancer activity. HPTLC has become a valuable and effective tool for the estimation of botanical materials. It offers a better resolution for phytoconstituents and has high efficiency and cost-effectiveness. The developed chromatographic method has gained popularity in the pharmaceutical and cosmetic industries for evaluating the quality of raw materials as well as finished goods. There are several genuine reasons for its increasing adoption, such as the requirement of smaller amounts of mobile phase and standard compounds and short analysis time. Moreover, flexibility concerning solvent systems, scanning wavelengths, and multiple-sample analysis in a single run are peculiar features of this technique [34][35][36][37][38].

Conclusions
In conclusion, we observed that LAHE exhibited potential cytotoxic effects against various cancer cell lines. Betulinic acid obtained from the bioactive hexane fraction proved to be a potent anticancer compound, whereas β-sitosterol was less active. Betulinic acid, which was isolated from L. acaciae for the first time, exerted its anticancer effects by inhibiting cell viability and inducing apoptosis. The presence of betulinic acid in LAHE at suitable levels (as evaluated by HPTLC) also supported the excellent anticancer activity of LAHE. This is the first report on the isolation and identification of betulinic acid and β-sitosterol from the aerial parts of L. acaciae. The established chromatographic method can be applied for the concurrent analysis of betulinic acid and β-sitosterol in any plant species or marketed herbal formulations containing these two compounds.