1. INTRODUCTION:

Cancer is a disease that can damage the cells caused by uncontrolled cells proliferation and differentiation which result to cell death. Plants has the source of anti-cancer drugs whereby 50% of the trial anticancer drugs were originated from the natural products whereas 25% of prescribed anti-tumor drugs come from plants [1].

According to Shoemaker (2006), many in vitro studies had proven that plant extracts and isolated compounds have the capability to be anticancer agents [2]. Meanwhile xanthine oxidase is a form of xanthine oxidoreductase, which is an enzyme that creates reactive oxygen species such as superoxide radicals and hydrogen peroxide. According to Tang (2013), it expedites the oxidation of hypoxanthine to xanthine and xanthine to uric acid [3]. From this point, XO is a key enzyme between purine and free radicals metabolism. The end product of purine metabolism uric acid and superoxide radicals result in cellular structure damage and may lead to cancer [4]. Annonaceae family is part of the Angiosperma flowering plants that was described to be one of the largest in the family. They comprise of approximate 128 genera and around 3,000 species [5]. Alphonsea is a genus that is labeled as part of the Annonaceae family. Alphonsea were commonly discovered in Hainan and South Yunnan, China while in the Asian regions, they can be discovered around Cambodia, Sri Lanka, Papua New Guinea, India, Thailand, Myanmar, Laos, Vietnam, Malaysia, and Indonesia [6]. In Malaysia, A. cylindrica King, or locally known as “mempisang”, is mainly located in the lowland forest and it is not widely dispersed [7]. A. cylindrica can grow up to 20 meter tall and 0.18 meter in diameter. Meanwhile, when the outer bark of this plant matured, the bark would be in greyish or brownish color while the inner bark is in pale-yellow or brown. There were only a few phytochemical studies on Alphonsea species have been reported so far. A number of chemical compounds including alkaloids, terpenoids and essential oil has been analyzed. Most of the phytochemical studies were conducted on the bark, leaves and stem bark of the plants. Different parts of Alphonsea species have been traditionally used by the natives. For example, in the Andamas, A. ventricosa timber is used by the Indian for making boat [8]. Moreover, previous studies had reported that Alphonsea species portray various purposes such as antioxidant [9], anticancer [10] and antibacterial [11]. In our previous work, we reported two new rare bisbenzylisoquinoline and oxoaporphine alkaloids from bark of A. cylindrica [12]. We believe that different types of interesting alkaloids have yet to be extracted from this species. Therefore, the present work aims to discover bioactive compounds from the plant. In this paper, four compounds have been isolated for the first time from A. cylindrica.

2. RESULTS AND DISCUSSION:

The crude extract of A. cylindrica bark was isolated using open column chromatography (CC) to produce eleven known compounds (Figure 1). The known compounds were identified by spectroscopic techniques and comparison with previously reported data; these compounds stigmasterol (1) [13], isoursuline (2) [14], cyathocaline (3) [15], kinabaline (4) [16], muniranine (5) [11], isooncodine (6) [17], iraqiine (7) [12], O-methylmoschatoline (8) [13], kareemine (9) [12], atherospermidine (10) [18] and N-methylouregidione (11) [18].

Stigmasterol (1); White steroid, Chemical formula: C₂₉H₄₈O, HRESIMS: m/z 413.2686 [M]⁺; IR v_max cm^-1 : 3325(OH), 2935 and 2864 (C-H), 1456 and 1047 (CH₂). UV (DCM) lmax 248 nm. ¹H-NMR (CDCl₃, 500 MHz) δ (ppm): 0.64-0.90 (m, 15H), 0.91-0.98 (m, 2H), 0.99-1.02 (m, 6H), 1.04-1.11 (m, 1H), 1.13-1.19 (m, 2H), 1.25‐1.29 (m, 2H), 1.49‐1.56 (m, 8H), 1.81‐1.88 (m, 3H), 1.94‐2.06 (m, 3H), 2.21-2.32 (m, 2H), 3.49-3.55 (m, 1H), 4.98-5.03 (m, 1H), 5.12-5.17 (m, 1H), 5.34-5.35 (m, 1H). ¹³C-NMR (CDCl₃ with 1% v/v TMS, 125 MHz) δ (ppm): 37.32 (C-1), 31.72 (C-2), 71.90 (C-3), 42.29 (C-4), 140.82 (C-5), 121.82 (C-6), 31.97 (C-7), 31.97 (C-8),50.19 (C-9), 36.58 (C-10), 21.20 (C-11), 39.74 (C-12), 42.36 (C-13), 56.83 (C-14), 24.38 (C-15), 29.03 (C-16), 56.00 (C-17), 12.13 (C-18), 19.49 (C-19), 40.62 (C-20), 21.15 (C-21), 138.42 (C-22), 129.33 (C-23), 51.32 (C-24), 32.50 (C-25), 21.30 (C-26), 19.06 (C-27), 25.51 (C-28), 12.36 (C-29).

Isoursuline (2); Yellow amorphous solid crystal, Chemical Formula C₁₄H₁₁NO₃, HRESIMS m/z 242.0832 [M]⁺; IR v_max cm^-1 : 3734 (OH), 1704 (C=O). UV (DCM) lmax at 252, 293, 306 and 385 nm. ¹H-NMR (CDCl₃ with 1% v/v TMS, 500 MHz) δ (ppm): 8.29 (1H, d, J = 5.2 Hz, H-3), 7.27 (1H, d, J = 8.0 Hz, H-8), 6.92 (1H, d, J = 5.2 Hz, H-2), 6.77 (1H, d, J = 8.0 Hz, H-7), 3.96 (3H, s, OCH₃-6), 2.60 (3H, s, CH₃-1); ¹³C NMR (CDCl₃ with 1% v/v TMS, 125 MHz) δ (ppm): 191.32 (C-9), 165.30 (C-4a), 153.92 (C-6), 151.34 (C-3), 147.79 (C-1), 143.16 (C-5), 127.59 (C-4b), 126.51 (C-8a), 125.65 (C-9a), 125.22 (C-2), 117.80 (C-8), 112.49 (C-7), 56.51 (OCH₃-6), 17.33 (CH₃-1).

Cyathocaline (3); Yellow amorphous solid crystal, Chemical Formula C₁₄H₁₁NO₄, HRESIMSm/z 258.0755; IR v_max cm^-1 : 3240 (OH), 1698 (C=O). UV (DCM) lmax at 261, 248 and 311 nm. ¹H-NMR (CDCl₃ with 1% v/v TMS, 500 MHz) δ (ppm): 8.16 (1H, d, J = 5.15 Hz, H-3), 6.88 (1H, s, H-8), 6.81 (1H, d, J = 5.15 Hz, H-2), 4.12 (3H, s, OCH₃-6), 2.55 (3H, s, CH₃-1); ¹³C-NMR (CDCl₃ with 1% v/v TMS, 125 MHz) δ (ppm): 191.66 (C-9), 166.49 (C-4a), 139.22 (C-6), 150.9 (C-3), 147.42 (C-1), 145.9 (C-5), 129.75 (C-4b), 119.87 (C-8a), 124.47 (C-9a), 124.24 (C-2), 104.38 (C-8), 151.99 (C-7), 61.01 (OCH₃-6), 17.15 (CH₃-1).

Isooncodine (6); Orange amorphous solid crystal, Chemical Formula C₁₄H₁₁NO₃, HRESIMS m/z 240.0662 [M-]; IR v_max cm^-1 :3210 (OH), 1710, (C=O). UV (DCM) lmax at 265, 278, 326 and 340 nm. ¹H-NMR (CDCl₃ with 1% v/v TMS, 500 MHz) δ (ppm): 8.29 (1H, d, J = 5.15 Hz, H-3), 7.34 (1H, s, H-5), 7.19 (1H, s, H-8), 6.85 (1H, d, J = 5.15 Hz, H-2), 3.96 (3H, s, OCH₃-7), 2.58 (3H, s, CH₃-1); ¹³C-NMR (CDCl₃ with 1% v/v TMS, 125 MHz) δ (ppm): 192.35 (C-9), 165 (C-4a), 152.06 (C-6), 152.23 (C-3), 146.2 (C-1), 106.32 (C-5), 127.94 (C-4b), 138.75 (C-8a), 126.51 (C-9a), 125.2 (C-2), 107.64 (C-8), 148.52 (C-7), 56.51 (OCH₃-6), 17.06 (CH₃-1).

DCM, methanol, and hexane crude extracts from the barks of A. cylindrica was experimented onto MCF-7 human breast cancer cells. Based on the experiments, the percentage of the hexane’s IC₅₀ , DCM, and methanol crude extracts was unable to be estimated and the numbers of the treated cells are higher than the control ones whereby the estimated percentage is more than 100 percent. Furthermore, these samples were claimed to be appropriated for regenerative testing due to the presence of phytochemicals in the extracts. In this study, we studied the cytotoxic impacts of eight alkaloids and one steroid compounds isolated from the barks of A. cylindrica onto MCF-7 human breast cancer cells. The results revealed that there have been high cell viability inhibitory effects in the breast cancer cell lines after 48 hours of treatment. The values of IC₅₀ are tabulated in Table 1. Based on the investigation, isoursuline revealed to be most potent with IC₅₀ 33 μg/ml while other compounds showed moderate inhibitory effect on MCF-7 human breast cancer cells. In contrast, cyathocaline and muniranine compounds did not exhibit any activity to proof the inhibition of cancer cells. Additionally, the stigmasterol has exhibited anticancer activity on the MCF-7 human breast cancer cells.

The activity of XO towards xanthine as a substrate was assayed spectrophotometrically at 290 nm. Eight compounds from the barks of A. cylindrica was evaluated for their xanthine oxidase inhibitory activity. The inhibitory activities of each compound against XO are shown in Table 1. The compounds exhibited moderate xanthine oxidase inhibitory effect with IC₅₀ < 100 μM. This is due to the position of substitution groups that are present in the compounds and also to the geometry of the compounds as it was claimed that the compound with planar structure is more active than the non-planar structure. Therefore, the position of the functional groups and the geometry in the compounds are crucial to the inhibition of XO [19, 20].

Table 1: cytotoxic and xanthine oxidase inhibitory activities of A. cylindrica King

Compounds	Anticancer activity (IC₅₀ μg/mL)	Xanthine oxidase inhibition (μg/mL)
Stigmasterol	73	5.04 ± 1.58
Isoursuline	33	ND
Cyathocaline	NA	46.90 ± 4.73
Isooncodine	ND	31.97 ± 1.12
Kinabaline	62	50.72 ± 4.13
Muniranine	NA	32.94 ± 0.86
Atherospermidine	89	46.29 ± 5.73
O-methylmoschatoline	56	14.36 ± 4.11
Iraqiine	85	ND
Kareemine	98	ND
N-methylouregidione	ND	42.10 ± 1.31

ND: not determined, NA: no activity (concentration range 0-100 μg/ml for cytotoxicity).

3. EXPERIMENTAL:

3.1 Plant materials:

A. cylindrica King bark (KL 5379), was collected from Kechau Tui, Kuala Lipis, Pahang. The species were identified by the phytochemical group of Chemistry Department, University of Malaya, Kuala Lumpur,.

3.2 Extraction and isolation:

Dried and powdered bark of A. cylindrica King (2.0 kg) was extracted by cold extraction procedure using three different solvents; hexane, dichloromethane (DCM) and methanol respectively. The extracts were concentrated by using a rotary evaporator to produce 8.5g of DCM crude extract. The DCM extract (8.5g) was introduced to Column Chromatography over a column of silica gel (250g, dc 4.5 cm in diameter), using DCM/MeOH as a solvent system with ratio 100:0-0:100. The ratio of the solvents were (100:0, 99:1; 98:2, 96:4,93:7, 92:8, 90:10, 85:15, 80:20 and 50:50) yielding 83 fractions.

Meanwhile fractions 6–8 from first column were combined and pour into CC over silica gel the solvent system was EA/hexane with ratio (30:70, v/v) to afford 41 sub fractions. (sFr.1–sFr.3) were subjected to CC over silica gel using EA/hexane (10:90, v/v) to afford 5 fractions, sub-fractions 2-4 were combine and elucidated as (1) (4.2mg).

Furthermore, fractions 9 and 10 were subjected to column chromatography over a column of silica gel using DCM/MeOH (100: 0 to 95: 5, v/v) to afford 46 sub fractions, sub-fraction 4 (sFr. 4) was identified as (2) (3.5mg).

In other hand, fractions 11 and 12 were subjected to CC on silica gel (KA, 3 cm in diameter) using EA/hexane as mobile phase with ratio (25 : 75 to 50 : 50, v/v) to afford 180 sub-fractions. Sub-fractions 11-14 from KA column were subjected to CC on silica gel using DCM/MeOH (99.5 : 0.5, v/v) to afford 10, sub-fractions 3-8 (sFr.3–sFr.8) were subjected to CC over silica gel using DCM/MeOH (99.5 : 0.5, v/v) to give the pure compound and was elucidated as (3) (14mg)

Sub-fractions 102–123 from KA column were subjected to CC on silica gel using DCM/EA solvent system with ratio (90: 10, v/v) to afford 20 fractions, sub-fractions 17 -19 (sFr.17–sFr.19) were subjected to CC over silica gel using DCM/MeOH (99: 1, v/v) to give 9 fractions, fractions number 2-5 was saved and investigated as (6) (4mg).

3.3 Cytotoxic Assay:

Three crude extracts (hexane, DCM and methanol) of bark of A. cylindrica as well as the isolated compounds; 1, 2, 3, 4, 5, 7, 8, 9 and 10 were evaluated for their cytotoxicity against the MCF-7 breast cancer cell line according to the method that reported by Ahmed (2018) [13]. MCF-7 breast cancer cells were cultured to active stage. Once cells reached active stage, they were transferred into 96-well tissue culture plate at 1 x 10⁴ cells/well with 100 μl of cell suspension for each well. The culture was pre-incubated for 24 hours in a humidified carbon dioxide (CO2) incubator. After 24 hours, the culture was observed under the inverted-light microscope in order to check the morphology and density of cell growth. Once the cell attachment and growth were confirmed, samples and negative control were added to each designated well at 10 μl. The culture was incubated for another 48 hours to allow the effect of the samples towards MCF-7 cells to take place. Next, 10 μl of Cell Count Reagent SF was added to each well and the culture was subjected to 2 hours of incubation followed by the absorbance measurement at 450 nm using the microplate reader. Upon completion, culture plate was discarded and the absorbance data were analyzed using Equation 1 in order to generate decay curve for the 50 % inhibition concentration (IC₅₀) value estimation.

(_AbsSample-_AbsBlank/_AbsNegative control-_AbsBlank)

Percentage Of Controlled Cell Growth = ----------×100%

Equation 1

3.4 Xanthine Oxidase Inhibitory Activity:

The XO activity was measured for 1, 3, 4, 5, 6, 8, 10 and 11 according to the methods that reported by Aldulaimi (2018) [18]. The assay mixture consisted of 1.0 ml of test solution, 2.9 ml of 1/15 M phosphate buffer (pH 7.5) and 0.1 ml of enzyme solution. After preincubation of the mixture at 25 °C for 15 min, the reaction was initiated by adding 2.0 ml of substrate solution. This assay mixture was incubated at 25 °C for 30 min. The reaction was stopped by adding 1.0 ml of 1N HC1, and the absorbance of the assay mixture at 290 nm was measured spectrophotometrically. A blank was prepared in the same way, but the enzyme solution was added to the assay mixture after adding 1 N HC1. One unit of XO was defined as the amount of enzyme producing 1/µmmol of uric acid per min at 25 °C.

XO inhibitory activity was expressed as the percentage inhibition of XO in the above assay system, calculated as

(1 - B/A) x 100

Where A is the activity of the enzyme without test material and B is the activity of the enzyme with test material.

4. CONCLUSION:

This study reported the first time isolated of compounds 1, 2, 3 and 6 from bark of A. cylindrica King. These findings showed that A. cylindrica plant is rich with an alkaloids and steroids. The anticancer and xanthine oxidase inhibitory activities of compounds 2, 3, 5, 6, 7 and 9 were reported for the first time and its possessed potent of cytotoxic and xanthine oxidase effects.

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Received on 27.02.2019 Modified on 18.04.2019

Research J. Pharm. and Tech 2019; 12(9):4129-4134.

DOI: 10.5958/0974-360X.2019.00713.3