Phytoconstituents and bioactivities of the bark of Pleiogynium timorense (DC.) Leenh (Anacardiaceae)

Introduction The production of drugs from plant origin aims to avoid the complications of synthetic origin agents. Pleiogynium timorense (DC.) Leenh. (Anacardiaceae) is native to Australia, with the common name of Gambozia and is described as an ornamental tree (1,2). Anacardiaceae contains many plants with edible seeds and fruits (e.g., Cashew nuts, Pistachio nuts and mango (3). The fruits of P. timorense (DC.) Leenh. can be eaten and used in making jams and jellies (4). Rutin, myricetin, hyperin, quercitrin, quercetin, β-sitosterol and lupeol have been isolated from the leaves which possessed potent antimicrobial effects against Staphylococcus aureus and Bacillus subtilis (5). Another study showed that the alcoholic extract of the leaves had antioxidant, anti-inflammatory and hypoglycemic effects. Moreover, myricetin-3-O-α-L-rhamnopyranosid, kaempferol, kaempferol-3-O-β-D-glucopyranoside, quercetin -3-O-β-D-glucopyranoside 1,3,4,6-tetra-Ogalloyl-β-D-glucopyranose, 1,4,6-tri-O-galloyl-β-Dglucopyranose, 3,5-di-O-galloylquinic acid,quercetin3-O-β-D-galactopyranoside, gallic acid, kaempferol-3O-β-D-6”-methyl glucuronopyranoside, kaempferol3-O-β-D-galactopyranoside and kaempferol-3-O-β-Dglucuronopyranoside were isolated and identified from http://www.herbmedpharmacol.com doi: 10.15171/jhp.2020.03


Introduction
The production of drugs from plant origin aims to avoid the complications of synthetic origin agents. Pleiogynium timorense (DC.) Leenh. (Anacardiaceae) is native to Australia, with the common name of Gambozia and is described as an ornamental tree (1,2). Anacardiaceae contains many plants with edible seeds and fruits (e.g., Cashew nuts, Pistachio nuts and mango (3). The fruits of P. timorense (DC.) Leenh. can be eaten and used in making jams and jellies (4). Rutin, myricetin, hyperin, quercitrin, quercetin, β-sitosterol and lupeol have been isolated from the leaves which possessed potent antimicrobial effects against Staphylococcus aureus and Bacillus subtilis (5). Another study showed that the alcoholic extract of the leaves had antioxidant, anti-inflammatory and hypoglycemic effects. Moreover  (6). Cyanidin-3-glucoside was isolated from Pleiogynium fruits which showed a potent antioxidant activity (7). In our previous work, rutin, catechin, quercetrin and quercetin were isolated from the pericarp of the plant, as well as the methanol extracts of the pericarp and seeds and showed anti-inflammatory, antioxidant, analgesic, hepatorenal protective effects (8). Moreover, GC/MS analysis of the lipoidal matter of the seeds showed that 1-heptene was the major compound in unsaponifiable matter, while linoleic acid was the major fatty acid (9). Furthermore, dichloromethane extract of the bark showed a significant activity against the A2780 human ovarian cancer cell line due to the presence of three new trihydroxy alkylcyclohexenones which were isolated from this extract (10). In our recent work the constituents of P. timorense pericarp and seeds were identified by using high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (11). Moreover, the volatile constituents of P. timorense fruits were identified which showed a moderate cytotoxic effect on human hepatoma cells and a powerful cytotoxic effect against laryngeal carcinoma and breast adenocarcinoma human tumor cell lines (12). The present study aims to the methanol extract of the bark of this plant as antioxidant, antihyperglycaemic, cytotoxic and hepatorenal protective agent with the aim of producing a natural drug.

Plant identification and collection
The bark of P. timorense was prepared from Zoo garden, Giza, Egypt. The plant was identified by Dr. M. El-Gebaly, the taxonomist at the Department of Botany, National Research Centre (NRC), Giza, Egypt. A voucher specimen (possessing number 2001) was kept in NRC.

Phytochemical screening
The constituents of the methanolic extract of the bark were identified by standard procedures as previously described (13,14).

HPLC determination of phenolics and flavonoids
The identification and quantification of flavonoids and phenolics in 70% methanol extract of P. timorense bark were performed by HPLC according to the previously described methods (15,16).

Extraction and isolation
The air dried powdered bark of P. timorense (1 kg) was extracted with petroleum ether (60-80°C) for defatting. The defatted powder was extracted with 70% methanol by percolation, then the concentrated extract (70 g) was phytochemically screened using the standard procedures which were previously described (13) to identify its constituents. The extract (40 g) was subjected to polyamide CC and eluted with gradient water: methanol (10:0) to (0:10) to give five fractions. Fraction 1, one compound which was eluted with water: methanol (90:10), was purified on Sephadex LH-20 CC and was eluted with methanol to give compound 1. Fraction 2, two compounds which were eluted with water: methanol (80:20), was applied on Sephadex LH-20 C using methanol as eluent to give compounds 2 and 3. Fraction 3, two compounds which were eluted with water: methanol (70:30), was applied on Sephadex LH-20 C to give compounds 4 and 5. Fraction 4, one compound which was eluted with water: methanol (60:40), was purified on Sephadex LH-20 C to give compound 6. Fraction 5, one compound which was eluted with water: methanol (50:50), was purified on Sephadex LH-20 C to give compound 7.
Cytotoxicity assay procedures Human tumor cell lines Human breast carcinoma (MCF-7), normal melanocytes (HFB-4), human hepatocellular liver carcinoma (HepG2) and human colon carcinoma (HCT116) cell lines were obtained from the American Type Culture Collection and were maintained by serial sub-culturing in the National Cancer Institute, Cairo, Egypt.

Animals
Sprague Dawley rats (130-150 g) were selected and kept in the controlled environmental conditions with free access to water and diet. The handlings with animals were complied with the ethical guidelines of the Medical Ethical Committee of the National Research Centre in Egypt and in accordance with the guidelines of the International Association for the Study of Pain Committee for Research and Ethical Issues.
Acute toxicity study The LD 50 of methanol (70%) extract of P. timorense bark was determined using Karber method that was previously described (18).
Antioxidant, hepatoprotective, and renal-function protective effects Rats were classified into normal and damaged liver (received carbon tetra chloride (CCl 4 )) groups. Each group was classified into 4 different groups (4 rats in each); group 1 (control) received distilled water; group 2 received silymarin (50 mg/kg), while groups 3 and 4 received 150 and 300 mg/kg tested extract of P. timorense, respectively, for 15 days. At the end of experimental period, the rats were anaesthetized as previously described (19). Blood samples were collected, centrifuged at 3000 rpm for 15 minutes and stored at -20°C before they were analyzed.
Analytical methods Serum total antioxidant capacity (TAC) level was determined as previously described (20). Serum alanine amino transferase (ALT) and aspartate amino transferase (AST) activities were determined colorimetrically (21). The level of uric acid was determined using Barham and Trinder method (22), while creatinine was measured according to Bartles and Bohmer method (23).

Antihyperglycaemic effect
The methanol extract of P. timorense bark was evaluated for the antihyperglycaemic activity using the method that was described previously (24).

Statistical analysis
One-way ANOVA was used to analyze the data. The result was considered statistically significant at P value < 0.05.

Phytochemical screening
The results of phytochemical analysis of the bark of P. timorense revealed the presence of carbohydrate, terpenoids, coumarins, saponins flavonoids and tannins, while alkaloids were absent (Table 1). HPLC analysis of flavonoids and phenolic compounds in 70% methanol extract of Pleiogynium timorense bark Fourteen flavonoidal compounds were identified representing 36.97 mg/g of the total content, the major flavonoid was quercetin (5.31 mg/g) followed by naringenin (5.12 mg/g). On the other hand, 16 phenolic compounds were identified representing 25.85 mg/g of the total content. The major phenolic compound was catechin (4.56 mg/g) followed by ρ-hydroxy benzoic acid (3.26 mg/g) ( Table 2).

Compound 3
Compound 3, (9 mg), was isolated as yellow powder with R f = 0.93 in S1 and 0.15 in S2. It gave yellow color under UV light which was turned to yellow with ammonia vapor. UV spectral data: λ max nm in methanol; 261, 367, with     Acute toxicity study LD 50 was found to be 4.8 g/kg body weight, this relatively high LD 50 indicates low toxicity.
Antioxidant, hepato-protective and renal function protective effects TAC was significantly increased in the two doses of the bark extract compared with that of the control group. Liver enzymes (ALT and AST) were significantly decreased in the two doses of the bark extract compared with both control and silymarin groups (Table 5). Creatinine and uric acid concentrations were significantly decreased in the two doses of the bark extract compared with control and silymarin treatment groups (Table 5). CCl 4 treatment showed a significant decrease in TAC serum content ( Table 6). The two doses of the bark extract showed significant increase in TAC level in carbon tetrachloride hepatic damaged rats compared with   carbon tetrachloride treatment alone. Liver enzymes were significantly increased in CCl 4 -toxicated groups. The two doses of the bark extract showed a significant decrease in the elevated level of liver enzymes that was caused by CCl 4 . Uric acid and creatinine were significantly elevated by CCl 4 treatment. The bark extract in the two dose levels showed a significant decrease in creatinine and uric acid values compared with both control and silymarin groups ( Table 6).
Antihyperglycaemic test Serum glucose level of the rats was significantly decreased by treating with the two doses of the bark extract in a dose-dependent manner ( Table 7).

Discussion
Phytochemical screening of the bark of P. timorense revealed the presence of various phytoconstituents which are responsible for the bioactivities of the plant. So. the current study attempted to discover the relation between the phytoconstituents of P. timorense bark and the tested bioactivities. HPLC analysis confirmed the presence of extensive number of phenolic compounds with reasonable amounts. Recently, many studies have examined the cytotoxicity, antioxidant and antihyperglycemic activities of phenolic compounds (25)(26)(27). Chromatographic examination of the 70% methanol extract of P. timorense bark resulted in the isolation and identification of seven compounds for the first time from the bark. UV spectrum of compound 1 gave a bathochromic shift in band I in addition of NaOMe with an increase in intensity which indicated the presence of the free hydroxyl group at 4' . Band II showed bathochromic shift on addition of sodium acetate which indicated the presence of free hydroxyl group at position 7. The hypsochromic shift with HCl after addition of AlCl 3 confirmed the presence of orthodihydroxy pattern in ring B. Acid hydrolysis showed that the quercetin was aglycone, while sugars were rhamnose and glucose. 1 H-NMR spectrum of compound 1 showed a doublet at δ 6.6 (J= 8.3Hz) for H-5′, a doublet of doublet at δ 7.47 (J = 2.1, 8.3 Hz) for H-6′. It showed a doublet at δ 7.4 (J = 2.2 Hz) for H-2′, while H-6 and H-8 appeared at δ 6.1 and δ 6.3, respectively as meta coupling protons. The anomeric glucose proton H-1′′ showed doublet at δ 5.3 with β-linkage, while those at δ 4.2 and at δ1.2 for rhamnose with α-linkage. These data were confirmed with the data which were reported for rutin (28).
UV spectrum of compound 2 gave a bathochromic shift in band I in addition of NaOMe with a marked increase in the intensity which confirmed free hydroxyl group at 4 -. On addition of sodium acetate, band II showed a bathochromic shift which indicated the presence of free hydroxyl group at position 7. With addition of HCl after AlCl 3 , the spectrum showed hypsochromic shift that confirmed the presence of orthodihydroxy pattern in ring B. 1 H-NMR spectrum of compound 2 showed doublet for H-5' at δ 6.84 (J = δ 8.9 Hz) as a result of ortho-coupling with H-6' , and multiplet at δ 7.25 for H-2' and H-6' , and for H-8 and H-6, peaks at δ 6.3 and 6.1 appeared, respectively with J = 2.4 Hz due to meta coupling. Anomeric proton of the rhamnose showed signal at δ 5.3 with J = 2.1 Hz due to α-configuration and rhamnose showed doublet at δ 0.77 with J = 6 Hz for its methyl group. After acid hydrolysis, the sugar showed to be rhamnose while aglycone was quercetin. These data were confirmed with the data which were reported for quercitrin (28).
For compound 3, on addition of sodium methoxide, band I showed a bathochromic shift with a marked increase in intensity which indicated the presence of free hydroxyl group at 4' . On addition of sodium acetate, band II showed a bathochromic shift that confirmed that hydroxyl group at position 7 was free. The presence of free hydroxyl group at position 5 was indicated after addition of AlCl3/HCl due to bathochromic shift in band Ia when it was compared with that in methanol. The presence of ortho-dihydroxyl pattern at ring-B was indicated after addition of sodium acetate with boric acid, as band I showed a bathochromic shift when it was compared with that in methanol. 1 H-NMR spectrum of compound 3 showed doublet at δ 7.73 for H-2' with J= 2.1 Hz for metacoupling with proton at H-6' that showed signal at δ 7.54. For H-5' , it showed a doublet at δ 6.93 with J= 8.1 Hz that showed ortho-coupling with H6' . The two meta-coupling protons for H-8 and H-6 appeared at δ 6.5 and δ 6.17 with J= 2.1 Hz. These data were confirmed with the data which were reported for quercetin (28).
The UV spectrum of compound 4 showed a bathochromic shift in band I on addition of NaOMe and the intensity was increased which proved that hydroxyl group at position 4' was free. Band II showed a bathochromic shift on addition of sodium acetate that confirmed the   presence of free hydroxyl group at position 7. The absence of orthodihydroxy pattern in ring B was confirmed by the absence of hypsochromic shift with AlCl3/HCl. These data were confirmed with the data which were reported for kaempferol (28). 1 H-NMR spectrum of compound 5 gave doublet of doublet peak at δ 6.75 for proton H-6 with ortho-coupling with H-5′ and meta-coupling with H-2′, doublet peak at δ 5.88 for proton H-6 which formed with H-8 meta coupling, doublet peak at δ 6.79 for proton H-5 with ortho-coupling with H6′, doublet peak at δ 6.87 for proton H-2′ with meta-coupling with H-6. H-8 showed peak at δ 6.03 as doublet with meta coupling with H-6, while proton H-4e gave peak at δ 2.92, proton H-4a showed peak at δ 2.56, multiple at δ 4.02 for H-3, doublet peak at δ4.54 as for H-2 and multiple peaks at δ 8.03 for phenolic protons. 13 C-NMR spectrum of compound 5 gave peak at δ 93.6 for carbon (C-6), (C-8) showed peak at δ 95.3, peak at δ 80.7 for carbon (C-2), peak at δ 66.1 for carbon (C-3), peak at δ 27.4 for carbon (C-4) and showed peaks at δ 156. 6 (29), compound 5 was identified as catechin. 1 H-NMR spectrum of compound 6 gave a sharp singlet peak at δ 7.09 ppm for two protons H-2, 6. 13 C-NMR spectrum of compound 6 showed the carbon of carbonyl of carboxylic group at δ 169.18, δ 144.96 for C-3,5, δ 138.17 for C-4, δ 120.76 for C-1 and δ 108.7 for C-2,6. So, compound 6 was identified as gallic acid. 1 H-NMR spectrum of compound 7 showed a triplet peak at δ: 6.43 for H-5 and a doublet peak at 6.26 for two protons H-4, 6. 13 C-NMR spectrum of compound 7 gave a peak at δ: 107.6 for C-4 & C-6, peak at 119.5 for C-5, peak at 133.5 for C-2 and peak at 147.07 for C-1,3. By comparing with the previously published data (30), compound 7 was identified as pyrogallol.
As the antioxidant activity of the isolated compounds were previously reported (31,32), so they played an important role in appearing the bioactivities of the extract. Recently, dichloromethane extract of the bark showed a significant activity against the A2780 human ovarian cancer cell line due to the presence of three new trihydroxy alkylcyclohexenones which were isolated from this extract (10). This reported research is the first study regarding the bark of P. timorense. Therefore, we aimed to continue the study of the cytotoxic effect of the methanol extracts and the isolated compounds against different cell lines. The methanol extract showed a promising cytotoxic activity against HepG2 cell line (IC 50 =4.39 µg/mL) comparing with that of Doxorubicin (IC 50 =3.86 µg/mL). We expected that this potent activity might be due the phenolic compounds. So, the cytotoxic activity of the isolated compounds was assessed in vitro against HepG2 cell line in comparison with Doxorubicin. Both catechin and gallic acid possessed moderate cytotoxic activity against HepG2 cell line (IC 50 = 6.4, & 9.6 µg/mL, respectively, by comparing with Doxorubicin (IC 50 = 3.86 µg/mL). We can conclude that the methanol extract of P. timorense bark has more potent in vitro cytotoxicity against HepG2 cell line (IC 50 = 4.39 µg/mL) than that of the isolated compounds. This might be due to the synergistic effect of the phenolic contents in the methanol extract which play a vital role in the bioactivities. The two doses of the 70% methanol extract of P. timorense bark (150 & 300 mg/kg) showed significant increase in TAC and significant decrease in the liver enzymes (ALT& AST), urea and creatinine levels compared with control and silymarin groups in both normal and hepatotoxic groups. Moreover, the two doses of the 70% methanol extract significantly decreased the blood glucose level. These results revealed that the 70% methanol extract of P. timorense bark had promising antioxidant, hepatorenal protective and antihyperglycemic effects, and the phytoconstituents in the methanol extract were responsible for these activities.

Conclusion
The present study revealed the positive effects of methanol extract of the bark of P. timorense (DC.) Leenh for use as antioxidant, hepatorenal protective, antihyperglycemic and anticancer. Detailed information on the phytoconstituents and the above mentioned biological activities were discussed in this research.

Funding/Support
This research had no financial support.