Secondary Metabolites from Leaves of Polyalthia lateriflora and Their Antimicrobial Activity

Saripah Salbiah Syed Abdul Azziz; Ahmed Kareem Obaid Aldulaimi; Saadon Abdulla Aowda; Yuhanis Mhd Bakri; Ali Arkan Majhool; Rawdha Mohammed Ibraheem; Tamara Kareem Obaid Aldulaimi; Hamidah Idris; Chee Fah Wong; Khalijah Awang; Marc Litaudon; Fauziah Abdullah

doi:https://doi.org/10.26452/ijrps.v11i3.2652

Secondary Metabolites from Leaves of Polyalthia lateriflora and Their Antimicrobial Activity

Saripah Salbiah Syed Abdul Azziz ¹ , Ahmed Kareem Obaid Aldulaimi ✉ ² , Saadon Abdulla Aowda ³ , Yuhanis Mhd Bakri ¹ , Ali Arkan Majhool ⁴ , Rawdha Mohammed Ibraheem ⁵ , Tamara Kareem Obaid Aldulaimi ⁶ , Hamidah Idris ⁴ , Chee Fah Wong ⁴ , Khalijah Awang ⁷ , Marc Litaudon ⁸ , Fauziah Abdullah ⁹

1 Department of Chemistry, Faculty of Science and Mathematics, Sultan Idris Education University, 35900 Tanjong Malim, Perak, Malaysia

2 Department of Pharmacy, Al-Zahrawi University college, Karbala, Iraq

3 Department of Chemistry, Faculty of Science, University of Babylon, Babylon, Iraq

4 Department of Biology, Faculty of Science and Mathematics, Sultan Idris Education University, 35900 Tanjong Malim, Perak, Malaysia

5 Medical City, Surgical Hospital, Baghdad, Iraq

6 Faculty of Pharmacy, University of Babylon, Babylon, Iraq

7 Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

8 Centre de Recherche de Gif, Institute de Chimie des Substances Naturelles, CNRS, 1, Avenue de la Terrasse, 91198, Gif-sur-Yvette Cedex, France

9 Forest Research Institute Malaysia, 52109 Kepong, Selangor, Malaysia

Abstract

The study aimed to isolate and identify the phytochemical components of Polyalthia lateriflora leaves and evaluate the antimicrobial activity. Six well-known compounds, including three triterpene lupeol (1) betulinic acid (2), β-Sitosterol-β-D-glucoside (3) and three oxoaporphine alkaloids O-methylmoschotaline (4), liriodenine (5) and atherosperminine (6). Structural elucidation of compounds were established through spectroscopic techniques such as 1D and 2D NMR (¹H and ¹³C, DEPT, COSY, NOESY, HMBC, HMQC), IR and LC-MS. The isolated compounds and crude extracted were tested for their antibacterial potential against several microorganisms including P. aeruginosa, E. coli, s, S. aureus, B. subtilis and Saccharomyces cerevisiae and its showed significant inhibition toward the organisms species with different concentration range.

Keywords

Alkaloid, Triterpene, Antimicrobial, Bacterial, Yeast

Introduction

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 (Lopes, Chatrou, Mello-Silva, Rudall, & Sajo, 2018). Annonaceae family has long been used as traditional medicines to treat diarrhea,dysentery, fever and rheumatism (Bele, Focho, Egbe, & Chuyong, 2011; Moghadamtousi et al., 2015). Polyalthia lateriflora (Bl.) King belongs to the genus Polyalthia flowering plant, of Annonaceae family are often dispersed in the tropics and subtropics regions and known to have large genus of shrubs and trees. They have approximately around 120 species and is predominantly dispersed in the Old World tropics with major species in Malaysia and south-east Asia (Taylor, Brophy, Goldsack, & Forster, 2001).

Polyalthia genus has been investigated as source of many potential compounds and was reported to have biological applications such as anticancer activity (Nahata, 2017), antibacterial activity (Barman et al., 2016; Negi & Sharma, 2010), anti fungal activity (Barman et al., 2016) and antioxidant activity (Adaramola et al., 2017).

The chemical investigation studies of Polyalthia species showed various types of secondary metabolites, such as alkaloids (Shono et al., 2016), terpenes (Yu et al., 2016), flavones (Ghani, Ahmat, Ismail, & Zakaria, 2011) and chalcone (Ahmat, Ghani, Ismail, Zakaria, & Zawawi, 2012). According to the literature, this study is the first report on phytochemical components of P. lateriflora leaves.

Experimental

Instruments

The NMR (¹H, ¹³C, DEPT, COSY, NOESY, HMQC, HMBC) were studied using 500 MHz JEOL ECX system at (UPSI) instrument. An Agilent LC–MS analysis was performed using the Accela^TM UHPLC System (Thermo Scientific, San Jose, USA) equipped with quarternary pump, A Phemomenex Kinetex RP C₁₈ column (3 µm, 2.2 mm I.D. x 150 mm), was recorded at FRIM, Malaysia. FTIR Model 6700 spectrophotometer was used for IR study (FRIM). Thermo Scientific BIOMATE 3S UV-Visible Spectrophotometer (UPSI).

Plant sample

Leaves of P. lateriflora (KL 5255) were collected and identified by University of Malaya group, Department of Chemistry, Kuala Lumpur, Malaysia, from Perak, Chemor, Bukit Kinta, Hutan Simpan.

Extraction and isolation

Through the cold extraction procedure, the dried leaves of P.lateriflora that weigh 2.0kg were extracted by three different solvent systems; hexane, dichloromethane (DCM) and methanol (MeOH) to produce crude extracts. Rotary evaporator was used to concentrate the extracts in order to produce 37g of MeOH, 25g of DCM and 35g of hexane. The DCM crude extract (25g) was fractionized using open CC silica gel, eluted with different ratio of DCM/MeOH and yielding 90 fractions. Fractions 1–3 were poured in CC on SiO₂ eluted with (100:00 to 80:20 v/v) hexane/EA to produced 40 sub-fractions. Sub-fractions 6-20 were further purified by CC over SiO₂ using (95:5 v/v) hexane/EA to produced 30 fractions; fractions 18-26 identified as compound 1 (3.3mg).

The sub-fractions 21-40 were purified by CC on SiO₂ using (95:5 to 50:50 v/v) ratio of hexane/EA as mobile phase and afforded 22 sub-fractions. Sub-fraction number 5-14 were further placed on CC on SiO₂ eluted with (90:10 v/v) of hexane/DCM and produced 36 fractions; fraction number 12-16 were identified as compound 2 (5.2mg). The fraction 13-25 from first column were grouped and sequentially submitted to purification through CC on SiO₂ using DCM/MeOH as eluted solvent (100:00 to 90:10 v/v) to yield compound 4 (4mg), compound 5 (2.3mg) and compound 6 (3.6mg). The fractions 26-35 from first column were chromatographed over CC on SiO₂ with DCM/MeOH (100:00 to 80:20 v/v) as mobile phase to produce compound 3 (4.8mg)

Antimicrobial Screening

According to (Nascimento, Locatelli, Freitas, & Silva, 2000), the microorganisms were grown in the BHI at 37°C temperature. Each microorganism was inoculated on agar plates surface (MH) with concentration of 10⁶ cells/mL after 6 hours of growth. As a result, the filter discs with 6mm diameter had saturated with the crude extracts and isolated compounds (50 µL) placed on the surface of the inoculated plates. Each sample was implanted concurrently in a hole of plates. Incubated the plates for 24 hours at the temperature of 37°C. The plates were left incubated for 24 hours at the temperature of 37°C. As time ended, observation of the inhibition zone took place that was measured using a ruler. The testing materials were dissolved in MeOH. The solvents were controlled for tested microorganism in the study, there had been no sign of inhibition. The methanol solvents were used as positive control. Meanwhile, samples that appeared bioactivity were applied to estimate the MIC for each microorganism.

In nutrient broth for 6 hours, five bacterial samples known as P. aeruginosa , S. aureus, Saccharomyces cerevisiae, and E. aerogenes were grown. Later, different concentration ranging from 25-250 µL to the extracts and isolated compounds were inoculated with nutrient broth. After 24 hours at the temperature of 37°C, by measuring the optical density the MIC from each sample was determined using the spectrophotometer (620 nm). All tests were carried out in duplicates.

Results and Discussion

Results and discussion

The known compounds (Figure 1) were investigated by spectroscopic NMR and LC-MS techniques and comparison with literature; these compounds include; lupeol (1) (Obaid et al., 2018), betulinic acid (2) (Haque, Siddiqi, Rahman, Hasan, & Chowdhury, 2013), β-Sitosterol-β-D-glucoside (3) (Peshin & Kar, 2017) and three oxoaporphine alkaloids O-methylmoschotaline (Aldulaimi et al., 2019) (4), liriodenine (5) (Kareem et al., 2018) and atherosperminine (6) (Obaid et al., 2018).

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/b757f63c-b105-4a40-a741-cf62af9f3939/image/a1029d6f-c639-46cd-9f00-123c53b56c73-upicture1.png — **Figure 1: The structures of 1-6 isolated compounds**

Table 1: Antimicrobial activity of extracts and isolated compounds

Compound		Gram positive		Gram negative		Yeast
		S. aureus	B. subtilis	E. coli	P. aeruginosa	Saccharomyces cerevisiae
1	IZ	11.1	7.2	9.5	7.3	8.5
1	MIC	100	150	250	100	250
2	IZ	8.1	10.2	11.5	12.2	7.4
2	MIC	150	250	150	100	50
3	IZ	8.7	9.8	7.3	10.5	15.3
3	MIC	200	200	50	100	150
4	IZ	9.3	10.8	12.5	12.6	15.3
4	MIC	100	100	250	200	150
5	IZ	12.5	11.8	13.5	12.6	12.9
5	MIC	150	100	250	100	100
6	IZ	7.7	9.2	21.0	8.5	9.3
6	MIC	100	150	100	250	100
Methanol crude	IZ	14.2	12.5	12.2	15.2	7.4
Methanol crude	MIC	100	100	150	50	100
DCM crude	IZ	12.2	13.1	13.5	7.2	7.5
DCM crude	MIC	150	200	100	100	50
Hexane crude	IZ	11.2	11.6	8.2	7.1	12.5
Hexane crude	MIC	100	100	250	200	100

IZ = Inhibition Zone (mm) ± SE (0.5-1.8), MIC = Minimum Inhibition Concentration (µg/mL)

Lupeol (1)

White steroid, Chemical Formula: C₃₀H₅₀O, HRESIMS: 409.3894 [M+H-18]⁺; IR v_max cm^- ¹ : 3361 (OH), 2939 and 2864 (C-H), 1456 and 1042 (CH₂). UV (DCM) lmax 320 nm. ¹H-NMR (CDCl₃, 500 MHz) ppm (δ): 1.68 (3H, s, H-30), 4.64 (1H, s, H-29), 4.52 (1H, s, H-29), 0.76 (3H, m, H-28), 0.90 (3H, m, H-27), 1.04 (3H, m, H-26), 0.82 (1H, m, H-25), 0.72 (1H, m, H-24), 0.96 (3H, s, H-23), 1.16 (1H, m, H-22), 1.31 (1H, m, H-21), 2.37 (1H, m, H-19), 1.32 (1H, t, H-18), 1.39 (1H, m, H-16), 1.06 (1H, m, H-15), 1.65 (1H, m, H-13), 1.09 (1H, m, H-12), 1.23, 1.38 (1H each, m, H-11), 1.29 (1H, d, J = 3.50 Hz, H-9), 1.42 (1H, m, H- 7), 1.31, 1.50 (1H each, m, H-6), 0.64 (1H, d, J = 10.0 Hz, H-5), 3.15 (1H, m, H-3), 1.57 (1H, m, H-2) and 0.85 (1H, m, H-1). ¹³C-NMR (CDCl₃, 125 MHz) δ (ppm): 20.0 (C-30), 110.2(C-29), 18. 9 (C-28), 16.4 (C-27), 16.0 (C-26), 17.2 (C-25), 15.9 (C-24), 29.0 (C-23), 40. 8 (C-22), 29.7 (C-21), 151.0 (C-20), 47.0 (C-19), 48.2 (C-18), 43.5 (C-17), 35.4 (C-16), 26.5 (C-15), 42.9 (C-14), 38.0 (C-13), 25.2 (C-12), 20.3 (C-11), 37.2 (C-10), 50.6 (C-9), 40.4 (C-8), 34.2 (C-7), 18.9 (C-6), 55.8 (C-5), 38.0 (C-4), 78.8 (C-3), 27.1 (C-2) and 38.5 (C-1).

Betulinic acid (2)

Needle crystalls, HRESIMS: [M+H]⁺457.3637. ¹H-NMR (CDCI₃, 500 MHz) ppm (δ): 0.82, 0.76 (each 3H, s, H-25, 24), 0.92, 0.98, 0.90 (each 3H, s, H-26, 27, 23), 1.67 (3H, s, H-30), 2.94 (1H, d, J = 11.0, H-19), 3.17 (1H, d, J = 5.0, Hz, H-3) and 4.69, 4.57 (each 1H, s, H-29); ¹³C-NMR (CDCI₃, 125 MHz) ppm (δ): 19.0 (C-30), 109.2 (C-29), 177.0 (C-28), 13.5 (C-27), 16.6 (C-26), 15.4 (C-25), 15.8 (C-24), 29.2 (C-23), 29.9 (C-22), 37.9 (C-21), 151.8 (C-20), 44.1 (C-19), 46.7 (C-18), 56.1 (C-17), 33.4 (C-16), 30.0 (C-15), 41.8 (C-14), 38.2 (C-13), 25.4 (C-12), 21.4 (C-11), 37.4 (C-10), 50.2 (C-9), 40.9 (C-8), 36.5 (C-7), 19.1 (C-6), 55.3 (C-5), 38.9 (C-4), 77.3 (C-3), 26.6 (C-2) and 38.2 (C-1)

β-Sitosterol-β-D-glucoside (3)

White steroid, HRESIMS: 575.4266 [M+H]⁺; ¹H-NMR (CDCl₃, 500 MHz) ppm (δ): 1.03 (3H, s, H-29), 1.35 (3H, m, H-28), 0.97 (3H, d, J = 7.00 Hz, H-27), 0.88 (3H, d, J = 7.00 Hz, H-26), 1.65 (3H, m, H-25), 0.97 (3H, m, H-24), 1.28 (3H, m, H-23), 1.22 (1H, m, H-22), 0.91 (3H, d, J = 6.50 Hz, H-21), 1.45 (1H, m, H-20), 0.94 (3H, s, H-19), 0.65 (3H, s, H-18), 1.22 (1H, m, H-17), 1.28 (1H, m, H-16), 1.08 (1H, m, H-15), 0.97 (1H, m, H-14), 1.51 (1H, m, H-12), 1.43 (1H, m, H-11), 0.89 (1H, m, H-9), 1.27 (1H, m, H-8), 1.75 (1H, m, H- 7), 5.28 (1H, d, J = 4.55 Hz, H-6), 2.32 (1H, m, H-4), 2.96 (1H, m, H-3), 1.60 (1H, m, H-2), 0.98 (1H, m, H-1), 5.02 (1H, m, H-‘6), 3.07 (1H, t, H-‘5), 3.00 (1H, t, H-‘4), 3.24 (1H, t, H-‘3), 2.87 (1H, t, H- ‘2), 4.24 (1H, d, J = 8.00 Hz, H-‘1), 3.45 (OH, t, H-‘5), 3.45 (OH, t, H-‘4), 3.65 (OH, d, J = 4.50 Hz, H-;3) and 3.65 (OH, d, J = 4.50 Hz, H-‘2). ¹³C-NMR (CDCl₃, 125 MHz) ppm (δ): 12.3 (C-29), 23.4 (C-28), 19.6 (C-27), 20.2 (C-26), 28.2 (C-25), 45.9 (C-24), 26.7 (C-23), 32.1 (C-22), 19.3 (C-21), 34.1 (C-20), 19.2 (C-19), 12.2 (C-18), 56.3 (C-17), 28.2 (C-16), 24.4 (C-15), 56.8 (C-14), 42.5 (C-13), 40.3 (C-12), 20.1 (C-11), 39.9 (C-10), 50. 9 (C-9), 31.9 (C-8), 32.1 (C-7), 121.5 (C-6), 141.2 (C-5), 39.8 (C-4), 74.2 (C-3), 29.7 (C-2), 37.4 (C-1), 61.9 (C-‘6), 77.8 (C-‘5), 71.31 (C-‘4), 77.2 (C-‘3), 74.1 (C-‘2) and 101.6 (C-‘1).

O -methylmoschatoline (4)

Orange amorphous. HRESIMS 322.1092 m/z [M+H]⁺; IR v_max cm^-1 : 1660 (C=0). UV (DCM) lmax 433 and 272 nm. ¹H-NMR (CDCl₃, 500 MHz) ppm (δ): 8.20 (1H,d,J=5.5 Hz, H-4), 8.96 (1H,d,J=5.5 Hz,H-5), 8.53 (1H,d,J=8.0 Hz,H-8), 7.51 (1H, t, H-9), 7.70 (1H, t, H-10), 9.09 (1H,d,J=8.0 Hz,H-11), 4.04 (3H, s, OCH₃-1), 4.09 (3H, s, OCH₃-2) and 4.15 (3H, s, OCH₃-3); ¹³C-NMR (CDCl₃, 125 MHz) ppm (δ): 127.7 (C-11), 134.6 (C-11a), 134.4 (C-10), 128.2 (C-9), 128.9 (C-8), 182.2 (C-7), 131.5 (C-7a), 145.5 (C-6a), 144.6 (C-5), 119.2 (C-4), 122.9 (C-3b), 131.1 (C-3a), 148.5 (C-3), 147.4 (C-2), 115.7 (C-1a), 156.5 (C-1), 61.9 (OCH₃-3), 61.6 (OCH₃-2), 61.1 (OCH₃-1).

Liriodenine (5)

Yellow amorphous, HRESIMS m/z 276.0674 [M+H]⁺; IR v_max cm^-1 : 1661 (C=0), 1447, 1310 and 1260 OCH₂O. UV (DCM) lmax at 261, 316 and 410 nm. ¹H-NMR (CDCl₃, 500 MHz) δ (ppm): 8.63 (1H,d,J=8.0 Hz,H-11), 7.74 (1H, t, H10), 7.55 (1H, t, H-9). 8.55 (1H,d,J=8.0 Hz,H-8), 8.92 (1H,d,J=5.0 Hz, H-5), 7.75 (1H,d,J=5.0 Hz,H-4), 7.22 (1H, s, H-3), 6.37 (2H, s, O-CH₂-O), ¹³C-NMR (CDC1₃, 125 MHz) ppm (δ): 127.4 (C-11), 132.9 (C-11a), 134.0 (C-10), 128.9 (C-9), 128.7 (C-8), 182.5 (C-7), 131.27 (C-7a), 145.2 (C-6a), 144.8 (C-5), 124.4 (C-4), 103.3 (C-3), 123.3 (C-3a), 108.1 (C-3b), 151.9 (C-2), 148.1 (C-l), 135.9 (C-la) and 102.6 (1-O-CH₂O-2).

Atherospermidine (6)

Orange amorphous powder, HRESIMS m/z 306.0652 [M+H]⁺; IR v_max cm^-1 : 1715 (C=0), 1040 and 940 (OCH₂O). UV (DCM) lmax 428 and 280 nm. ¹H-NMR (CDCl₃, 500 MHz) ppm (δ): 8.48 (1H,d,J=8.0 Hz,H-11), 7.66 (1H, t, H-10), 7.48 (1H, t, H-9), 8.43 (1H,d,J=8.0 Hz,H-8), 8.89 (1H,d,J=5.5 Hz,H-5), 8.12 (1H,d,J=5.5 Hz,H-4), 6.31 (2H, s, 1-OCH₂O-2) and 4.29 (3H, s, OCH₃-3). ¹³C-NMR (CDC1₃, 125 MHz) ppm (δ): 60.2 (OCH₃-3), 102.4 (1-OCH₂O-2), 126.7 (C-11), 133.2 (C-11a), 134.0 (C-10), 127.7 (C-9), 128.7 (C-8), 182.6 (C-7), 130.6 (C-7a), 145.0 (C-6a), 144.3 (C-5), 119.4 (C-4), 136.5 (C-3), 130.7 (C-3a), 122.9 (C-3b), 136.3 (C-2), 149.7 (C-l) and 102.6 (C-la).

In vitro antimicrobial activity was estimated for the samples using disc diffusion and MIC. Table 1 showed the antimicrobial activity of secondary metabolites and crude extracted of P. lateriflora leaves. The methanol crude extract showed the higher antimicrobial activity compare with isolated compounds for all tested microorganism, that may refer to presence of high polar bioactive compounds.

Conclusion

This study represented the first report on the phytochemical components from leaves of P. lateriflora. All six compounds isolated are known and were identified in comparison to their spectral data with those previously published. The crude extracts and isolated compounds showed antimicrobial activity all the pathogenic organisms.

Ethical Clearance

The Research Ethical Committee at scientific research by ethical approval of both MOH and MOHSER in Iraq

Conflict of Interest

None

Funding Support

Self-funding

[1] Lopes, J.C., Chatrou, L.W., Mello-Silva, R., Rudall, P.J. & Sajo, M.G. . 2018. Phylogenomics and evolution of floral traits in the Neotropical tribe Malmeeae (Annonaceae) Molecular Phylogenetics and Evolution 118(8):379–391.

[2] Bele, M Y, Focho, D A, Egbe, E A & Chuyong, B G . 2011. Ethnobotanical survey of the uses of Annonaceae around mount Cameroon. African Journal of PlantScience 5(4):237–247.

[3] Moghadamtousi, Soheil, Fadaeinasab, Mehran, Nikzad, Sonia, Mohan, Gokula, Ali, Hapipah & Kadir, Habsah . 2015. Annona muricata (Annonaceae): A Review of Its Traditional Uses, Isolated Acetogenins and Biological Activities. International Journal of Molecular Sciences 16(7):15625–15658.

[4] Taylor, P, Brophy, J. J., Goldsack, R. J. & Forster, P. I. . 2001. Leaf Oils of the Australian Species ofPolyalthia (Annonaceae) Journal of Essential Oil Research 13(1):5–7.

[5] Nahata, A . 2017. Anticancer agents: a review of relevant information on important herbal drugs. Int J Clin Pharmacol Toxicol 6(2):250–255.

[6] Barman, Harikamal, Roy, Aniruddha, Das, Shaon Kumar, Singh, N. U., Dangi, D. K., Dahun, & Tripathi, A. K. . 2016. Antifungal properties of some selected plant extracts against leaf blight (Alternaria alternata)in tomato. Research on Crops 17(1):151.

[7] Negi, R S & Sharma, B . 2010. In Vitro Antibacterial Activity Of Plants Available In Semiarid Region Of Rajasthan. II. J. Phytol. Rres 1(8):41–45.

[8] Adaramola, Banji, Otuneme, Oluchi, Onigbinde, Adebayo, Orodele, Kunle, Aleshinloye, Abimbola, David, Jegede, Ogunnowo, Ayodele & Adewumi, Aderiike . 2017. Phytochemical Analysis and in vitro Antioxidant Activity of Fractions of Methanol Extract of Polyalthia longifolia var. Pendula Leaf. European Journal of Medicinal Plants 21(1):1–10.

[9] Shono, Takumi, Ishikawa, Naoki, Toume, Kazufumi, Arai, Midori A., Masu, Hyuma, Koyano, Takashi, Kowithayakorn, Thaworn & Ishibashi, Masami . 2016. Cerasoidine, a Bis-aporphine Alkaloid Isolated from Polyalthia cerasoides during Screening for Wnt Signal Inhibitors. Journal of Natural Products 79(8):2083–2088.

[10] Yu, Z.-X, Fu, Y.-H, Chen, G.-Y, Song, X.-P, Han, C.-R, Li, X.-B & Chen, S.-C . 2016. New clerodane diterpenoids from the roots of Polyalthia laui. Fitoterapia 111:36–41.

[11] Ghani, N A, Ahmat, N H, Ismail, & Zakaria, . 2011. Flavonoid constituents from the stem bark of Polyalthia cauliflora var cauliflora. Australian Journal of Basic and Applied Sciences 5(8):154–158.

[12] Ahmat, N., Ghani, N.A., Ismail, N.H., Zakaria, I. & Zawawi, N.K.N.A. . 2012. Chemical Constituents and Cytotoxic Activity of Polyalthia cauliflora var. cauliflora. Research Journal of Medicinal Plant 6(1):74–82.

[13] Nascimento, Gislene G. F., Locatelli, Juliana, Freitas, Paulo C. & Silva, Giuliana L. . 2000. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian Journal of Microbiology 31(4):247–256.

[14] Obaid, Ahmed Kareem, Azziz, S S S, Bakri, Y M, Nafiah, M A, Awang, K, Saadon Abdulla Aowda, , Hassan, N M, Has-Yun, Y Z & Abdullah, F . 2018. Antioxidative and cytotoxic activities of crude and isolated compounds of P. Lateriflora (bl.) King. Journal of Pharmaceutical Sciences and Research 10(11):2718–2721.

[15] Haque, Ayesha, Siddiqi, M Mahboob Ali, Rahman, AFM Mustafizur, Hasan, Choudhury M & Chowdhury, AM Sarwaruddin . 2013. Isolation of Betulinic Acid and 2,3-Dihydroxyolean-12-en-28-oic Acid from the Leaves of Callistemon linearis. Dhaka University Journal of Science 61(2):211–212.

[16] Peshin, Tania & Kar, H . 2017. Isolation and Characterization of β-Sitosterol-3-O-β-D-glucoside from the Extract of the Flowers of Viola odorata. British Journal of Pharmaceutical Research 16(4):1–8.

[17] Obaid Aldulaimi, Ahmed Kareem, Abdul Azziz, Saripah Salbiah Syed, Bakri, Yuhanis Mhd, Nafiah, Mohd Azlan, Aowda, Saadon Abdulla, Awang, Khalijah & Litaudon, Marc . 2019. Two New isoquinoline alkaloids from the bark of Alphonsea cylindrica King and their antioxidant activity. Phytochemistry Letters 29:110–114.

[18] Ahmed Kareem, Aldulaimi, Azziz, S S S A, Bakri, Y M, Nafiah, M A, Awang, K, Abdulla Aowda, Saadon, Litaudon, M, Hassan, N M, Naz, H, Abbas, P, Hashim, Y Z H & Majhool, A A . 2018. Alkaloids from Alphonsea Elliptica Barks and Their Biological Activities. Journal of Global Pharma Technology 10(08):270–275.