F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.130477.1 Research Article Articles Anti-angiogenic activity of Ficus deltoidea L. Jack silver nanoparticles using chorioallantoic membrane assay

[version 1; peer review: 1 approved with reservations]

 Aryani Retno Conceptualization Formal Analysis Writing – Original Draft Preparation Writing – Review & Editing 1 Nugroho Rudy Agung Conceptualization Methodology Supervision Validation Writing – Original Draft Preparation Writing – Review & Editing a 2 Manurung Hetty Funding Acquisition Methodology Validation Writing – Review & Editing https://orcid.org/0000-0003-0161-6459 3 Rulimada Muhammad Hafidz Conceptualization Validation Writing – Original Draft Preparation Writing – Review & Editing 4 Maytari Erin Conceptualization Formal Analysis Project Administration Writing – Review & Editing 4 Siahaan Angeline Formal Analysis Investigation Writing – Original Draft Preparation Writing – Review & Editing 4 Rudianto Rudianto Formal Analysis Project Administration Validation Writing – Review & Editing https://orcid.org/0000-0003-0909-2848 2 Jati Wibowo Nugroho Formal Analysis Writing – Original Draft Preparation Writing – Review & Editing 5 Anatomy and Microtechnique Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Mulawarman, Samarinda, East Kalimantan, 75123, Indonesia Animal Physiology, Development, and Molecular Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Mulawarman, Samarinda, East Kalimantan, 75123, Indonesia Plant Physiology and Development Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Mulawarman, Samarinda, East Kalimantan, 75123, Indonesia Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Mulawarman, Samarinda, East Kalimantan, 75123, Indonesia Faculty of Technobiology, Universitas Atma Jaya Yogyakarta, Yogyakarta, Special Region of Yogyakarta, 55281, Indonesia rudyagung.nugroho@fmipa.unmul.ac.id

No competing interests were disclosed.

 24 5 2023 2023 12 544 31 3 2023 Copyright: © 2023 Aryani R et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Ficus deltoidea L. Jack is a folk medicinal plant known for its pharmacological properties, including anti-inflammatory, anticancer and anti-angiogenic. This study aimed to evaluate the anti-angiogenic properties of silver nanoparticles biosynthesized using F. deltoidea leaf extract (AgNPs-Fd).

Methods: The AgNPs-Fd were synthesized by mixing 100 mL 1 mM aqueous silver nitrate (AgNO 3) and 100 mL 0.1% F. deltoidea ethanolic leaf extract. The resulting AgNPs-Fd were observed for color change and Tyndall effects. Reaction mixture color change from pale brown to reddish brown was observed at 48 h at 37°C. The characterization of AgNPs-Fd was completed with UV–Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. For quantitative analysis of the vascular network in the chorioallantoic membrane (CAM) assay, AngioTool open-source software was used.

Results: The plasmon resonance peak for AgNPs-Fd at 430 nm was visible in the UV-Visible spectrum, indicating the formation of AgNPs-Fd. The F. deltoidea extract and nanoparticles interacted well according to FTIR analysis. The AgNPs-Fd morphology of 20 nm particle sizes was observed using TEM. The chromatographic analysis of AgNPs-Fd identified potential anti-angiogenic compounds, such as phytol, stigmasterol, lupeol and sitosterol. The angiogenic inhibition properties of AgNPs-Fd were tested using the CAM assay. The 90 mg dose AgNPs-Fd treatment in CAMs demonstrated significant anti-angiogenesis, indicating effectiveness in controlling vessel formation.

Conclusions: The present study suggests that eco-friendly work and the “green” process of AgNPs-Fd is potentially applicable for nanobiotechnology in anti-angiogenic fields.

 anti-angiogenic Ficus deltoidea green synthesize nanomaterials Kementerian Pendidikan, Kebudayaan, Riset, dan Teknologi 464/UN17.L1/HK/2022 223/E5/PG.02.00.PT/2022 The authors received funding from Ministry of Education, Culture, Research and Technology, Indonesia for grant funding contract number: 223/E5/PG.02.00.PT/2022 and 464/UN17.L1/HK/2022. This has been approved by the Institute for Research and Community Service, Mulawarman Universitas, Samarinda, East Kalimantan, Indonesia The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Ficus deltoidea is a well-known medicinal plant that has been historically used by various ethnic groups in Indonesia. This plant, from the Moraceae family, is an epiphyte with a shrub habit that is found in secondary forests. The F. deltoidea leaf is used to treat diabetes mellitus, 1 for anti-inflammation, 2 as an antibacterial 3 and for cancer inhibition. 4 Additionally, F. deltoidea is reported to be antinociceptive, anti-inflammatory, antidiabetic, anti-obesity, anti-melanogenic, antioxidative and a free radical scavenger. 5

Furthermore, F. deltoidea leaf extract may suppress cancer development through three mechanisms: apoptosis via the intrinsic route, migration and invasion inhibition, and angiogenesis inhibition. 6 , 7 The aqueous and ethanol extracts of F. deltoidea are known to have anticancer activities on human ovarian carcinoma cells. The F. deltoidea aqueous extract has an IC 50 of 224.39±6.24 g/mL, while the F. deltoidea ethanolic extract has an IC 50 of 143.03±20.21 g/mL. However, cell growth is affected differently by exposure to the different extracts. Cell release is triggered with the aqueous extract, while cell growth is reduced with the ethanol extract. 8

Recently, technology surrounding nanoparticles, which can be biologically synthesized, has grown, since the nano-size items exhibit different characteristics of its bulk components due to variations in their surface–volume ratio. 9 Most nanomaterials have demonstrated outstanding quantum confinement with unique catalytic features in many biological reactions and a variety of applications in various sectors that enhance the approach for electrical, environmental and medicinal objectives. 10 Nanomaterials are employed in biological imaging, diagnostics, biosensing, gene therapy, and antibacterial and anticancer drugs. 11

“Greener” nanoparticle synthesis is advantageous over previous approaches as it is simple, inexpensive, and relatively repeatable, typically resulting in more stable compounds. Nanoparticles can also be created using microorganisms. However, the rate of synthesis is slower, and the process is limited regarding accessible sizes and forms when compared with approaches employing plant-based components. The green synthesis process does not require high pressure, energy, temperature, or harmful ingredients; as a result, many scientists have abandoned synthetic approaches. Moreover, plants generate more stable nanoparticles than other technologies and are easily scaled up. 11

Previous studies revealed that silver nanoparticles synthesized using Ficus deltoidea extract (AgNPs) have been biosynthesized using various plant extracts, including Salvia spinosa, 12 Origanum vulgare L., 13 Araucaria angustifolia, 14 Myrmecodia sp bulb, 15 Eleutherine americana, 16 and Citrus medica. 17 The biosynthesis of AgNPs using various plant extracts is a simple and rapid method, with its success easily determined by color changes and UV-Vis spectrophotometry. Furthermore, to characterize the resulting AgNPs, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) are employed. 18

Several studies using plant derived AgNPs involving cancer and angiogenesis have been performed. Previous research reported that the green synthesis of AgNPs using rapeseed flower pollen potentially reduced angiogenesis. 19 Similar findings were also described by Vimalraj and Ashokkumar, 20 who stated that green synthesis of gold nanoparticles mediated by Mangifera indica seed water extracts demonstrated anti-angiogenic properties. Baharara and Namvar 21 revealed that AgNPs generated from Achillea biebersteinii flower extracts were well-dispersed and stable using green techniques and exhibited potential therapeutic advantages against angiogenesis. In addition, the biosynthesized-AgNPs successfully reduced angiogenesis in an embryonated chicken model, and this anti-angiogenic property of AgNPs can be investigated as a potential therapeutic against pathological angiogenesis and solid tumors by targeting the vasculature. 22

Various in vivo angiogenesis assays have been conducted to study the angiogenic processes and to discover new therapeutic agents that inhibit or trigger angiogenesis. One of the most common laboratory methods used to study angiogenesis and anti-angiogenesis is the chorioallantoic membrane (CAM) assay of chicken embryos. The CAM methodology has several advantages, such as a high embryo survival rate, simple procedure, not requiring high sterility, low cost and a high level of reproducibility and reliability. The CAM assay was first performed by Judah Folkman and colleagues for testing angiogenic activity in tumor tissue. 23 – 25

Though several studies of AgNPs and anti-angiogenic properties of plant extracts have been conducted, the anti-angiogenic properties of AgNPs biosynthesized using F. deltoidea (AgNPs-Fd) has yet to be defined. Thus, the present research aimed to evaluate the anti-angiogenic properties of AgNPs-Fd using the CAM method. The biosynthesis of AgNPs-Fd was characterized by observing the color change, Tyndall effect, UV-Vis spectrophotometry, TEM, XRD and FTIR. Phytochemical analysis of AgNPs-Fd was also conducted using gas chromatography-mass spectrometry (GC-MS). For quantitative analysis of the vascular network in the CAM assay, AngioTool open-source software was used. 26

Methods

Present methods has been deposited step-by-step on protocols.io with DOI: dx.doi.org/10.17504/protocols.io.14egn2nj6g5d/v1.

Ethical statement

The Ethics Research and Community Service Mulawarman Universitas approved the use of chicken embryos to be used in the chorioallantoic membrane assay (Contract Number: 464/UN17.L1/HK/2022; 10 May 2022). This study is reported in line with ARRIVE guidelines. 53

Plant extract preparation

Leaves of the F. deltoidea were collected from a local farmer at East Kalimantan, Indonesia. The collected leaves were identified using a floras identification manual. The dry leaves were cut into small pieces and ground using a mechanical blender. The leaf powder was extracted using ethanol, concentrated using a rotary evaporator and stored at 4°C until further use.

 Biosynthesis of AgNPs-Fd

Aqueous silver nitrate (AgNO 3) (100 mL, 1 mM) was combined with 100 mL 0.1% F. deltoidea leaf extract. The pH of the solution was adjusted to 7.0, and it was subsequently incubated on a rotary shaker at 150 rpm for 48 h at 28°C. The fabrication of AgNPs-Fd was observed macroscopically by color change and the Tyndall effect.

 Characterization of AgNPs-Fd

The resulting AgNPs-Fd were confirmed using UV-Vis spectrophotometry (Shimadzu, UV-1280, Japan) in the range of 350–750 nm. The TEM (MIRA3 model, Czech Republic) was operated to display the surface morphology of the AgNPs-Fd. XRD was applied to evaluate the chemical characterization of AgNPs-Fd. To determine phytochemicals surrounding the AgNPs-Fd, FTIR spectroscopy (Agilent, Cary 630 model, US) was performed. 27

Phytochemical analysis

Phytochemical analysis of the AgNPs-Fd was performed using GC-MS (HP-5MS UI, Agilent, USA) to evaluate the chemical compounds that potentially serve as anti-angiogenic. For this, samples of AgNPs-Fd were dissolved in ethanol in a microtube, vortexed and centrifuged for three minutes at 9,500 rpm. The resulting supernatant was used for identification and injected into the GC-MS apparatus. The condition of the GC-MS is as follows: column: HP-5MS UI, gas carrier: helium UHP (He), injector temperature: 290°C, split flow: 10 ml/min, split ratio: 10; front inlet flow: 1.00 ml/min, MS transfer line temp: 230°C, ion source temp: 200°C, mass list range (amu): 40–500, purge flow: 3 ml/min, gas saver flow: 5 ml/min, and gas saver time: five minutes.

 CAM assay

To analyze AgNPs-Fd for anti-angiogenic properties, a CAM assay was performed following previous methods by Ribatti, 24 Camposano and Torre, 28 and Gamallo and Espere. 29 In total, 24 chicken eggs were collected in preparation for the CAM analysis and were dosed with F. deltoidea extract (Fd) or AgNPs-Fd. The doses used in the paper disk for the CAM assay were as follows: negative control (30 ng basic fibroblast growth factor (bFGF); positive control (30 μg cortisone acetate with 30 ng bFGF); treatment groups (30 ng bFGF with AgNPs-Fd at 45, 60, 75 and 90 μg, respectively). A basic fibroblast growth factor (bFGF-Thermo Fisher Scientific, USA) is a group of proteins secreted by tissues to regulate cell metabolism, proliferation, differentiation, and survival. The bFGF was used to induce neovascularization. 30

Before incubation, eggshells were cleansed with 70% alcohol. The eggs were incubated in for six days at 37−39°C with 50−60% humidity. The boundaries of the air space, which indicated the location of the embryo, were marked on all eggshells with a pencil (1 × 1 cm). Candling, with egg binoculars, was used to determine the location of the embryo. By using a povidone-iodine solution, the eggshell was cleaned at the pole containing the air space and the part above the embryo. Using a needle, a small hole was made in the air chamber and vacuumed until the air moved from the pole to the top of the egg.

Next, a window measuring 1×1 cm was made on the marked area using a mini drill. Paper discs for each treatment were then embedded onto the CAM through this window. The holes in the polar regions and 1×1 cm windows were sealed with paraffin film after they had been planted according to the treatment. The eggs were then incubated for 48 h at 37−39°C with 50−60% humidity. After, the eggs were removed and subsequently killed by freezing for 24 h. The eggs were then opened by cutting the eggshell into two parts, beginning with the part closest to the air cavity. The egg contents were slowly and carefully removed, keeping the CAM attached to the shell. Each CAM was photographed and analyzed using AngioTool 0.6 software (RRID:SCR_016393).

AngioTool is a small, easy-to-use application that allows for the quick, hands-free and reproducible measurement of vascular networks in micrographs. AngioTool calculates a variety of morphological and geographical data, such as the area covered by a vascular network, the number of vessels, vessel length, vascular density and lacunarity. AngioTool also computes the “branching index” (branch points/unit area), which quantifies specimen sprouting activity. 26

Data analysis

Data from the AgNPs-Fd characterization were evaluated as graphs and images. Notations and arrow marks were added to the images, and they were cropped and the resolution was increased to 300 dpi, all of which were performed using Adobe photoshop (RRID:SCR_014199) (Adobe Photoshop, adobe, Inc., USA). Meanwhile, data from the CAM assays, such as vessel area, total number of junctions, junction density, total vessel length, average vessel length and total number of end points, were presented as the mean±standard errors. Significant differences among the treatment groups were assessed using analysis of variance (ANOVA) with IBM SPSS Statistics (RRID:SCR_016479) ver. 22 (IBM Corp., USA). A Duncan’s multiple range test (DMRT) was performed if significant differences were detected from ANOVA analysis. Significance was defined as p<0.05.

 Results Biosynthesis of AgNPs-Fd

The solution began to change color once the plant extract was added to the AgNO 3 solution. When the reaction began, the solution was light brown. After 30 minutes of constant stirring, the solution gradually darkened ( Figure 1). 52 Further, Tyndall scattering was performed to confirm the biosynthesis of AgNPs-Fd. As depicted in Figure 2, the solution of colloidal AgNPs-Fd ( Figure 2b) demonstrated the Tyndall effect (beam of light visible from the side) due to the presence of lyophobic sol particles large enough for light dispersion, while genuine solutions ( Figure 2a and c) did not exhibit light dispersion. Thus, the presence of AgNPs-Fd in aqueous solution as colloids was deduced.

Color change of biosynthesized AgNPs-Fd.

(A) Plant extract; (B) colloidal AgNPs-Fd; (C) AgNO 3 solution. AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract; AgNO 3, aqueous silver nitrate.

 Tyndall effects of biosynthesized AgNPs-Fd.

(A) Plant extract; (B) colloidal AgNPs-Fd; (C) AgNO 3 solution. AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract; AgNO 3, aqueous silver nitrate.

 AgNPs-Fd analysis using UV-Vis Spectrophotometer

A peak in the UV-Vis absorption spectra of AgNPs-Fd at 430 nm with respect to 2.25 absorbance was observed, whereas the ethanol extract of the F. deltoidea leaf absorbed at 490 nm ( Figure 3). This finding is similar to previous studies reporting that the UV-Vis maximum absorbance of AgNPs was 430 nm when mediated by the Pedalium murex leaf extract, 31 Alternanthera dentata leaf extract 32 and Tecomella undulata leaf extract. 33

Absorbance spectrum of biosynthesized AgNPs-Fd.

The highest absorbance of AgNPs-Fd was observed at 430 nm with an absorbance of 2.25. AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract.

 AgNPs-Fd morphology and size analysis using TEM

The morphology and size of the AgNPs-Fd were analyzed using TEM images. Figure 4 shows a TEM image of AgNPs biosynthesized from ethanolic extract of F. deltoidea leaves. The AgNPs-Fd shape was spherical in nature. The AgNPs-Fd were surrounded by a faint, thin layer of other substances, which were assumed to be organic material from the F. deltoidea extract. Few particles were agglomerated among the 20 nm nanoparticles that were produced. A recent study performed by Ref. 34 mentioned that the TEM results of the AgNPs mediated using Terminalia arjuna leaf extract have spherically-shaped nanoparticles approximately 10–50 nm in size.

TEM image of the AgNPs-Fd produced by ethanolic extract of <italic toggle="yes">F. deltoidea</italic> leaves.

Yellow markings and notations in the TEM image have been made by using Adobe Photoshop. TEM, transmission electron microscope; AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract.

 XRD analysis

The crystalline nature of the biosynthesized AgNPs-Fd was evaluated using XRD. The XRD pattern obtained revealed that AgNPs-Fd had a face-centered spherical structure in nature ( Figure 5), and there were several characteristic diffraction peaks indexed from 21.386 to 76.655.

The XRD of the AgNPs-Fd biosynthesized using ethanolic extract of <italic toggle="yes">F. deltoidea</italic> leaves.

XRD, X-ray diffraction; AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract.

 FTIR spectroscopy

Biomolecules for the capping and effective stability of the metal nanoparticles produced using the ethanolic extract of F. deltoidea leaves were identified through FTIR analysis. Figure 6 presents the FTIR spectrum of AgNPs-Fd. The bands at 3,356, 3,200, and 3,243 cm -1 correspond to H-bonded alcohols and phenols with O-H stretching. The signals at 2,917 and 2,849 cm -1 correspond to carboxylic acids with an O-H stretch. The assignments in 1,711, 1,610, 1,516, and 1,449 cm -1 correspond to primary amines with a bent N-H. The peak at 1,376 cm -1 relates to C-N stretching of an aromatic amine group, and the band at 1,242 cm -1 pertains to a C-N bond, which is either an amine or an amide molecule.

The FTIR spectrum of AgNPs-Fd biosynthesized using ethanolic extract of <italic toggle="yes">F. deltoidea</italic> leaves.

FTIR, Fourier transform infrared spectroscopy; AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract.

 GC-MS analysis

Phytosterols appear to suppress carcinogen generation, cancer cell development, angiogenesis, invasion, and metastasis, as well as promote apoptosis in malignant cells through a variety of methods. Phytosterol consumption potentially increases the activity of antioxidant enzymes, lowering oxidative stress. 35 The present study found several phytosterols in the AgNPs-Fd biosynthesized using ethanolic extract of Ficus deltoidea leaves ( Figure 7 and Table 1).

Chromatogram of AgNPs-Fd biosynthesized using <italic toggle="yes">F. deltoidea</italic> ethanolic leaves extract.

AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract.

 Bioactive compounds in AgNPs-Fd biosynthesized using <italic toggle="yes">F. deltoidea</italic> ethanolic leaves extract.

AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract.

No Bioactive compound Chemical formula Molecular weight
1 Phenol,2-propyl- C 9H 12O 136
2 2H-Indeno[1,2-b]furan-2-one, 3,3a,4,5,6,7,8,8b-octahydro-8,8-dimethyl C 13H 18O 2 206
3 Hexadecanal C 16H 32O 240
4 13-Heptadecyn-1-ol C 17H 32O 252
5 2-Heptadecanone C 17H 34O 254
6 1-Hexadecanol,2-methyl- C 17H 36O 256
7 Estra-1,3,5(10)-trien-17fl-ol C 18H 24O 256
8 1-Hexadecanol,2-methyl- C 17H 36O 256
9 n-Hexadecanoic acid C 16H 32O 2 256
10 Acetamide, N-methyl-N-[4-[2-acetoxymethyl-1-pyrrolidyl]-2-butynyl]- C 14H 22N 2O 3 266
11 7-Methyl-Z-tetradecen-1-ol acetate C 17H 32O 2 268
12 Hexadecanoic acid, ethyl ester C 18H 36O 2 284
13 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C 20H 40O 296
14 Phytol C 20H 40O 296
15 Ethyl 14-methyl-hexadecanoate C 19H 38O 2 298
16 Z,Z-3,15-Octadecadien-1-ol acetate C 20H 36O 2 308
17 Ethyl 9.cis.,11.trans.-octadecadienoate C 20H 36O 2 308
18 cis-13-Eicosenoic acid C 20H 38O 2 310
19 (E)-9-Octadecenoic acid ethyl ester C 20H 38O 2 310
20 Ethanol, 2-(9-octadecenyloxy)-, (Z)- C 20H 40O 2 312
21 Heptadecanoic acid, 15-methyl-, ethyl ester C 20H 40O 2 312
22 2-[4-methyl-6-(2,6,6-trimethylcyclohex-1-enyl)hexa-1,3,5-trienyl]cyclohex-1-en-1-carboxaldehyde C 23H 32O 324
23 Cyclopropanebutanoic acid, 2-[[2-[[2-[(2-pentylcyclopropyl)methyl]cyclopropyl]methyl]cyclopropyl]methyl]-, methyl ester C 25H 42O 2 374
24 Ingol 12-acetate C 22H 32O 7 408
25 Stigmasterol C 29H 48O 412
26 Sitosterol C 29H 50O 414
27 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3fl,5Z,7E)- C 27H 44O 3 416
28 Tocopherol C 28H 48O 2 416
29 4,4,6a,6b,8a,11,11,14b-Octamethyl-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-octadecahydro-2H-picen-3-one C 30H 48O 424
30 fl-Amyrin C 30H 50O 426
31 a-Amyrin C 30H 50O 426
32 Lupeol C 30H 50O 426
33 Friedelan-3-one C 30H 50O 426
34 (+)Tocopherol, O-methyl- C 29H 50O 2 430
35 Ethyl iso-allocholate C 26H 44O 5 436
36 Lanosta-8,24-dien-3-ol, acetate, (3fl)- C 32H 52O 2 468
37 Lup-20(29)-en-3-ol, acetate, (3fl)- C 32H 52O 2 468
38 3',8,8'-Trimethoxy-3-piperidyl-2,2'-binaphthalene-1,1',4,4'-tetrone C 28H 25NO 7 487
39 Cholest-22-ene-21-ol, 3,5-dehydro-6-methoxy-, pivalate C 33H 54O 3 498
40 7,8-Epoxylanostan-11-ol, 3-acetoxy- C 32H 54O 4 502
41 17-(1,5-Dimethyl-3-phenylthiohex-4-enyl)-4,4,10,13,14-pentamethyl-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopent(a)phenanthren-3-ol C 36H 54OS 534
42 1-Heptatriacotanol C 37H 76O 536
43 9-Desoxo-9-x-acetoxy-3,8,12-tri-O-acetylingol C 28H 40O 10 536
44 1-Heptatriacotanol C 37H 76O 536
45 Rhodopin C 40H 58O 554
46 Oleic acid, 3-(octadecyloxy) propyl ester C 39H 76O 3 592
47 Cholesterol margarate C 44H 78O 2 638
CAM analysis

The CAM assay is commonly used as an in vivo testing system for anti-angiogenesis research. It has the advantage of being cost-efficient and lends itself to large-scale screening by testing the efficacy of an inhibitor using various stimulators alone or in combination with an anti-angiogenic medication. The present study used AgNPs-Fd as an anti-angiogenic compound and found that AgNPs-Fd at 90 μg in the CAM analysis resulted in higher anti-angiogenic activities than other treatment groups. The anti-angiogenic activities were demonstrated through the reduction of vessel area, total number of junctions, junction density, total vessel length, average vessel length and the total number of endpoints ( Table 2).

Anti-angiogenic activity of AgNPs-Fd mediated by <italic toggle="yes">F. deltoidea</italic> on the CAM of chicken embryos.
Parameters Control (-) Control (+) AgNPs-Fd (μg)
45 60 75 90
Vessel area 427.78±5.24 e 95.34±0.10 a 246.09±6.87 d 253.55±1.86 d 214.04±9.91 c 170.85±5.25 b
Total number of junctions 1,235.00±35.28 e 614.00±35.38 a 981.66±23.38 d 947.67±10.17 d 853.33±32.46 c 738.67±27.14 b
Junction density 1.49±0.04 e 0.75±0.04 a 1.18±0.04 d 1.09±0.02 cd 1.02±0.06 c 0.89±0.03 b
Total vessel length 1,468.98±20.49 f 477.63±7.83 a 937.09±10.72 e 829.98±5.93 d 766.07±10.27 c 670.21±29.64 b
Average vessel length 6.36±0.83 c 1.87±0.08 a 3.75±0.12 b 3.20±0.18 b 3.04±0.06 b 2.63±0.08 ab
Total number of endpoints 1,168.67±23.69 d 556.33±37.71 a 851.33±10.26 b 937.00±32.05 c 853.00±28.04 b 560.67±13.91 a

Note: Negative control (30 ng bFGF); Positive control (cortisone acetate 30 μg + 30 ng bFGF); treatment groups (30 ng bFGF added with AgNPs-Fd at doses: 45; 60; 75; and 90 μg). Mean±standard error followed by different superscript (a, b, c, d, e, f) at same column in the same parameter/row, indicated significantly different at P<0.05. AgNPs-Fd, silver nanoparticles synthesized using Ficus deltoidea extract; CAM, chorioallantoic membrane; bFGF, basic fibroblast growth factor.

 Discussion

The green synthesis of nanoparticles using biological systems, particularly plant extracts, is an emerging subject in nanotechnology. Various plant extracts and several methodologies have been investigated to explore the benefits of these nanomaterials. In the field of medicine, specifically angiogenesis, which is an essential physiological process and important in several pathological diseases, including tumor development and metastasis, the utilization of plant extracts is a low-cost and environmentally friendly method for producing nanoparticles. The present work aimed to investigate the anti-angiogenic properties of AgNPs biosynthesized using the ethanolic extract of F. deltoidea leaves on chick CAMs.

In the biosynthesis of the nanoparticles, the color change in is an indicator of AgNPs-Fd formation. Similar findings were revealed by Anju and Parvathy, 36 who mentioned that the visual confirmation of AgNPs generation via green synthesis is possible by evaluating the color shift. The color shift is caused by the reduction of silver ions (Ag +) to AgNPs 37 and is facilitated by bioactive phytochemicals in the F. deltoidea leaf extract, such as flavonoids. 38 Flavonoids may be responsible for the rapid reduction and capping of Ag + into AgNPs-Fd observed in this research. The flavonoids in F. deltoidea leaf extracts are potent reducing agents, suggesting that AgNPs-Fd may be synthesized by AgNO 3 reduction. The flavonoid components of F. deltoidea ethanolic extract may actively be involved in and liable for Ag + to silver (II) oxide (Ag 0) reduction. 39 Another investigation also found that water-soluble flavonoids were involved in the reduction of metal ions utilizing plant extracts. 40

Meanwhile, the Tyndall effect was especially relevant to colloidal suspensions at room temperature, with the production of AgNPs-Fd exhibiting an immediate pale brown color. It also demonstrated that the AgNPs-Fd were strongly formed and stable in the aqueous phase, with no precipitation. 41 Furthermore, previous reports on AgNPs revealed that absorbance at approximately 430 nm UV-Vis spectrophotometer is a hallmark of the noble metal particles. 42 It was noticed that the surface plasmon resonance at 430 nm indicated the retention of AgNPs-Fd in the colloidal solutions due to the emergence of the pale brown color for particle stabilization.

Characterization of resulting AgNPs-Fd using TEM found few particles had a size of about 20 nm. A past study performed previously 34 mentioned that the TEM results of the AgNPs mediated using Terminalia arjuna leaf extract have spherically shaped nanoparticles approximately 10–50 nm in size. In addition, the crystalline nature of the biosynthesized AgNPs-Fd detected using XRD revealed that AgNPs-Fd have a face-centered spherical structure in nature, and there were several characteristic diffraction peaks indexed from 21.386 to 76.655, which contained some functional groups.

Amines/proteins and metabolites comprising functional groups of alcohols, ketones, aldehydes and carboxylic acids surrounded the generated AgNPs-Fd. The carbonyl group of amino acid residues and proteins has the highest capacity to bind metal, suggesting that proteins may be able to encapsulate metal nanoparticles, including AgNPs, to avoid agglomeration and stabilize the medium. 43 The present study identified an assortment of chemicals compounds of AgNPs-Fd that were detected through GC-MS analysis. From Figure 7 and Table 1, the GC-MS of AgNPs-Fd contains several bioactive compounds that have anti-angiogenic and anticancer activities, such as phytol (PHY), stigmasterol, lupeol and sitosterol.

Previous studies revealed that PHY, or 3,7,11,15-tetramethylhexadec-2-en-1-ol, has anticancer properties and a variety of pharmacological features, including toxicity and cytotoxicity. Furthermore, PHY modulates pro-carcinogens as well as produced genotoxicity and death in breast cancer cells. It has also demonstrated DNA damage repair capabilities in mouse lymphocytes. 44 Additionally, PHY increases the activity of natural killer cells, which identify and eliminate cancer cells, and stimulates macrophage roles in immunity. 45 Phytol exhibits anti-angiogenic properties by inducing apoptosis in cancer cells, such as lung adenocarcinoma cells. 46

Meanwhile, stigmasterol and lupeol are potent compounds that exhibit anti-angiogenic properties. Stigmasterol and lupeol are two major phytosterols found in many herbal plants, with anti-inflammatory properties and have been proposed as anticancer agents. Though their mechanisms in inhibiting cancer are unclear, past studies report that cell survival, migration, and morphogenesis of human umbilical vein endothelial cells, but not cholangiocarcinoma cells, were inhibited by stigmasterol and lupeol. Both compounds lowered the transcript level of tumor necrosis factor-α considerably in expression studies. 47

Furthermore, a growing body of research suggests that β-sitosterol (BSS) may have anti-angiogenic properties. BSS impedes synovial angiogenesis by inhibiting the proliferation and migration of endothelial cells. 48 Current findings align with previous research performed by Nurhidayati et al. 49 who used the ethanol extract of Ficus septica Burm. f. leaves, revealing that the number of new blood vessels had been decreased as the dose of the extract increased. The anti-angiogenic effects of AgNPs-Fd also involved the phytochemical contents in the nanomaterial, which was produced during the biosynthesis of the AgNPs.

Further, the application of 90 μg AgNPs-Fd in the CAM assay had similar effects of anti-angiogenic activities to the positive control (cortisone acetate 30 μg and 30 ng bFGF), which confirms its anti-angiogenic properties. Cortisone acetate acts as an inhibitor of angiogenesis in CAM chicken embryos. Cortisone acetate is a corticosteroid compound in the glucocorticoid class, which is typically used in the treatment of angiogenesis-related diseases such as diabetic retinopathy and solid tumors. Meanwhile, glucocorticoids also regulate angiogenesis through several mechanisms, namely suppressing the proliferation, migration and growth of endothelial cells and reducing the secretion or expression of cytokines. 50 , 51

Conclusions

F. deltoidea leaves can be used as a reductor agent for the biosynthesis of AgNPs . Color change and Tyndall effects were an indicator of green synthesis of AgNPs-Fd. The surface plasmon resonance, which was identified at 430 nm, is the optimum peak of AgNPs-Fd. Several phytochemically active groups were identified in AgNPs-Fd using FTIR analysis, and it was observed that the morphology of the AgNPs-Fd had particle sizes of 20 nm, based on TEM characterization. The GC-MS analysis of the Fd leaf extract and AgNPs-Fd highlighted potential anti-angiogenic compounds, such as phytol, stigmasterol, lupeol and sitosterol. Fd or AgNPs-Fd treatments at 90 μg doses demonstrated significant anti-angiogenesis in CAM chicken embryo analysis, indicating greater effectiveness in controlling vessel formation. The AgNPs-Fd displayed anti-angiogenesis properties as indicated by the reduction of vessels in the CAM model. The anti-angiogenic properties of AgNPs-Fd in the CAM model were exhibited through the parameters of vessel area, total number of junctions, junction density, average vessel length and total number of end points. The 90 μg of AgNPs-Fd provided optimal anti-angiogenic effects. From a technical standpoint, the resulting AgNPs-Fd offer a simple technique to synthesize AgNPs. This method contains various benefits, such as cost-effectiveness, suitability for medical and pharmaceutical applications, and large-scale commercial manufacturing potential. The present research also suggested that AgNPs mediated by F. deltoidea leaf ethanolic extract have potential as anti-angiogenic agents.

 Data availability Underlying data

Figshare: Raw data antiangiogenic 2022, https://doi.org/10.6084/m9.figshare.22256071. 52

This project contains the following underlying data:

Raw data GCMS (Table 1 and Figure 7).zip

Raw data FTIR (Figure 6).zip

Spectrophotometer data (Figure 3).xlsx

Raw image TEM Tbat barito.tif

Raw data for XRD (Figure 5).zip

Reporting guidelines

Figshare: ARRIVE checklist for ‘Anti-angiogenic activity of Ficus deltoidea L. Jack silver nanoparticles using chorioallantoic membrane assay’, https://doi.org/10.6084/m9.figshare.22090673. 53

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

 Acknowledgements

All authors thank Ministry of Education, Culture, Research and Technology, Indonesia for grant funding financial year: 2022. Our gratitude expressed to Department of Biology Faculty of Mathematics and Natural Sciences, Mulawarman University, Samarinda, East Kalimantan, Indonesia for all support.

 References Ashraf K Haque MR Amir M : An overview of phytochemical and biological activities: Ficus deltoidea Jack and other Ficus spp. J. Pharm. Bioallied Sci. 2021;13(1):11–25. 34084044 10.4103/jpbs.JPBS_232_19 PMC8142919 Chitalia M Adimoolam S Jun BH : Comprehensive Review on Ficus Deltoidea Effervescent Mouthwash Formulation in Treating Oral Pathogens. Int. J. Healthc. Med. Sci. 2021;7(3):63–75. Janatiningrum I Lestari Y : Enzyme production, antibacterial and antifungal activities of actinobacteria isolated from Ficus deltoidea rhizosphere. Biodiversitas Journal of Biological Diversity. 2022;23(4). 10.13057/biodiv/d230429 Ismail F Zakaria Y Hassan NFN : Malaysian herbs as potential natural resources of anticancer drugs: From folklore to discovery. Asia Pac. J. Mol. Biol. Biotechnol. 2022;62–89. 10.35118/apjmbb.2022.030.1.06 Manurung H Kustiawan W Kusuma IW : Total flavonoid content and antioxidant activity of tabat Barito ( Ficus deltoidea Jack) on different plant organs and ages. J. Med. Plants Stud. 2017;5(6):120–125. Shafaei A Muslim NS Nassar ZD : Antiangiogenic effect of Ficus deltoidea Jack standardised leaf extracts. Trop. J. Pharm. Res. 2014;13(5):761–768. 10.4314/tjpr.v13i5.16 Hanafi MM Afzan A Yaakob H : In vitro pro-apoptotic and anti-migratory effects of Ficus deltoidea L. plant extracts on the human prostate cancer cell lines PC3. Front. Pharmacol. 2017;8:895. 29326585 10.3389/fphar.2017.00895 PMC5736988 Bunawan H Amin NM Bunawan SN : Ficus deltoidea Jack: a review on its phytochemical and pharmacological importance. Evid. Based Complement. Alternat. Med. 2014;2014:1–8. 24772185 10.1155/2014/902734 PMC3977116 Yaqoob AA Ahmad H Parveen T : Recent advances in metal decorated nanomaterials and their various biological applications: a review. Front. Chem. 2020;8:341. 32509720 10.3389/fchem.2020.00341 PMC7248377 Maduraiveeran G Sasidharan M Ganesan V : Electrochemical sensor and biosensor platforms based on advanced nanomaterials for biological and biomedical applications. Biosens. Bioelectron. 2018;103:113–129. 29289816 10.1016/j.bios.2017.12.031 Oves M Ahmar Rauf M Aslam M : Green synthesis of silver nanoparticles by Conocarpus lancifolius plant extract and their antimicrobial and anticancer activities. Saudi J. Biol. Sci. 2022;29(1):460–471. 35002442 10.1016/j.sjbs.2021.09.007 PMC8716933 Pirtarighat S Ghannadnia M Baghshahi S : Green synthesis of silver nanoparticles using the plant extract of Salvia spinosa grown in vitro and their antibacterial activity assessment. J. Nanostructure Chem. 2019;9(1):1–9. 10.1007/s40097-018-0291-4 Shaik MR Khan M Kuniyil M : Plant-extract-assisted green synthesis of silver nanoparticles using Origanum vulgare L. extract and their microbicidal activities. Sustainability. 2018;10(4):913. 10.3390/su10040913 Zamarchi F Vieira IC : Determination of paracetamol using a sensor based on green synthesis of silver nanoparticles in plant extract. J. Pharm. Biomed. Anal. 2021;196:113912. 33581590 10.1016/j.jpba.2021.113912 Nugroho RA Hindryawati N Aryani R : Biosynthesis of silver nanoparticles from aqueous extract of Myrmecodia pendans bulb. AIP Conference Proceedings. AIP Publishing LLC.;2021. Manullang JR Nugroho RA Rohmah M : Plant-extract-mediated biosynthesis of silver nanoparticles using Eleutherine americana bulb extract and its characterization. 2021. Chandhirasekar K Thendralmanikandan A Thangavelu P : Plant-extract-assisted green synthesis and its larvicidal activities of silver nanoparticles using leaf extract of Citrus medica, Tagetes lemmonii, and Tarenna asiatica. Mater. Lett. 2021;287:129265. 10.1016/j.matlet.2020.129265 Salayová A Bedlovičová Z Daneu N : Green synthesis of silver nanoparticles with antibacterial activity using various medicinal plant extracts: Morphology and antibacterial efficacy. Nanomaterials. 2021;11(4):1005. 33919801 10.3390/nano11041005 PMC8070782 Hajebi S Tabrizi MH Moghaddam MN : Rapeseed flower pollen bio-green synthesized silver nanoparticles: a promising antioxidant, anticancer and antiangiogenic compound. J. Biol. Inorg. Chem. 2019;24(3):395–404. 30927090 10.1007/s00775-019-01655-4 Vimalraj S Ashokkumar T Saravanan S : Biogenic gold nanoparticles synthesis mediated by Mangifera indica seed aqueous extracts exhibits antibacterial, anticancer and anti-angiogenic properties. Biomed. Pharmacother. 2018;105:440–448. 29879628 10.1016/j.biopha.2018.05.151 Baharara J Namvar F Ramezani T : Green synthesis of silver nanoparticles using Achillea biebersteinii flower extract and its anti-angiogenic properties in the rat aortic ring model. Molecules. 2014;19(4):4624–4634. 24739926 10.3390/molecules19044624 PMC6271002 Khandia R Munjal A Bangrey R : Evaluation of silver nanoparticle mediated reduction of neovascularisation (angiogenesis) in chicken model. Adv. Anim. Vet. Sci. 2015;3(7):372–376. 10.14737/journal.aavs/2015/3.7.372.376 West DC Thompson WD Sells PG : Angiogenesis assays using chick chorioallantoic membrane. Angiogenesis protocols. Springer;2001; pp.107–129. Ribatti D : The chick embryo chorioallantoic membrane as an in vivo assay to study antiangiogenesis. Pharmaceuticals. 2010;3(3):482–513. 27713265 10.3390/ph3030482 PMC4033966 Ribatti D : History of research on angiogenesis. Angiogenesis, Lymphangiogenesis and Clinical Implications. Vol.99. Basel, Karger,2014; pp.1–14. 10.1159/000353311 Zudaire E Gambardella L Kurcz C : A computational tool for quantitative analysis of vascular networks. PLoS One. 2011;6(11):e27385. 22110636 10.1371/journal.pone.0027385 PMC3217985 Barabadi H Mojab F Vahidi H : Green synthesis, characterization, antibacterial and biofilm inhibitory activity of silver nanoparticles compared to commercial silver nanoparticles. Inorg. Chem. Commun. 2021;129:108647. 10.1016/j.inoche.2021.108647 Camposano J Torre GD Laxamana J : Screening for the anti-angiogenic activity of selected Philippine medicinal plants using chorioallantoic membrane assay. Mahidol. Univ. J. Pharm. Sci. 2016;43:173–182. Gamallo JPM Espere G Carillo DMC : Evaluation of antiangiogenic property of Ocimum basilica ethanolic leaf extract by using duck embryo chorioallantoic membrane (cam) assay and its morphometric analysis. Int. J. Herb. Med. 2016;4(4):22–26. Benington L Rajan G Locher C : Fibroblast growth factor 2—A review of stabilisation approaches for clinical applications. Pharmaceutics. 2020;12(6):508. 10.3390/pharmaceutics12060508 Anandalakshmi K Venugobal J Ramasamy V : Characterization of silver nanoparticles by green synthesis method using Pedalium murex leaf extract and their antibacterial activity. Appl. Nanosci. 2016;6(3):399–408. 10.1007/s13204-015-0449-z Kumar DA Palanichamy V Roopan SM : Green synthesis of silver nanoparticles using Alternanthera dentata leaf extract at room temperature and their antimicrobial activity. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2014;127:168–171. 24632169 10.1016/j.saa.2014.02.058 Chaudhuri SK Chandela S Malodia L : Plant Mediated Green Synthesis of Silver Nanoparticles Using Tecomella undulata Leaf Extract and Their Characterization. Nano Biomed. Eng. 2016;8(1). 10.5101/nbe.v8i1.p1-8 Raj S Singh H Trivedi R : Biogenic synthesis of AgNPs employing Terminalia arjuna leaf extract and its efficacy towards catalytic degradation of organic dyes. Sci. Rep. 2020;10(1):1–10. 10.1038/s41598-020-66851-8 Woyengo TA Ramprasath VR Jones PJH : Anticancer effects of phytosterols. Eur. J. Clin. Nutr. 2009;63(7):813–820. 10.1038/ejcn.2009.29 Anju T Parvathy S Veettil MV : Green synthesis of silver nanoparticles from Aloe vera leaf extract and its antimicrobial activity. Materials Today: Proceedings. 2021;43:3956–3960. 10.1016/j.matpr.2021.02.665 Mohammadi F Yousefi M Ghahremanzadeh R : Green synthesis, characterization and antimicrobial activity of silver nanoparticles (AgNPs) using leaves and stems extract of some plants. Adv. J. Chem. A. 2019;2(4):266–275. Rufatto LC Gower A Schwambach J : Genus Mikania: chemical composition and phytotherapeutical activity. Rev. Bras. 2012;22:1384–1403. 10.1590/S0102-695X2012005000099 Zuas O Hamim N Sampora Y : Bio-synthesis of silver nanoparticles using water extract of Myrmecodia pendan (Sarang Semut plant). Mater. Lett. 2014;123:156–159. 10.1016/j.matlet.2014.03.026 Prabhu N Raj DT Yamuna GK : Synthesis of silver phyto nanoparticles and their antibacterial efficacy. Dig. J. Nanomater. Biostructures. 2010;5(1). Maung MM Lwin NN May KA : Phyto-mediated synthesis of gold nanoparticles (AuNP) by watery extract of Terminalia catappa L. (Almond) leaf. 8th IUPAC International Conference on Green Chemistry Organized by The Chemical Society of Thailand under the Patronage of Professor Dr. HRH Princess Chulabhorn;2018. Vilchis-Nestor AR Sánchez-Mendieta V Camacho-López MA : Solventless synthesis and optical properties of Au and Ag nanoparticles using Camellia sinensis extract. Mater. Lett. 2008;62(17-18):3103–3105. 10.1016/j.matlet.2008.01.138 Sadeghi B Rostami A Momeni S : Facile green synthesis of silver nanoparticles using seed aqueous extract of Pistacia atlantica and its antibacterial activity. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2015;134:326–332. 25022505 10.1016/j.saa.2014.05.078 Alencar MVOB de Islam MT Lima RMT de : Phytol as an anticarcinogenic and antitumoral agent: An in vivo study in swiss mice with DMBA-Induced breast cancer. IUBMB Life. 2019;71(2):200–212. 30394663 10.1002/iub.1952 Jeong SH : Inhibitory effect of phytol on cellular senescence. Biomed. Dermatol. 2018;2(1):1–9. Sakthivel R Malar DS Devi KP : Phytol shows anti-angiogenic activity and induces apoptosis in A549 cells by depolarizing the mitochondrial membrane potential. Biomed. Pharmacother. 2018;105:742–752. 29908495 10.1016/j.biopha.2018.06.035 Kangsamaksin T Chaithongyot S Wootthichairangsan C : Lupeol and stigmasterol suppress tumor angiogenesis and inhibit cholangiocarcinoma growth in mice via downregulation of tumor necrosis factor-α. PloS one. 2017;12(12):e0189628. 29232409 10.1371/journal.pone.0189628 PMC5726636 Qian K Zheng XX Wang C : β-Sitosterol Inhibits Rheumatoid Synovial Angiogenesis Through Suppressing VEGF Signaling Pathway. Front. Pharmacol. 2021;12:816477. 10.3389/fphar.2021.816477 Nurhidayati LG Nugroho AE Retnoaji B : Antiangiogenesis Activity of Awar-Awar Leaf Extract ( Ficus Septica Burm. F.) In Chorioallantoic Membrane Assay. Indonesian J. Pharm. 2021;32(1). 10.22146/ijp.607 Liu B Goodwin JE : The effect of glucocorticoids on angiogenesis in the treatment of solid tumors. J. Cell. Signal. 2020;1(3):42–49. 32728672 10.33696/Signaling.1.011 Pietras RJ Weinberg OK : Antiangiogenic steroids in human cancer therapy. Evid. Based Complement. Alternat. Med. 2005;2(1):49–57. 15841278 10.1093/ecam/neh066 PMC1062159 Nugroho R : Raw data antiangiogenic 2022.[Dataset]. figshare. 2023. 10.6084/m9.figshare.22256071 Nugroho R : ARRIVE.[Dataset]. figshare. 2023. 10.6084/m9.figshare.22090673 10.5256/f1000research.143239.r295259 Reviewer response for version 1 Soto-Hernández Ramón Marcos 1 Referee Colegio de Postgraduados en Ciencias Agrícolas Campus Montecillo, Montecillo, Mexico

Competing interests: No competing interests were disclosed.

 6 7 2024 Copyright: © 2024 Soto-Hernández RM 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The manuscript describes the anti-angiogenic activity of F. deltoidea L. Jack silver nano particles using  chorioallalntoic membrane assay, and apparently seems quite interesting, but the authors should consider some details to increase its quality. Some of them are:

1. In the extraction process, there is a lack of some details as the duration of the extraction: what method was used to conduct the process (maceration, Soxhlet, ultrasound, etc).

In the analysis by GC-EM also there are missing some details as retention times, Kovax index, relative abundance.

These data are important because they could be related to the biological activity detected.

The conclusions should be re written considering these details.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

No

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

No

Reviewer Expertise:

Phytochemistry, natural products isolation

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.