Anticancer and antiretroviral activities of methanolic extract from <i>Theobroma cacao</i> L pod husk: focusing on the ethyl acetate partition

Mustanir Yahya; Binawati Ginting; Nurdin Saidi

doi:10.12688/f1000research.128048.1

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Research Article

Anticancer and antiretroviral activities of methanolic extract from Theobroma cacao L pod husk: focusing on the ethyl acetate partition

[version 1; peer review: 2 approved with reservations]

Mustanir Yahya ¹, Binawati Ginting¹, Nurdin Saidi¹

PUBLISHED 28 Nov 2022

Author details Author details

¹ Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, BAnda Aceh, 23111, Indonesia

Mustanir Yahya
Roles: Conceptualization, Funding Acquisition, Resources, Supervision, Writing – Original Draft Preparation

Binawati Ginting
Roles: Conceptualization, Data Curation, Investigation, Project Administration, Writing – Review & Editing

Nurdin Saidi
Roles: Methodology, Validation, Visualization, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Many researchers have paid attention to Theobroma cocoa pod husk for its bioactive phytoconstituents which have several medicinal benefits. Herein, we aim to evaluate the methanolic extract from T. cocoa pod husk and its partitions for their anticancer and antiretroviral activities.
Methods: The T. cocoa pod husk was macerated using methanol, and then sequentially partitioned with n-hexane and ethyl acetate. MCF-7 and HeLa cells were used to assess the anticancer activities, while the simian retrovirus-2 (SRV-2)-infected A549 cells were used for antiretroviral study. The ethyl acetate partition (TCEA) was then fractionated and screened for in-vitro antioxidant and cytotoxicity. The most active fraction was sub-fractionated and analyzed using gas chromatography-mass spectroscopy (GC-MS).
Results: The results suggested that TCEA had moderate and weak activities against MCF-7 (IC₅₀=53.91 μg/mL) and HeLa cells (IC₅₀=120.71 μg/mL), respectively. TCEA 125 μg/mL had higher anti-SRV-2 activity in comparison with lamivudine 25 μg/mL after 1—7 days of incubation. The GC-MS analysis of the polyphenol-predominated sub-fraction from the most active fraction revealed the presence of lupeol, syringaresinol, catechol, and squalene.
Conclusions: TCEA derived from the methanolic extract of T. cacao pod husk had moderate activity against MCF-7 cells and weak activity against HeLa cells. Antiretroviral study suggests that TCEA 125 μg/mL had higher inhibitory activity against SRV-2 replication as compared to lamivudine 25 μg/mL.

Keywords

Cocoa pod husk; Simian retrovirus-2; MCF-7; HeLa; phytochemical profile

Corresponding author: Mustanir Yahya

Competing interests: No competing interests were disclosed.

Grant information: This research was funded by Kementrian Pendidikan, Kebudayaan, Riset, dan Teknologi under the Penelitian World Class Research (WCR) Scheme No.2/UN11.2.1/PT.01.03//DPRM/2022.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Yahya M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Yahya M, Ginting B and Saidi N. Anticancer and antiretroviral activities of methanolic extract from Theobroma cacao L pod husk: focusing on the ethyl acetate partition [version 1; peer review: 2 approved with reservations]. F1000Research 2022, 11:1395 (https://doi.org/10.12688/f1000research.128048.1) First published: 28 Nov 2022, 11:1395 (https://doi.org/10.12688/f1000research.128048.1) Latest published: 28 Nov 2022, 11:1395 (https://doi.org/10.12688/f1000research.128048.1)

Introduction

Theobroma cocoa pod husk, a cacao by-product, has intrigued many researchers to study its potential as natural medicine. A review article has highlighted its bioactivities as an anticarcinogenic, antibacterial, antiviral, antidiabetic, neuroprotective, and anti-inflammatory agent.¹ This is due to the fact that T. cocoa pod husk is rich in flavonoids and polyphenols which have been associated with a series of medicinal properties of plant extracts.²^–⁴ Several methods to extract the active phytoconstituents have been carried out previously, including those using supercritical CO₂ and microwave assistance.⁵^,⁶ A simple maceration technique has also been carried out using different solvents namely methanol:acetone,⁷ ethyl acetate,⁸ and n-hexane solvents.⁹ In our recently published work, we extracted the T. cocoa pod husk using distilled water and found its potential as a source for dietary antioxidants.¹⁰ Nonetheless, the research on methanolic extract of T. cacao pod husk has been scarcely reported. Some studies suggest the methanolic extraction could attract a wide-range of bioactive compounds.¹¹^–¹³

In previous research, T. cacao was found to be active in inhibiting the growth of Caco-2 colon cancer, HL-60 leukemia, SH-SY5Y neuroblastoma, MCF-7 breast cancer, HepG2 hepatoma, and Int-407 intestinal cancer cell lines.² Polyphenols from T. cacao were responsible for the downregulation of transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) that are involved in oncogenic process.¹⁴^,¹⁵ Under the same light, the phytoconstituents of T. cacao may also have a role in immunomodulation. In fact, these biological activities have been utilized to reactivate latent human immunodeficiency virus type 1 (HIV-1).¹⁶ Herein, we investigated the methanolic extract from T. cacao pod husk and its partitions for their anticancer and antiretroviral activities. We used MCF-7 breast cancer and HeLa cervical cell lines to investigate the anticancer activities as these cells have been used for anticancer screening of natural products.¹⁷^–²⁰ Simian retrovirus-2 (SRV-2)-infected A549 cell line was used to study the antiretroviral activity as this method has been developed to culture the Indonesian strain.²¹ The active extract sample was fractionated for in-vitro antioxidant and cytotoxicity screenings and further sub-fractionated for phytoconstituents analysis.

Methods

Materials

Materials used in this research included technical methanol, technical n-hexane, technical ethyl acetate, silica gel G₆₀ F₂₅₄, He gas, 2,2-diphenyl-1-picrylhydrazyl (DPPH) powder, ascorbic acid, pro-analytical methanol, gallic acid, and quercetin. Reagents used for the phytochemical screening included Folin-Ciocalteu reagent, Na₂CO₃ 10%, AlCl₃, and potassium acetate. Dulbecco’s Modified Eagle Medium (DMEM) from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) was used for cell culture. In addition, NaHCO₃, fetal bovine serum (FBS) (Invitrogen, USA), Penicillin-Streptomycin (Invitrogen, USA), trypsin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), trypan blue (Sigma, USA), PBS (phosphate buffer saline) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), dimethyl sulfoxide (Sigma, St. Louis, MO, USA), CO₂ incubator, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA), and pro-analytical ethanol (Merck, Germany) were also used. Kimwipes® tissue (Kimberly-Clark, Jawa Barat, Indonesia), Direct-zolTM RNA PreWash, RNA Wash Buffer, RNase-Free Water, primers SRV-2 5737 U19 and SRV-2 5943 L20, SSoFast Evagreen Mastermix (Bio-Rad Laboratories, Hercules, CA, USA), and nuclease-free water (NFW) were used for the real-time polymerase chain reaction (RT-PCR) protocol.

Plant specimen and bioindicators

Theobroma cacao L. specimen was collected from Desa Lawe Loning, Lawe Sigala-Gala District, Aceh Tenggara Regency, Aceh Province, Indonesia on 20 August 2021. The plant specimen was identified at the Department of Biology, Universitas Syiah Kuala, Indonesia with a voucher number of 795/UN11.1.28.1/DT/2008. The cocoa pod husk was washed with distilled water, cut into small pieces, and air-dried at 25±1°C for 7—10 days before crushed into powder.

The bioindicators MCF-7 cell line, HeLa cell, A549 cell, and Simian retrovirus-2 (SRV-2) were obtained from the Pusat Studi Satwa Primata-Lembaga Penelitian dan Pengabdian pada Masyarakat, Institut Pertanian Bogor (PSSP LPPM-IPB), Indonesia.

Cocoa pod husk extraction

Previously prepared cacao pod husk powder (5 kg) was macerated using methanol for 24 h, performed repeatedly until the filtrate became clear. The methanolic filtrate was concentrated via rotary evaporation to produce the methanolic extract which was then labeled as TCM. Thereafter, the TCM was partitioned sequentially using n-hexane and ethyl acetate, where each sample was labeled as TCH and TCEA, respectively.

MTT Assay

MCF-7 and HeLa cancer cell lines (5x10³ cells/well) were respectively grown in 96 well plate for 24 h (37°C; 5%CO₂) with DMEM medium which had been priorly added with FBS 5% and Penicillin-Streptomycin 1% antibiotic. On the following day, each well was added with DMSO-dissolved extracts (TCM, TCH, and TCEA) with concentrations of 500, 250, 125, 62.5, and 31.25 μg/mL. The cell cultures were then incubated for 48 h before being added with 10 μL MTT and re-incubated for 4 h. Subsequently, as much as 100 μL ethanol 96% was added and optical density (OD) reading was carried out on a microplate reader (ELISA reader) at 595 nm. The procedure was performed in triplicate for each sample.

A549 cell culture for anti-SRV-2

The concentration range for anti-SRV-2 studies was determined by MTT assay (as explained previously), but the non-infected A549 lung cancer cell line was used instead. For the antiretroviral study, SRV-2-infected A549 cell culture (50x10³) was firstly sub-cultured in a 12-well plate and incubated for 24 h at 37°C, 5% CO₂. Afterward, extract samples (2 mL) with a concentrations range that was non-cytotoxic against A549 cells (inhibition percentage < 50%) were added to each well (performed in duplicate). Cell receiving no treatment was taken as control. For the positive control, we used lamivudine at 25 μg/mL. Thereafter, the cells were incubated until day-7, where the supernatant was harvested on day-1, -3, -5, and -7. In each harvesting, the supernatant was taken as much as 500 μL, and into the cell culture, 500 μL sample solution was re-added. The harvested supernatant was stored at -80°C.

RT-PCR

The viral RNA was extracted from the supernatant using QIAamp Viral RNA Mini Kits (Qiagen, Hilden, Germany) which was then reverse transcripted using Superscript^TM III First-Strand Synthesis System for RT-PCR (Invitrogen) to obtain the viral cDNA. Primers used in the RT-PCR were SRV-2 5737U19 and SRV-2 5943L20 (collections of PSSP LPPM-IPB) which had been amplified with envelope gene gp70. As much as 1 μL of each primer was added to the master mix (PCR reaction mixture) containing 10 μL Sofast Evagreen Master Mix, 6 μL Nuclease Free Water dan 2 μL cDNA template. The PCR was performed in an iCycler Thermal cycler with iQ5 multicolor real time PCR detection system at 95°C for 30 seconds, followed by 40 cycles consisting of: denaturation (95°C; 30 second), annealing (47°C; 20 seconds), and DNA extension (72°C; 20 seconds). Standard used for the quantification of SRV-2 was SRV-2 plasmids with a concentration range of 10¹—10⁶. This protocol was performed in duplicate, following the suggestion from a previously published study.²²

Fractionation using gravitational column chromatography

Extracts with anticancer and antiretroviral activities were further isolated using gravitational column chromatography with silica gel G₆₀ F₂₅₄. The extract was treated with gradient elution starting with non-polar solvent n-hexane (100%) up to semi-polar solvent ethyl acetate:methanol (1:1). Fractions with the similar patterns from the thin layer chromatography were combined into subfraction, and then tested for antioxidant and cytotoxic activities.

The subfraction was re-chromatographed using a column with silica gel G₆₀ F₂₅₄ based on gravitational force. The obtained subfraction was tested on thin layer chromatography and sprayed with vanillin sulfate and FeCl₃ 5% to identify the polyphenol contents. Subfraction with the most dominant polyphenol content was investigated on gas chromatography-mass spectroscopy (GC-MS) for phytoconstituents profiling.

DPPH assay

Antioxidant activities were based on the inhibition of DPPH radical (molecular weight=394.32 g/mol). As much as 11.82 mg DPPH powder was dissolved in methanol using 75-mL volumetric flask. The extract sample and positive control (ascorbic acid) were prepared in concentrations of 3, 6, 9, 12, and 15 μg/mL. Into each solution, DPPH 0.4 mM was drop-wised (1 mL), and the volume was increased with methanol until it reached 5 mL. The container was then sealed with aluminum foil to avoid the light exposure. Thereafter, the mixture was homogenized with vortex mixer and incubated for 30 minutes at 37°C. The mixture solution was measured for its absorbance at 517 nm using UV-vis spectrophotometer.

BSLT assay

The cytotoxicity was determined by brine shrimp lethality test (BSLT) using Artemia salina larvae. The larvae (±50-100 mg) were firstly incubated in a covered container with saline water for 48 h. On the other hand, the extract sample (30 mg) was diluted with 3 drops of DMSO 5% and added with 30 mL saline water, and subsequently sonicated until homogenous. The concentrations of the prepared extract sample ranged from 1000 to 1 μg/mL, and a combination of saline water and DMSO 5% was taken as the negative control. A glass container was filled with the prepared extract sample and then added with 10 brine shrimps before incubated (24 h; room temperature). Dead brine shrimps were then counted in each glass container.

Results

Anticancer activities

TCH, TCEA, and TCM were screened for their anticancer activities against MCF-7 breast cancer and HeLa cervical cancer cell lines, where the results have been presented in Figure 1. At 31.25 μg/mL, TCH has a higher inhibition than TCEA (33.29% versus 18.93%). Meanwhile, no inhibition was observed in sample treated with TCM 31.25—62.5 μg/mL until its concentration increased to 125 μg/mL (inhibition=28.35%). Interestingly, at 250 μg/mL, TCM appeared to yield the highest inhibition (97.62%) as compared to TCEA (95.66%) and TCH (93.85%). Concentration-dependent increment of the MCF-7 inhibitions were observed when the extract concentrations ranged from 31.25 to 250 μg/mL. Hence, the IC₅₀ values were calculated based on the polynomial regression on the concentration range of 31.25—250 μg/mL (Table 1). The calculation of the IC₅₀ against MCF-7 revealed TCH as the most potent extract (IC₅₀=45.36 μg/mL) among others (IC₅₀=53.91 and 161.53 μg/mL for TCEA and TCM, respectively) (Table 1).

Figure 1. Anticancer activities of methanol extract of T. cacao (TCM), ethyl acetate soluble of T. cacao (TCEA) and n-hexane soluble of T. cacao (TCH) against MCF-7 and Hela cell lines.

Table 1. IC₅₀ values of methanol extract of T. cacao (TCM), ethyl acetate soluble of T. cacao (TCEA) and n-hexane soluble of T. cacao (TCH) against MCF-7 and HeLa cell lines based on the equations obtained from polynomial regression.

Sample	Equation	R²	IC₅₀ (μg/mL)
MCF-7
TCH	y= -11.43x²+133.89x-294.46	0.952	45.36
TCEA	y= -13.41x²+156.76x-361.81	0.999	53.91
TCM	y=34.32x²-254.75x+458.11	0.999	161.53
HeLa
TCH	y=6.46x²-46.68x+130.12	0.948	82.44
TCEA	y=2.27x²+5.91x-30.43	0.997	120.71
TCM	y=8.93x²-60.10x+106.19	0.928	272.58

A similar trend of anticancer activities was also observed for the HeLa cancer cell line, where highest to the lowest, the order was as follows: TCH, TCEA, and TCM with inhibition percentages of 44.15, 17.5 and 1.39%, respectively. For TCH and TCM, the increase of concentration from 31.25 to 62.5 caused a rapid elevation of inhibition. As for TCEA, the rapid increasing trend of inhibition percentage was observed until 125 μg/mL. Due to this nature of the curves, we employed polynomial regression for the IC₅₀ calculation so that R²>0.90 could be obtained (Table 1). The lowest IC₅₀ was obtained by TCH (82.44 μg/mL). Meanwhile, TCEA and TCM have IC₅₀ against HeLa cervical cancer cell line of 120.71 and 272.58 μg/mL, respectively.

Antiretroviral activities

Screening of anticancer activities revealed that TCEA and TCH fractions had higher activities as compared to TCM. Herein, we tested the antiretroviral activity of TCEA and further continued to analyze its subfractions along with antioxidant, cytotoxicity, and phytochemical screenings. The investigation on TCH fraction is currently being carried out and will be published separately later. For the antiretroviral investigation, we calculate the non-cytotoxic concentration of TCEA against A549 cell line since it was used for the viral replication (Figure 2). From the study, we obtained 438.99 μg/mL as the IC₅₀ value which was then set as the cytotoxic concentration.

Figure 2. Inhibition (%) and IC₅₀ of ethyl acetate soluble of T. cacao (TCEA) against A549 lung cancer cell line.

The antiretroviral activity of TCEA against SRV-2 with concentration range of 31.25—125 μg/mL has been presented in Table 2. On Day-1 the SRV-2-infected cells receiving TCEA 125 μg/mL and positive control experienced replication inhibitions, but not in TCEA 62.5 and 31.25 μg/mL groups. Viral replications of lower than that of the negative control (indicating the inhibitory activity) were significantly observed in TCEA 62.5 and 31.25 μg/mL only after 5 days of incubation. TCEA with a concentration of 125 μg/mL was considered as having more potent antiretroviral activity compared to lamivudine (positive control).

Table 2. Antiretroviral activity of ethyl acetate soluble of T. cacao (TCEA) against Simian retrovirus-2 (SRV-2).

Incubation time (days)	SRV-2 replication (x10⁵)
	TCEA			Positive control	Negative control
	125 μg/mL	62.5 μg/mL	31.25 μg/mL	Positive control	Negative control
0					7.03
1	0.44	267.92	247.27	2.37	90.31
3	4.12	558.94	653.29	83.72	611.04
5	7.89	186.74	258.54	70.99	2225.19
7	1.27	467.67	110.86	61.29	1393.61

Isolation of the TCEA

As many as 581 fractions were obtained from the column chromatography of TCEA, where those with the same stain patterns were combined one into a single subfraction. As many as 9 subfractions were obtained from the procedure and each of the subfractions was labeled with TCEA 1 to 9. The nine subfractions were analyzed with thin layer chromatography, and the results have been presented in Figure 3. Observation using vanillin sulfate revealed that the TCEA 1—4, TCEA 4—7, and TCEA 7—9 were predominated by terpenoids (purplish blue), flavonoids (yellow), and tannins (brownish color), respectively. Meanwhile, the FeCl₃ 5% confirmed the presence of polyphenols in all samples as indicated by the dark green color.

Figure 3. Chromatogram of ethyl acetate soluble of T. cacao (TCEA) 1—4 (a), TCEA 4—6 (b), and TCEA 7—9 (c).

From left to right: UV light at 254 nm; UV light at 366 nm; vanillin sulfate; and FeCl₃ 5%.

DPPH Antioxidant profiles of the TCEA 1—9

Antioxidant activities of TCEA 1—9 based on the radical DPPH inhibition have been presented in Figure 4. TCEA was found to have the weakest antioxidant power with IC₅₀ of the DPPH inhibition only reaching 137.511 μg/mL. TCEA 6 has the lowest 7.64 IC₅₀ of 6.49 μg/mL, hence the most potent antioxidant among others. TCEA 5 was only seconds after TCEA 6 with IC₅₀ of 7.64 μg/mL. It is worth noting that both TCEA 5 and 6 were obtained from column chromatography using n-hexane:ethyl acetate (3:7) eluent. Meanwhile, as a comparison, the IC₅₀ obtained from the ascorbic acid was almost twice lower than TCEA 6 (3.25 μg/mL versus 6.49 μg/mL).

Figure 4. DPPH antioxidant activities of ethyl acetate soluble of T. cacao (TCEA) 1—9 and positive control (ascorbic acid).

BSLT cytotoxicity profiles of the TCEA subfractions

The cytotoxicity profiles of TCEA 1—9 based on the brine shrimp mortality are presented in Table 3. TCEA 8, 9, and 1 had the lowest LC₅₀ with the values being 411, 394, and 252, respectively. Similar to the antioxidant test, we found TCEA 5 and 6 being the two most cytotoxic subfractions (LC₅₀=5 and 2 μg/mL, respectively). The lethal cytotoxicity of these two subfractions could be observed even when the concentration was as low as 1 μg/mL (3 and 4% mortality for TCEA 5 and 6, respectively). TCEA 6 reached 100% mortality when the concentration was 500 μg/mL. Meanwhile, the same level of mortality was reached by TCEA 5 when its concentration was 1000 μg/mL. Other than TCEA 5 and 6, none of the subfractions could reach 100% mortality even when the concentration was as high as 1000 μg/mL.

Table 3. Results from BSLT assay indicating the cytotoxicity of ethyl acetate soluble of T. cacao (TCEA) 1—9.

Sample	Mortality (%)					LC₅₀ (μg/mL)
Sample	1 μg/mL	10 μg/mL	100 μg/mL	500 μg/mL	1000 μg/mL
TCEA 1	0	1.33	3	5	8.33	252
TCEA 2	1	3	4.67	8	9.33	44
TCEA 3	2	2.67	4	5.33	7	187
TCEA 4	2.33	3	4.33	7	8.33	53
TCEA 5	3	5.67	9	9.67	10	5
TCEA 6	4	6.67	9.33	10	10	2
TCEA 7	2	3	4	6	8	89
TCEA 8	2	3	3.67	5	6	411
TCEA 9	0	1	2	4	7	394

Re-chromatography

Due to its high antioxidant and cytotoxicity activities, TCEA 6 was selected for re-chromatography to obtain sample with higher purity. The re-chromatography was carried out using gradient elution of n-hexane and ethyl acetate combination resulting in another four subfractions (TCEA 6.1—6.4). The phytoconsituents of each subfraction were qualitatively analyzed using thin layer chromatography as presented in Figure 5. When sprayed using vanillin sulfate, the presence of flavonoids (yellow) was observed and more pronounced in TCEA 6.3 and 6.4. It was confirmed by FeCl₃ 5% test, where a dark green color was observed indicating the high content of polyphenols. We were interested in further analyzing TCEA 6.3 through GC-MS for phytoconstituents profiling due its green color being the most intense among others.

Figure 5. Chromatogram of ethyl acetate soluble of T. cacao (TCEA) 6.1—6.4.

From left to right: UV light at 254 nm; UV light at 366 nm; vanillin sulfate; and FeCl₃ 5%.

Phytochemical profile

A list of phytoconsituents identified by the GC-MS analysis from sample TCEA 6.3 has been presented in Table 4. As many as 39 compounds were identified from the 40 peaks in the chromatogram where benzoic acid, 4-hydroxy-3,5-dimethoxy- has the widest peak area (21.56%). Lupeol was identified twice, assigned for peaks 4 and 5, but the former had lower similarity than the latter (75.3 and 93.9%). Bioactive compounds which had been recognized for their anticancer and/or antioxidant activities were identified, namely lupeol, syringaresinol, catechol, and squalene. Among the four aforementioned compounds, catechol had the highest quantity (peak area = 9.29%) with similarity over 93.9%.

Table 4. Results from chromatography-mass spectroscopy (GC-MS) analysis on ethyl acetate soluble of T. cacao (TCEA) sub-fraction.

Peak	Compounds (Formula)	Retention time (minutes)	Molecular weight (g/mol)	Area (%)	Similarity (%)
1.	3-Hexanol, 3-methyl-(C₇H₁₆O)	4.514	116	4.53	83.5
2.	(R)-(-)-2-Methyl-2,4-pentanediol (C₆H₁₄O₂)	5.579	118	0.52	79.6
3.	Phenol, 2-methoxy-(C₇H₈O₂)	6.395	124	1.00	92.5
4.	Catechol (C₆H₆O₂)	8.970	110	3.37	75.3
5.	Catechol (C₆H₆O₂)	10.269	110	9.29	93.9
6.	Phenol, 5-ethenyl-2-methoxy-(C₁₈H₂₀O₂)	10.748	268	0.75	78.0
7.	Phenol,5-ethenyl-2-methoxy-(C₁₈H₂₀O₂)	11.742	268	1.24	74.6
8.	Phenol, 2,6-dimethoxy-(C₈H₁₀O₃)	12.966	154	3.30	88.1
9.	trans-Linalool oxide (furanoid) (C₁₀H₁₈O₂)	14.156	170	10.46	80.1
10.	α-Methyl-α-[4-methyl-3-pentenyl] oxiranemethanol (C₁₉H₂₄N₂)	14.935	94	1.97	68.1
11.	2-Furanmethanol, 5-ethenyltetrahydro- α, α,5-trimethyl-, cis-(C₁₀H₁₈O₂)	15.820	170	10.88	79.3
12.	Benzenemethanol, 3,4-dimethoxy-(C₉H₁₂O₃)	17.313	174	1.59	66.5
13.	Vanillicacid,1,1,1-trifluoro-2-propyl ester (C₈H₈O₄)	18.537	168	1.44	67.5
14.	Phenol,4-methoxy-3(methoxymethyl)-(C₉H₁₂O₃)	21.101	168	3.16	72.3
15.	2-Cyclohexen-1-one,3-(3-hydroxybutyl)-2,4,4-trimethyl-(C₁₃H₂₂O₃)	21.465	226	1.29	66.6
16.	2(1H)-Naphthalenone,1-(dimethoxymethyl)-3,4,5,6,7,8-hexahydro-(C₁₃H₂₀O₃)	22.305	224	0.30	67.4
17.	[1,1'-Bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester (C₂₁H₃₈O₂)	22.778	322	0.71	68.7
18.	6-Hydroxy-4,4,7α -trimethyl-5,6,7,7α tetrahydrobenzofuran-2(4H)-one (C₁₁H₁₆O₃)	23.363	196	2.37	74.7
19.	2,5-dimethoxy phenylethylsulfide (C₁₀H₁₄O₂S)	24.237	182	4.69	69.8
20.	Benzoic acid, 4-hydroxy-3,5-dimethoxy-(C₉H₁₀O₅)	25.798	198	21.56	74.0
21.	Spiro[4.5]decan-7-one,1,8-dimethyl-8,9-epoxy-4-isopropyl	26.754	151	0.00	74.0
22.	Cinnamic acid, 4-hydroxy-3-methoxy-, (5-hydroxy-2-hydroxymethyl-6-[2-(4-hydroxy-3-methoxy phenyl)ethoxy]-4-(6-methyl-3,4,5-trihydroxy tetrahydropyran-2-yloxy) tetrahydropyran-3-yl) ester (C₃₁H₄₀O₁₅)	26.982	652	1.12	72.1
23.	1b,4a-Epoxy-2Hcyclopenta [3,4]cyclopropa[8,9]cycloundec[1,2-b]oxiren-5(6H) -one, 7-(acetyloxy) decahydro-2,9,10-trihydroxy-3,6,8,8,10α-pentamethyl-(C₂₂H₃₂O₈)	28.832	424	0.47	73.0
24.	trans-13-Octadecenoic acid (C₁₈H₃₄O₂)	29.281	282	0.96	83.3
25.	Abscisic acid (C₁₅H₂₀O₄)	30.750	264	2.73	85.8
26.	1-Heptatriacotanol (C₃₇H₇₆O)	31.737	536	0.41	79.2
27.	Octan-2-yl palmitate (C₂₄H₄₈O₂)	34.794	368	0.65	75.4
28.	Curan, 16,17-didehydro-, (20.xi.)-(C₁₉H₂₄N₂)	35.845	280	1.36	67.0
29.	1-Heptatriacotanol (C₃₇H₇₆O)	36.944	536	0.62	70.3
30.	1,3-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester (C₂₄H₃₈O₄)	38.212	390	0.44	67.4
31.	5H-Cyclopropa[3,4] benz [1,2-e]azulen-5-one,9-(acetyloxy)-3-[(acetyloxy)methyl]-1,1a,1b,4,4a,7a, 7b,8,9,9a-decahydro-4a,7b,9a-trihydroxy -1,1,6,8-tetramethyl-,[1ar-(1aa,1bß,4aß,7aa, 7ba,8a,9ß,9aa)]-(C₂₆H₃₄O₁₀)	38.440	179	0.29	74.8
32.	Squalene (C₃₀H₅₀)	39.328	410	0.70	87.8
33.	4aa,4bß-Gibbane-1,1,10ß-tricarboxylic acid,4a,7-dihydroxy-8-methylene-, 1a,4a-lactone,dimethyl ester (C₁₉H₂₂O₇)	40.032	362	0.31	67.7
34.	2,4,6-Decatrienoic acid, 1a,2,5,5a,6,9,10,10a-octahydro-5,5a-dihydroxy-4-(hydroxy methyl)-1,1,7,9-tetramethyl-11-oxo-1H-2,8amethano cyclopenta [a] cyclopropa [e] cyclodecen-6-yl ester, [1aR(1aa,2a,5ß,5aß,6ß,8aa,9a,10aa)]-	41.212	312	0.25	74.5
35.	1H-Cyclopropa[3,4] benz[1,2-e]azulene-5,7b,9,9a-tetrol, 1a,1b,4,4a, 5,7a,8,9-octahydro-3-(hydroxymethyl)- 1,1,6,8-tetramethyl-, 9,9a-diacetate, [1aR-(1aa,1bß,4aß,5ß,7aa, 7ba,8a,9ß,9aa)]-	43.011	411	0.98	69.7
36.	1H-Cyclopropa[3,4] benz [1,2-e]azulene-3-carboxaldehyde, 9a-(acetyloxy)-1a,1b,4,4a, 5,7a,7b,8,9,9a-decahydro-4a,7bdihydroxy-1,1,6,8- tetramethyl-5,9-dioxo-,[1aR-(1aa,1bß, 4aß,7aa,7ba,8a,9aa)]-	44.178	360	0.73	69.2
37.	N-(2,4-Dinitrophenyl)-N'-[3-(2,6,6-trimethylcyclohex-1-enyl)-propylidene]-hydrazine (C₁₈H₂₄N₄O₄)	44.988	332	1.02	69.7
38.	Lupeol (C₃₀H₅₀O)	45.770	426	0.60	81.3
39.	Phenol, 2,6-dimethoxy-4-[tetrahydro-4-(4-hydroxy-3-methoxyphenyl)-1H,3H-furo[3,4-c]furan-1-yl]-(C₂₁H₂₄O₇)	47.096	389	0.29	73.6
40	Syringaresinol (C₂₂H₂₆O₈)	49.855	418	1.65	93.7

Discussion

Based on the IC₅₀ obtained herein, TCH was moderately active against MCF-7 and HeLa cancer line, whilst TCEA was only moderately active against MCF-7. Moderate activity could be ascribed to the presence of non-anticancer compounds in the extract, as suggested by a previous study investigating Calotropis gigantea extract against P388 leukemia cell lines.²³ In our previous study, a moderate anticancer activity against MCF-7 cell lines was also obtained from n-hexane extract of Myristica fragrans Houtt roots, where a lignan compound was isolated and identified.²⁰ The anticancer activity increased when the analysis was carried out on a fraction of the foregoing extract,¹⁹ indicating the activity is dependent on the bioactive compound concentrations. This is also the reason why TCEA and TCH were more active against the cancer cell lines than TCM because TCEA and TCH had higher purities as they were obtained from the fractionation of TCM.

In the case of T. cacao, its anticancer activities have been anticipated by researchers due to its rich content of dietary polyphenols.² A review article has summarized these phenolic compounds that include catechin, 3′-O-methyl epicatechin, epicatechin, procyanidin B2, and polymer procyanidins.³ Our data is in an agreement with that from a previously published study, where the methanolic extract from T. cacao pod husk had lower IC₅₀ on MCF-7 as compared to HeLa cell lines.²⁴ Previously, BSLT cytotoxic activities of n-hexane extract from T. cacao pod husk have been documented to yield LC₅₀<10 μg/mL.⁹

In this present study, the TCEA had a significantly higher inhibitory effect even when compared to lamivudine. Previous studies have reported that the NaOH extract from T. cacao pod husk mainly worked as antiviral by preventing the infection of human immunodeficiency virus (HIV) to the host immune cell including the syncytium formation.²⁵ The lignin-carbohydrate complex derived from NaOH extraction in combination with acid precipitation of T. cacao pod husk had anti-HIV activity and immunomodulating activities.²⁶ Moreover, the flavonoid compound from T. cacao could help the efficacy of antiretroviral therapy through the reactivation of latent HIV in human T-cells which was found correlated to NF-κB and MAPK signaling pathways.¹⁶ A review on the antiviral potential of T. cacao pod husk highlighted the inhibitory activity of its lignin fractions against cytopathic effects induced by influenza virus, in which this action could be synergized with ascorbic acid.¹

Following the investigation of the anticancer and antiretroviral activities, the TCEA was fractionated producing TCEA 1—9 fractions. The TCEA was revealed as the most potent antioxidant (IC₅₀=6.49 μg/mL) and cytotoxic (LC₅₀=2 μg/mL) fraction among others. This value is lower in comparison to that obtained from our previous study using aqueous extraction, where the highest antioxidant was IC₅₀=9.61 μg/mL and the highest cytotoxicity – 74 LC₅₀=μg/mL.¹⁰ However, more cytotoxically active compounds were obtained in the n-hexane extract of T. cacao pod husk with LC₅₀ as low as 0.29 μg/mL.⁹ The bioactivities of T. cacao pod husk could be associated with the phenolic compound contained in the extract as suggested previously.¹⁰

Further purification of fraction TCEA 6 in this present study produced 4 subfractions (TCEA6.1—6.4), with TCEA 6.3 being the most predominated with polyphenol (based on FeCl₃ 5% test). The GC-MS analysis on subfraction TCEA 6.3 revealed its phytoconsituents consisted of bioactive compounds lupeol, syringaresinol, catechol, and squalene among many other compounds. The structures of the foregoing compounds are presented in Figure 6. Lupeol itself has been named as a novel anti-inflammatory and anti-cancer agent by a review article in 2009.²⁷ Interestingly, lupeol has been found to specifically attack cell cancer in which a study reported its high toxicity against 451Lu and WM35 melanoma cells but not in normal human melanocyte cells.²⁸ As an antiviral, lupeol has been found active against herpes simplex virus type 1 (HSV-1) and even its acyclovir-resistant strains.²⁹ Syringaresinol isolated from Rhus javanica var. roxburghiana could inhibit the replication of tobacco mosaic virus.³⁰ The ability of syringaresinol to induce cancer cell death has been documented in a study using HL-60 leukemia cells.³¹ However, a recent study revealed that syringaresinol was not cytotoxic towards HepG2 and HT29 cells.³²

Figure 6. Bioactive phytoconstituents detected in ethyl acetate soluble of T. cacao (TCEA) 6.3 by chromatography-mass spectroscopy (GC-MS).

Catechol and its derivatives have been recognized as broad-spectrum antiviral and anticancer agents.³³^,³⁴ A study suggested that the antiviral and immunomodulating activities of catechol are dependent on the structure and position of the substituent group located on its aromatic skeleton.³⁵ A reputable natural antioxidant, squalene has long been associated with anticancer activities especially in the case of the absence of cancer in sharks.³⁶ Squalene has been witnessed to not only increase the toxicity of several commercial anti-cancers but also provide cytoprotective effect against normal cells.³⁷ A recent report revealed that intravenous squalene BID could significantly improve COVID-19 conditions without any adverse effect.³⁸^,³⁹ Taken altogether, the foregoing phytocompounds contained in TCEA 6.3 had a potential to act as anti-cancer and anti-retroviral agent.

Conclusions

TCEA derived from the methanolic extract of T. cacao pod husk had moderate activity against MCF-7 cells and weak activity against HeLa cells. Antiretroviral study suggests that TCEA 125 μg/mL had higher inhibitory activity against SRV-2 replication as compared to lamivudine 25 μg/mL. Further fractionation using gravitational column chromatography yielded TCEA fractions with relatively high DPPH antioxidant and BSLT cytotoxic activities. The phytoconsituents of the subfraction revealed the presence of lupeol, syringaresinol, catechol, and squalene which have been reported previously as anti-cancer and anti-viral. The investigation on TCH for its anti-cancer and anti-retroviral activities is warranted.

Author contributions

Conceptualization, M.Y. and B.G.; methodology, N.S.; validation, N.S.; formal analysis, M.Y.; investigation, B.G.; resources, M.Y.; data curation, B.G.; writing—original draft preparation, M.Y.; writing—review and editing, B.G. and N.S.; visualization, N.S.; supervision, M.Y.; project administration, B.G.; funding acquisition, M.Y. All authors have read and agreed to the published version of the manuscript.

Data availability

Underlying data

figshare: ‘Anticancer and Antiretroviral Activities of Methanolic Extract from Theobroma cacao L Pod Husk: Focusing on the Ethyl Acetate Partition’. https://doi.org/10.6084/m9.figshare.21483096.v4 ⁴⁰

This project contains the following underlying data:

- 1. MTT Test Design IC50 Calculation.xls [Table containing the raw data of the study].
- 2. OD Value of MCF-7, HeLa dan A549 Cells MTT Test.xlsx
- 3. Bar and Line Graphs represented MCF-7, HeLa & A549 Cells.xlsx
- 4. Data qRT PCR SRV.xlsx
- 5. TCEA Antioxidant Subfraction.xlsx
- 6. TCEA Cytotoxic Subfraction.xlsx
- 7. Supplementary Figures.zip [file containing supplementary figures of the study].

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

The authors appreciate the assistance from the Head of Lembaga Penelitian dan Pengabdian Kepada Masyarakat, Universitas Syiah Kuala and the Dean of Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala. A special acknowledgment is granted to Reka Nurmania, Ayu Rahmadani who, at the moment of this manuscript preparation, were the students of the Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala.

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Author details Author details

¹ Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, BAnda Aceh, 23111, Indonesia

Mustanir Yahya
Roles: Conceptualization, Funding Acquisition, Resources, Supervision, Writing – Original Draft Preparation

Binawati Ginting
Roles: Conceptualization, Data Curation, Investigation, Project Administration, Writing – Review & Editing

Nurdin Saidi
Roles: Methodology, Validation, Visualization, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This research was funded by Kementrian Pendidikan, Kebudayaan, Riset, dan Teknologi under the Penelitian World Class Research (WCR) Scheme No.2/UN11.2.1/PT.01.03//DPRM/2022.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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https://doi.org/10.12688/f1000research.128048.1

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Yahya M, Ginting B and Saidi N. Anticancer and antiretroviral activities of methanolic extract from Theobroma cacao L pod husk: focusing on the ethyl acetate partition [version 1; peer review: 2 approved with reservations]. F1000Research 2022, 11:1395 (https://doi.org/10.12688/f1000research.128048.1)

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Reviewer Report 11 May 2024

Shinta Rosalia Dewi, Universitas Brawijaya, Malang, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.140602.r232067

This is very good research that will be very beneficial to researchers working in anticancer and antiviral areas, as well as medical studies. The valorization of cacao pod husk waste into anticancer and antiviral agents is a highly intriguing idea.
... Continue reading

This is very good research that will be very beneficial to researchers working in anticancer and antiviral areas, as well as medical studies. The valorization of cacao pod husk waste into anticancer and antiviral agents is a highly intriguing idea.

The research study has been carried out systematically and the results are written coherently in the article. The Abstract has been written clearly and reflects the manuscript's content. However, some improvements are still needed to ensure the information presented in the manuscript is clear. Possibly some English revision should also be done, and the following other comments can be addressed:

1. English revision needs to be done to make the paper much pretty. For example, the term “as much as” is inappropriate (should be removed), or if required, replaced with “about”.

2. Introduction: Please clarify the originality and research aim of this study in the Introduction section. The authors have explained the potential of cacao pod husk as a medicine for anticancer, antiviral, etc. Various extraction methods and solvents that are widely used have also been mentioned, including what had been previously studied (extraction using water). However, the authors did not elaborate to provide clear explanations for why the maceration with methanol solvent was then chosen for this study (the research gap analysis was unclear).

3. Methods:

Material: instead of using “technical methanol, technical n-hexane, etc”, it is preferable to use “methanol, n-hexane, etc (mention all), with technical grade quality”. Also, for “pro-analytical ethanol, methanol, etc”, I would recommend using “methanol, ethanol, etc, with extra pure or analytical grade quality.
Cacao pod extraction: what is the solvent-to-sample ratio, number of repetitions, and total amount of solvent used? Introduce the abbreviation for the first time it appears in the manuscript: TCM, TCH.

4. Results:

Please write the information clearly for readers to grasp. Compare the %inhibition with IC50 results to conclude the antioxidant activity. For example, how does the antioxidant activity of TCH, which has the highest %inhibition, correspond with the lowest IC50 value?
Figure 1: Instead of placing the “MCF- and Hela” annotations within the picture, write the notations (a) and (b) in the figure title (bold text). Also, use consistent terminology: methanol extract, EA soluble, hexane soluble (all are extracts).
Figure 2: provide number information (1,2, 3, 4, and so on) (possibly) under each figure.

5. Discussion:

Please explain (based on the reference) the level of antioxidant activity (for example: very strong: <50 µg/mL; strong: 50-100 µg/mL, etc) to support your discussion. Additionally, compare your results to commercial antioxidant compounds, such as BHT, vitamin C, or others (included in Table 1) for a more interesting discussion. Also, to further highlight the anticancer potential of your extract, compare its anticancer activity to that of commercial anticancer drugs/compounds that are widely used.
Paragraph 4: which TCEA fraction explains in this sentence: “The TCEA was revealed as the most potent antioxidant (IC50=6.49 μg/mL) and cytotoxic (LC50=2 μg/mL) among others” ?
Figure 6: typo on GCMS, it should be “gas chromatography-mass spectroscopy”

6. Conclusion means something else than summary, so please include recommendations for applying your extract, for example in the medical field where might it be used as a natural alternative treatment/drug/supplement (???)

7. The is no statistical analysis or interpretation because the author did not explain the repetitions, either for experiments or analysis.

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

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

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

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

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biomass valorisation, Bioactive extraction, Microwave extraction

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.

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Reviewer Report 19 Oct 2023

Vijaya Anand Arumugam, Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India

Approved with Reservations

https://doi.org/10.5256/f1000research.140602.r216306

The authors have put effort to evaluate the “Anticancer and antiretroviral activities of methanolic extract from Theobroma cacao L pod husk: focusing on the ethyl acetate partition”. The authors describe the research undertaken with this in an organized manner, emphasizing ... Continue reading

The authors have put effort to evaluate the “Anticancer and antiretroviral activities of methanolic extract from Theobroma cacao L pod husk: focusing on the ethyl acetate partition”. The authors describe the research undertaken with this in an organized manner, emphasizing the results obtained by them. The article needs modification for better cohesion of information to achieve the goal and the shortcomings which need to be considered. Hence, the paper can be accepted after MAJOR REVISIONS are carried out.

The English need improvement since there are some grammatical and syntax errors in the manuscript. For example, the words “source for” may be as “source of”; “suggest the” as “suggest that the”; “in oncogenic” as “in the oncogenic”; “before crushed” as “before being crushed”; “with iQ5” as “with an iQ5”; “Standard” as “The standard”; “the similar” as “similar”; “75-mL” as “a 75-mL”; “the light” as “light”; “vortex” as “a vortex”; “brine” as “the brine”; “and added” as “added”; “in sample” as “in the sample”; “against HeLa” as “against the HeLa”; “on TCH” as “on the TCH”; “polynomial” as “the polynomial”; “against A549” as “against the A549”; “for the viral” as “for viral”; “with concentration” as “with a concentration”; “replications of” as “replications”; “as having” as “to have”; “results have been” as “results are”; “soluble of” as “soluble”; “6 being” as “6 are”; “obtain sample” as “obtain a sample”; “FeCl3 5%” as “the FeCl3 5%”; “due its” as “due to its”; “similarity over” as “a similarity of over”; “Moderate” as “The moderate”; “a moderate” as “moderate”; “that include” as “including”; “an agreement” as “agreement”; “as antiviral” as “as an antiviral”; “found correlated” as “found to correlated”; “Figure 5” as “in Figure 5”; “by influenza” as “by the influenza”; “of tobacco” as “of the tobacco”; “a potential” as “the potential”. The grammar mistakes which are not mentioned here are also to be checked and corrected properly.
There are some typing mistakes as well, and authors are advised to carefully proof-read the text. For example, the words “wide-range” may be as “wide range”; “sub-cultured” as “subcultured”; “Afterward,” as “Afterwards,”; “a concentrations” as “a concentration”; “real time” as “real-time”; “30 second” as “30 seconds”; “thin layer” as “thin-layer”; “extract sample” as “extracted sample”; “firstly incubated” as “first incubated”; “incubated” as “incubation”; “brine shrimps” as “brine shrimp”; “the rapid” as “the rapidly”; “Day-1” as “Day 1”; “phytoconsituents” as “phytoconstituents”; “review on” as “review of”; “cytoprotective effect” as “cytoprotective effects”; “retroviral agent” as “retroviral agents”; “investigation on” as “investigation of”. The typos not mentioned here are also to be checked and corrected properly.
Check the abbreviations throughout the manuscript and introduce the abbreviation when the full word appears the first time in the abstract and the remaining part of the manuscript (For example, simian retrovirus-2 (SRV-2), ethyl acetate partition (TCEA), DMSO, etc.,). Make a word abbreviated in the article that is repeated at least three times in the text, not all words to be abbreviated.
The full form of the species should be given when the first time appears in both the abstract and in the remaining part of the manuscript and it should be followed by only the first letter of the genus (For example, Theobroma cacao when the first time appear and followed by T. cacao).
The technical terms (Latin Phrase) "in-vitro " should be italic and it should be checked all over the manuscript.
The introduction part appears less informative about Theobroma cacao, thus this section should be indicated as detailed to understand the manuscript in clear since the main objectives is focused on Theobroma cacao. The botanical description, family name and different plants of the plant with it medicinal uses.
The authors may include the details of the quantity of fresh sample used and the quantity of the powder obtained (the ratio between fresh plant and powder) under the heading materials and methods.
In the materials and methods, the authors may cite references for standard protocol, instead of mentioning kid or manufacture instructions, if reference is given with it and the same should be added in the reference section.
The table and figure legends should be improved and a proper footnote should be given. All legends should have enough description for a reader to understand the tables and figures without and tables having to refer back to the main text of the manuscript. For example, the necessary abbreviations should be given.
The limitation of the present study may be given along with conclusion or under separate heading for understanding the concepts clearly.

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

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

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

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

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

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Phytotherapeutics, Clinical Biolchemistry, Medical Genetics

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.

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Version 1 28 Nov 22	read	read

Vijaya Anand Arumugam, Bharathiar University, Coimbatore, Tamil Nadu, India
Shinta Rosalia Dewi, Universitas Brawijaya, Malang, Indonesia

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4 Views

11 May 2024 | for Version 1

Shinta Rosalia Dewi, Universitas Brawijaya, Malang, Indonesia

4 Views Cite this report Responses(0)

Approved With Reservations

This is very good research that will be very beneficial to researchers working in anticancer and antiviral areas, as well as medical studies. The valorization of cacao pod husk waste into anticancer and antiviral agents is a highly intriguing idea.

The research study has been carried out systematically and the results are written coherently in the article. The Abstract has been written clearly and reflects the manuscript's content. However, some improvements are still needed to ensure the information presented in the manuscript is clear. Possibly some English revision should also be done, and the following other comments can be addressed:

1. English revision needs to be done to make the paper much pretty. For example, the term “as much as” is inappropriate (should be removed), or if required, replaced with “about”.

2. Introduction: Please clarify the originality and research aim of this study in the Introduction section. The authors have explained the potential of cacao pod husk as a medicine for anticancer, antiviral, etc. Various extraction methods and solvents that are widely used have also been mentioned, including what had been previously studied (extraction using water). However, the authors did not elaborate to provide clear explanations for why the maceration with methanol solvent was then chosen for this study (the research gap analysis was unclear).

3. Methods:

Material: instead of using “technical methanol, technical n-hexane, etc”, it is preferable to use “methanol, n-hexane, etc (mention all), with technical grade quality”. Also, for “pro-analytical ethanol, methanol, etc”, I would recommend using “methanol, ethanol, etc, with extra pure or analytical grade quality.
Cacao pod extraction: what is the solvent-to-sample ratio, number of repetitions, and total amount of solvent used? Introduce the abbreviation for the first time it appears in the manuscript: TCM, TCH.

4. Results:

Please write the information clearly for readers to grasp. Compare the %inhibition with IC50 results to conclude the antioxidant activity. For example, how does the antioxidant activity of TCH, which has the highest %inhibition, correspond with the lowest IC50 value?
Figure 1: Instead of placing the “MCF- and Hela” annotations within the picture, write the notations (a) and (b) in the figure title (bold text). Also, use consistent terminology: methanol extract, EA soluble, hexane soluble (all are extracts).
Figure 2: provide number information (1,2, 3, 4, and so on) (possibly) under each figure.

5. Discussion:

Please explain (based on the reference) the level of antioxidant activity (for example: very strong: <50 µg/mL; strong: 50-100 µg/mL, etc) to support your discussion. Additionally, compare your results to commercial antioxidant compounds, such as BHT, vitamin C, or others (included in Table 1) for a more interesting discussion. Also, to further highlight the anticancer potential of your extract, compare its anticancer activity to that of commercial anticancer drugs/compounds that are widely used.
Paragraph 4: which TCEA fraction explains in this sentence: “The TCEA was revealed as the most potent antioxidant (IC50=6.49 μg/mL) and cytotoxic (LC50=2 μg/mL) among others” ?
Figure 6: typo on GCMS, it should be “gas chromatography-mass spectroscopy”

6. Conclusion means something else than summary, so please include recommendations for applying your extract, for example in the medical field where might it be used as a natural alternative treatment/drug/supplement (???)

7. The is no statistical analysis or interpretation because the author did not explain the repetitions, either for experiments or analysis.

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

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

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

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

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biomass valorisation, Bioactive extraction, Microwave extraction

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.

Respond to this report

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Reviewer Report

14 Views

19 Oct 2023 | for Version 1

Vijaya Anand Arumugam, Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India

14 Views Cite this report Responses(0)

Approved With Reservations

The authors have put effort to evaluate the “Anticancer and antiretroviral activities of methanolic extract from Theobroma cacao L pod husk: focusing on the ethyl acetate partition”. The authors describe the research undertaken with this in an organized manner, emphasizing the results obtained by them. The article needs modification for better cohesion of information to achieve the goal and the shortcomings which need to be considered. Hence, the paper can be accepted after MAJOR REVISIONS are carried out.

The English need improvement since there are some grammatical and syntax errors in the manuscript. For example, the words “source for” may be as “source of”; “suggest the” as “suggest that the”; “in oncogenic” as “in the oncogenic”; “before crushed” as “before being crushed”; “with iQ5” as “with an iQ5”; “Standard” as “The standard”; “the similar” as “similar”; “75-mL” as “a 75-mL”; “the light” as “light”; “vortex” as “a vortex”; “brine” as “the brine”; “and added” as “added”; “in sample” as “in the sample”; “against HeLa” as “against the HeLa”; “on TCH” as “on the TCH”; “polynomial” as “the polynomial”; “against A549” as “against the A549”; “for the viral” as “for viral”; “with concentration” as “with a concentration”; “replications of” as “replications”; “as having” as “to have”; “results have been” as “results are”; “soluble of” as “soluble”; “6 being” as “6 are”; “obtain sample” as “obtain a sample”; “FeCl3 5%” as “the FeCl3 5%”; “due its” as “due to its”; “similarity over” as “a similarity of over”; “Moderate” as “The moderate”; “a moderate” as “moderate”; “that include” as “including”; “an agreement” as “agreement”; “as antiviral” as “as an antiviral”; “found correlated” as “found to correlated”; “Figure 5” as “in Figure 5”; “by influenza” as “by the influenza”; “of tobacco” as “of the tobacco”; “a potential” as “the potential”. The grammar mistakes which are not mentioned here are also to be checked and corrected properly.
There are some typing mistakes as well, and authors are advised to carefully proof-read the text. For example, the words “wide-range” may be as “wide range”; “sub-cultured” as “subcultured”; “Afterward,” as “Afterwards,”; “a concentrations” as “a concentration”; “real time” as “real-time”; “30 second” as “30 seconds”; “thin layer” as “thin-layer”; “extract sample” as “extracted sample”; “firstly incubated” as “first incubated”; “incubated” as “incubation”; “brine shrimps” as “brine shrimp”; “the rapid” as “the rapidly”; “Day-1” as “Day 1”; “phytoconsituents” as “phytoconstituents”; “review on” as “review of”; “cytoprotective effect” as “cytoprotective effects”; “retroviral agent” as “retroviral agents”; “investigation on” as “investigation of”. The typos not mentioned here are also to be checked and corrected properly.
Check the abbreviations throughout the manuscript and introduce the abbreviation when the full word appears the first time in the abstract and the remaining part of the manuscript (For example, simian retrovirus-2 (SRV-2), ethyl acetate partition (TCEA), DMSO, etc.,). Make a word abbreviated in the article that is repeated at least three times in the text, not all words to be abbreviated.
The full form of the species should be given when the first time appears in both the abstract and in the remaining part of the manuscript and it should be followed by only the first letter of the genus (For example, Theobroma cacao when the first time appear and followed by T. cacao).
The technical terms (Latin Phrase) "in-vitro " should be italic and it should be checked all over the manuscript.
The introduction part appears less informative about Theobroma cacao, thus this section should be indicated as detailed to understand the manuscript in clear since the main objectives is focused on Theobroma cacao. The botanical description, family name and different plants of the plant with it medicinal uses.
The authors may include the details of the quantity of fresh sample used and the quantity of the powder obtained (the ratio between fresh plant and powder) under the heading materials and methods.
In the materials and methods, the authors may cite references for standard protocol, instead of mentioning kid or manufacture instructions, if reference is given with it and the same should be added in the reference section.
The table and figure legends should be improved and a proper footnote should be given. All legends should have enough description for a reader to understand the tables and figures without and tables having to refer back to the main text of the manuscript. For example, the necessary abbreviations should be given.
The limitation of the present study may be given along with conclusion or under separate heading for understanding the concepts clearly.

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

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

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

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

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

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Phytotherapeutics, Clinical Biolchemistry, Medical Genetics

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.

Respond to this report

Responses (0)

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Anticancer and antiretroviral activities of methanolic extract from Theobroma cacao L pod husk: focusing on the ethyl acetate partition

Abstract

Keywords

Introduction

Methods

Materials

Plant specimen and bioindicators

Cocoa pod husk extraction

MTT Assay

A549 cell culture for anti-SRV-2

RT-PCR

Fractionation using gravitational column chromatography

DPPH assay

BSLT assay

Results

Anticancer activities

Figure 1. Anticancer activities of methanol extract of T. cacao (TCM), ethyl acetate soluble of T. cacao (TCEA) and n-hexane soluble of T. cacao (TCH) against MCF-7 and Hela cell lines.

Table 1. IC50 values of methanol extract of T. cacao (TCM), ethyl acetate soluble of T. cacao (TCEA) and n-hexane soluble of T. cacao (TCH) against MCF-7 and HeLa cell lines based on the equations obtained from polynomial regression.

Antiretroviral activities

Figure 2. Inhibition (%) and IC50 of ethyl acetate soluble of T. cacao (TCEA) against A549 lung cancer cell line.

Table 2. Antiretroviral activity of ethyl acetate soluble of T. cacao (TCEA) against Simian retrovirus-2 (SRV-2).

Isolation of the TCEA

Figure 3. Chromatogram of ethyl acetate soluble of T. cacao (TCEA) 1—4 (a), TCEA 4—6 (b), and TCEA 7—9 (c).

DPPH Antioxidant profiles of the TCEA 1—9

Figure 4. DPPH antioxidant activities of ethyl acetate soluble of T. cacao (TCEA) 1—9 and positive control (ascorbic acid).

BSLT cytotoxicity profiles of the TCEA subfractions

Table 3. Results from BSLT assay indicating the cytotoxicity of ethyl acetate soluble of T. cacao (TCEA) 1—9.

Re-chromatography

Figure 5. Chromatogram of ethyl acetate soluble of T. cacao (TCEA) 6.1—6.4.

Phytochemical profile

Table 4. Results from chromatography-mass spectroscopy (GC-MS) analysis on ethyl acetate soluble of T. cacao (TCEA) sub-fraction.

Discussion

Figure 6. Bioactive phytoconstituents detected in ethyl acetate soluble of T. cacao (TCEA) 6.3 by chromatography-mass spectroscopy (GC-MS).

Conclusions

Author contributions

Data availability

Underlying data

Acknowledgments

References

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Table 1. IC₅₀ values of methanol extract of T. cacao (TCM), ethyl acetate soluble of T. cacao (TCEA) and n-hexane soluble of T. cacao (TCH) against MCF-7 and HeLa cell lines based on the equations obtained from polynomial regression.

Figure 2. Inhibition (%) and IC₅₀ of ethyl acetate soluble of T. cacao (TCEA) against A549 lung cancer cell line.