Cocoa (Theobroma cacao L., family Sterculiaceae) is an important agricultural and economic crop that grows in several tropical areas such as West Africa, South America, and Central America.T. cacao is the most prominent in trade among about 20 species of the Theobroma genus^[1]. Cocoa beans, the main ingredient in the manufacture of chocolate, are the primary economic part of the cocoa fruit. Following removal of the beans from cocoa pods, large quantities of cocoa pod husks (CPHs) as under exploited by-product representing a serious disposal problem are generated. CPH weighs about 75% of the whole fruit^[2].

Research studies directed at reducing the CPH waste burden includelow-value use of CPH as soil manure for crops^[3] or as a component of poultry or animal feed^[4]. Waste-valorization studies for CPH also comprise its use as activated carbon and catalyst in biodiesel production^[5,6]. In a recent review article, Campos-Vega et al.^[7] expatiated on the valorization potentials of CPH (rich in fiber and bioactive polyphenols) toward generating new pharmaceuticals, neutraceuticals, medical or functional food products; remarking that its quality and functionality improvements over years have been through fermentation, enzymatic hydrolysis and combustion processing methods.

Combustion of dried CPH leads to CPH ash production. The ash has been studied as an extractive agent for carrageen an hydrocolloid from red seaweeds^[8], as an alkaline digester (cheaper than sodium hydroxide) for crude fiber content of poultry feed^[9], as a stabilizer component in production of compressed earth bricks^[10], as an ingredient for soap making^[11], and as corrosion inhibitor for carbon steel^[12]. CPH pectin extracted with hot aqueous citric acid has also been studied as nutraceutical and a potential pharmaceutical excipient^[13].

The aim of this study was to determine physicochemical properties of Theobroma cacao pod husk (CPH) ash methanolic extract and antibacterial activities of its aqueous solution; which could afford insights into the appropriate use and application of the extract for pharmaceutical products development.

Materials and Methods

Collection and identification of Theobroma cacao (cocoa) pod husk

Freshly harvested, ripe cocoa pods were procured from cocoa farmers residing in Ile-Ife Nigeria. A specimen of the cocoa pod and leaves was prepared, authenticated and assigned the catalogue number IFE 17464 at the Ife Herbarium, Department of Botany, ObafemiAwolowo University (OAU), Ile-Ife Nigeria, and deposited there.

Following removal of the beans, the broken cocoa pods were washed, sun-dried for 14 days, turning them over constantly to hasten the drying process. They were further dried in an hot-air oven (at 45°C) to a moisture content of about 10%, cut into smaller pieces and ground to powder in a hammer mill to obtain the cocoa pod husk (CPH) powder. This was stored in air tight glass bottles until needed for analyses.

Characterization of cocoa pod husk (CPH)

Proximate, mineral contents and phytochemical analyses of the CPH powder were carried out. Proximate composition of the CPH was determined according to the Association of Official Analytical Chemists methods^[14]. The parameters assayed for included crude protein, crude fiber, ash, moisture, crude fat (lipid) and crude carbohydrate (nitrogen-free extract). For crude protein, the total nitrogen of a 1 g sample of CPH was determined by the Kjeldahl method as described by Gul and Safdar^[15]. Similarly, crude fiber content of a 1 g CPH sample was determined using the Fibretec hot and cold extraction method^[14,15]. The AOAC methods^[14] as described by Nguyen^[16] were used for determination of moisture and ash contents of the CPH samples, while fat content of the samples was determined using Soxhlet type of the direct solvent extraction method. The solvent used was petroleum ether (boiling range 40-60°C). Carbohydrate content was determined by the difference method (i.e. 100 minus [% protein+ % fat+ % moisture+ % ash + % fiber]). All the proximate determinations were carried out in triplicates and the results reported in percentage values^[14].

Mineral content of the CPH was estimated by employing Atomic Absorption Spectroscopy (Perkin Elmer Aanalyst 400, Shelton, USA) for iron, magnesium, calcium, manganese, copper; and Flame Emission Photometry for sodium and potassium^[17].

Phytochemical analyses of cocoa pod husk (CPH)

The CPH samples were subjected to phytochemical studies for determination of antioxidant activity, total flavonoids, tannins, trypsin inhibitor, and total phenols. Antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging method as described by Magalhaes et al.^[18], while the percentage of DPPH scavenged was obtained according to the equation^[19]:

(eqn.1)

Total flavonoid present in 1 g CPH sample was determined using aluminium chloride colorimetric method of Lin and Tang^[20], and calculated using the following equation^[14]:

(eqn.2)

Where DF = dilution factor

The AOAC titrimetric methods for tannin content determination^[14] described by Khasnabis et al^[21]. and for trypsin inhibitor determination described by Van-Eys^[22], using the concentration versus absorbance calibration graphs of standard tannic acid and trypsin inhibitor solutions, respectively, were used for 1 g CPH sample, respectively. The values were calculated using the following relevant equations^[14]:

(eqn.3)

Where DF = dilution factor

(eqn.4)

where DF = dilution factor

The spectrophotometric determination of total phenolcontent on 1 g CPH sample using garlic acid, as described by Siddiqui et al^[23]., was used. Standard garlic acid solutions in distilled water at different concentrations (0.5, 1, 2, 4, 6, 8, and 10 mg / ml) were prepared and their absorbance read at 765 nm, for construction of the standard calibration graph of garlic acid concentration against the absorbance. The concentration of garlic acid in the CPH sample was extrapolated from the graph. The total phenol content was then calculated using the formula^[14]:

(eqn.5)

where DF = dilution factor

Ashing of cocoa pod husk and extraction of ash

Cocoa pod ash was obtained by burning oven-dried cocoa pod husks (CPHs) previously cut into small pieces in a furnace at 500ºC for 6 h. The resulting ash was collected, weighed and extracted with methanol and water, respectively.

Methanolic extraction

To a 150 g sample of the CPH ash placed in a 1 liter-capacity conical flask was added 700 ml of methanol. The dispersion was shaken intermittently at 15 min intervals over a 3 h period. The resulting liquid was decanted and 500 ml of fresh solvent (methanol) was added to the sediment, and shaken intermittently again at 15 min intervals over a 2 h period. The resulting mixture was also decanted. Both methanolic extract liquids were combined, filtered using Whatman No. 1 filter paper, and the filtrate concentrated to dryness using a rotary evaporator (Buchi, Switzerland) to yield the methanolic CPH ash extract (granular) powder.

Aqueous extraction

To a 150 g sample of CPH ash in a 1 L-capacity conical flask was added 700 ml of distilled water. This was shaken together, allowed to stand for 4 h and then percolated through a muslin cloth. The resulting percolate was boiled to concentrate the liquid to a volume of 150 ml and subsequently evaporated to dryness in a vacuum-enhanced hot-air oven (Isotemp^® vacuum oven, Model 282A, Fisher Scientific, USA), to yield the aqueous CPH ash extract powder.

Physicochemical testing of CPH ash extract

Determination of water solubility

Solubility in water of the methanolic and the aqueous extract powders of CPH ash was determined at the ambient temperature (30±1°C) following the Organization for Economic Cooperation and Development (OECD) guideline for water solubility testing of chemicals (no. 105 flask method, adapted)^[24]: Freshly distilled water was added drop-wise onto a 1.0 g sample of the CPH ash extract powder placed in a 10 ml test tube (having air tight stopper), thereby producing gradually increasing volume of aqueous dispersion or solution of the extract powder, and shaking the test tube for 2 min after each addition. The solubility (value) of the extract was determined from the total volume of water required for complete dissolution of the sample in triplicate tests.

Determinations of pH and of citric, salicylic acids equivalents of CPH ash extract solution

The pH value of 25 mg/ml aqueous solution of the methanolicCPH ash extract powder was determined with a digital pH meter (HM Digital Inc. California, USA) in triplicate tests.10.00 ml of the solution was titrated, separately, with citric acid solution (10 mg/ml) and with salicylic acid solution (2 mg/ml) in triplicate tests, adding each acid solution drop-wise from a burette into the CPH ash extract solution in a conical flask, while monitoring pH of the resulting liquid mixture with the pH meter, until neutral pH (7.0) was obtained. The quantity of each acid solution required to neutralize pH of the CPH ash extract solution was indicative of equivalence of the acid solution to that of the extract^[25].

Determinations of microbial purity of CPH extract powders

Ten percent (w/v) aqueous solution of methanolic CPH ash extract powder and of aqueous CPH ash extract powder were prepared aseptically by dissolving 1 g of extract in sufficient sterile water to produce a 10 ml solution in a sterile volumetric flask. The solution (0.5 ml aliquot) was surface-plated onto supportive over-dried growth medium for bacteria and fungi: nutrient agar and Sabouraud dextrose agar plates, respectively, in triplicate tests to reveal any possible viable contaminants in the CPH ash extract powders.

Antimicrobial activity testing of CPH ash extract

The antimicrobial activities of the methanolic extract and of the aqueous extract of CPH ash were determined against selected reference organisms namely: Staphylococcus aureus NCTC 6571, Bacillus subtilis NCTC 8263, Pseudomonas aeruginosa ATCC 10145, Escherichia coli ATCC 25922, and Candida pseudotropicalis NCYC 6, using the agar-well diffusion and micro-titer broth dilution assays.

Agar-well diffusion assay

The susceptibility of each organism to 667 mg/ml methanolicCPH ash extract, 227 mg/ml aqueous CPH ash extract (saturated solutions); and susceptibility of the bacterial organisms to 1.75 mg/ml solution of neomycin sulphate (Xinyu Pharm.Co. Ltd. China), and of the Candida organism to 10.0 mg/ml solution of acriflavine hydrochloride (Ankur Chemicals, India), positive controls; were tested using the agar-well diffusion assay. Twenty milliliters of melted and cooled nutrient agar plates (for bacteria)and Sabouraud dextrose agar plates (for yeast) were each seeded with 0.2 ml of an overnight broth culture (approx. 1×10⁸ cells per ml) of each test organism growing in nutrient broth (bacteria) or in Sabouraud dextrose broth (yeast), respectively, and allowed to set. Three 7 mm diameter wells were cut, equidistant, into each plate with a sterile cup borer. Then 100 µl of each test and control agent was pipetted into the agar wells. The tests were carried out in triplicates. The test substances were allowed one hour to diffuse into the agar, and the plates were incubated at 37°C for 24 h (bacteria), and 25°C for 48 h (Candida). The inhibition zone diameters were thereafter measured.

Micro-titer broth dilution assay

The methanolic CPH ash extract powder (at 667 mg/ml) and aqueous CPH ash extract powder (at 227 mg/ml) dissolved in Mueller-Hinton broth (MHB) for bacteria, and in Sabouraud dextrose broth (SDB) for yeast, were evaluated for inhibitory activities against the test organisms using the micro-titer broth dilution assay. Neomycin sulphate and acriflavine hydrochloride aqueous solutions (0.70 and 10.0 mg/ml), respectively, were employed as positive control antibacterial and antifungal agents, with neat MHB and SDB as the negative controls. The assay was carried out in duplicates.

The test sample (100 μl) was inoculated into the 1^st column wells of a 96 wells micro-titer plate containing 100 μl MHB for bacteria and 100 μl SDB for yeast,and was serially diluted two fold down to the 10th column wells to obtain concentration ranges of 333.5 – 0.65 and 113.5 – 0.22 mg/ml for the methanolic and aqueous CPH ash extracts, respectively; and concentration ranges of 0.35– 0.00068 and 0.50 – 0.00098 mg/ml for the positive controls:neomycin sulphate and acriflavine hydrochloride, respectively. Each of the wells was challenged with a 5 μl organism inoculum standardized at 2 × 10⁴ to 10⁵ cfu/ml ascertained from viable counts simultaneously carried out, for minimum inhibitory concentration (MIC) determination of the test agents^[26]. The positive control (11^th) well consisted of 200 μl of appropriate medium (MHB for bacteria and SDB for yeast) and 5 μl of the test organism, while the negative (sterility) control well (the 12^th) consisted of 200 μl of the appropriate medium but without inoculated organism.

The plates were covered with sterile lid and incubated at 37°C for 24 h and 25°C for 48 h for bacterial and yeast growth, respectively. Following incubation, the plates were observed for growth or inhibition to growth in all the wells and results recorded. A flamed and cooled multi-inoculator was then used to transfer the organisms onto recovery plates (over-dried nutrient agar and Sabouraud dextrose agar plates for bacteria or yeast, respectively). The micro-titer and recovery plates were incubated appropriately for microbial growth for a further 24 and 48 h periods, respectively. Growth on the recovery plates were noted and recorded. Cell viability in the wells was ascertained by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric test. Viable micro organisms caused a change of the yellow dye into a purple colour. The minimum inhibitory concentration (MIC) was taken as the lowest concentration of the test agent that inhibited microbial growth, while the minimum bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) was taken as the lowest concentration of the test agent to kill the test organism. The MICs, MBCs or MFCs were determined as the lowest sample concentrations that produced no change in colour, indicating inhibition of microbial growth.

Data analysis

The results of all determinations carried out in triplicate were expressed as mean ± SEM. The data were subjected to one-way analysis of variance (ANOVA). P values less than 0.05 (p<0.05) were regarded as statistically significant.

Results and Discussion

Proximate composition, mineral and chemical contents of CPH

The results from characterization of CPH studied are given in Tables 1 and 2. Proximate analysis (Table 1) revealed the crude fiber content to be in the highest relative proportion, followed by carbohydrate, moisture, protein, total ash, and lipid contents, respectively. The data obtained for carbohydrate and ash contents agreed with those earlier reported by Shodehinde and Adamson^[27]. Other research findings, however, indicated higher crude fiber contents (53.37 - 61.80 %) and lower protein (7.70 - 9.37%) and ash contents (9.30-10.85%) in different CPH meal samples^[28,29]. Moisture content of CPH was dependent on relative equilibrium moisture of the cocoa cultivation environment and the length of time of cocoa pods sun-drying after harvesting^[29].

The mineral composition of CPH in the present studyshowed high proportions of potassium, magnesium and calcium elements (Table 2). Earlier workers^[30] also similarly found sizeable amounts of potassium, magnesium, calcium, phosphorus, copper and zinc contained in all CPH samples studied. These were considered to enhance quality of plant-based raw materials in general, and particularly those used for animal nutrition such as livestock feed. High potassium content,for instance, is valuable for protein synthesis, starch formation and enzyme activation^[27].

Table 1: Proximate composition of fresh T. cacao pod husk

Table 2: Mineral contents of fresh T. cacao pod husk

*The data are expressed as Mean ± SEM

The phytochemical components of CPH (Table 3), known also as antinutrients, are biologically active secondary metabolites in plants, which are reported to perform immunological defense in man and animals, among their other biological functions^[27]. Flavonoids for instance, found to be the component of highest proportion in CPH (Table 3), are known to exert antioxidant effect by neutralizing and chelating oxidizing radicals (e.g. the superoxide and hydroxyl radicals) due to their ability to release an electron from their phenolic groups^[31]. From veterinary and livestock feed perspectives, these data (Tables 1-3) could enable an appraisal of the vitamin and mineral intake that CPH consumption provides with regard to recommended dietary prescriptions; and differences or variation of the proximate, mineral elements and phytochemical valuations of CPH from different places are attributable to operational or environmental factors such as the level of fertilizer applied on cultivated fields, soil pH and soil nutrients,all of which are amenable to advantageous modification^[30].

Table 3: Phytochemical contents of fresh T. cacao pod husk

*The data are expressed as mean ± SEM

Yield, physical characteristics, solubility and pH of cocoa pod husk ash extract

The yield of cocoa pod husk (CPH) ash methanolic extract was 13.6 ± 0.14 %w/w, while that of the aqueous extract was 12.6 ± 0.07 %w/w. The difference in these mean yield values was statistically significant (p<0.05). Each extract, evaporated dry, occurred as deliquescent, mostly white (or translucent), irregularly shaped crystalline solid; the methanolic extract being generally larger size (Figures 1 and 2).

Figure 1: Methanolic extract of Theobroma cacao pod husk ash in a Petri dish

Figure 2: Aqueous extract of Theobroma cacao pod husk ash in a Petri dish

At the ambient temperature (30±1°C), a 1.00 g dry methanolic CPH ash extract powder sample required the addition of 1.50 ± 0.003 ml of distilled water to dissolve, while the same weight of aqueous CPH ash extract powder sample required 4.40 ± 0.004 ml of distilled water to dissolve; giving the water-solubility values of 666.67 and 227.27 mg/ml determined for the methanolic and aqueous extracts, respectively. Thus, while both extract powders were ‘freely soluble’ in water (i.e. <10.0 mg/ml solubility^[32]), the methanolic CPH ash extract powder demonstrated a relatively higher yield and water-solubility values than the aqueous extract powder.

Each of the extracts produced a colourless, strongly alkaline solution on dissolution in water. The pH value of saturated aqueous solution of the CPH ash methanolic extract was 12.42 ± 0.48, while that of the saturated aqueous solution of CPH ash aqueous extract was 13.48 ± 0.52. These mean pH values of the different extract types were, however, not significantly different (p>0.05).

Citric acid was more efficient than salicylic acid to neutralize alkaline pH of CPH ash extract solution

When citric acid solution (10 mg/ml; pH=0.68±0.07) was titrated against CPH ash methanolic extract solution (10.00 ml; 25 mg/ml; pH=12.42±0.48), 18.00 ml of the acid solution was required to neutralize the pH of the extract, reducing it from the initial value to 6.88±0.13. However, 90.00 ml of salicylic acid solution (2 mg/ml; pH=2.98±0.04) was required to neutralize the pH of the CPH ash methanolic extract solution, reducing the pH from its initial value (12.42±0.48) to 6.87±0.19. A 20.0 ml volume of the salicylic acid solution (double of the volume of CPH ash extract solution titrated) only minimally reduced pH of the extract to 10.44±0.02, from the initial value.

Thus, citric acid demonstrated a more efficient neutralizing capacity for CPH ash extract solution than salicylic acid.The stronger neutralizing effect of citric acid solution alludes to its lower pH value than that of salicylic acid solution. Citric acid and salicylic acid are pharmaceutical ingredients employed in therapeutic skin products (balms, creams), sometimes to function as pH-modifier to obtain a suitable pH value in a product for diseased skin treatment^[33].

Microbiological quality and antimicrobial activities of CPH ash extract

The aqueous solutions of CPH ash methanolic and aqueous extract powders did not support microbial growth. Plated and spread on nutrient agar or Sabouraud dextrose agar, each extract in solution showed no growth of contaminant, whatsoever.

Contrariwise, the saturated solution of each extract (and the controls) demonstrated antimicrobial activities against selected test organisms as presented in Table 4.

Table 4: Antimicrobial activities of T. cacao pod husk (CPH) ash extract on selected organisms

Key:

* Data are presented as Mean ± SEM of inhibition zone diameters; Values with same superscript letters are not significantly different

** Positive control (antibacterial agent)

*** Positive control (antifungal agent)

‒ Testing was excluded for incongruity

In the agar-well diffusion assay, the methanolic CPH ash extract demonstrated significantly higher antibacterial activity against E. coli (p<0.05), similar activity (p>0.05) against B. subtilis and P. aeruginosa, and significantly lower activity against S. aureus (p<0.05) when compared to the antibacterial activities of neomycin (the positive control) against the respective organisms. The antifungal activity of the methanolic CPH ash extract against C. pseudotropicalis was also significantly greater than that of acriflavine (control) (p<0.05) (Table 4). On the other hand, the antibacterial activities of the aqueous CPH ash extract were significantly lower than those of neomycin sulphate against three (out of four) tested bacterial organisms (p<0.05); the exception being the extract’s activity against E. coli, which was not significantly different from that of the control (neomycin sulphate) (p>0.05). But the extract’s antifungal activity was significantly greater than that of acriflavine (p<0.05) (Table 4).

The fact that both the methanolic and aqueous CPH ash extracts demonstrated considerable antimicrobial activities comparable to the positive controls (neomycin and acriflavine) against the test organisms signifies the potential for advantageous use of the extracts in pharmaceutical formulations development. Very few literature had similarly reported antibacterial activity of T. cacao husk extract prepared in some other way, e.g. aerobically fermented CPH extract, which demonstrated inhibitory activities against pathogenic bacteria: Salmonella choleraesuis, Staphylococcus epidermidis and Pseudomonas aeruginosa implicated in hospital infections; the activity of which was attributed to phenolics, steroids and terpenes (bioactive components) present in the fermented husk^[34].

From results of the present study (Tables 4,5), the yeast C. pseudotropicalis was the most susceptible organism to the general antimicrobial action of the CPH ash extracts. Similarly, Santos et al.^[34] earlier reported fermented CPH extract as active against the yeast Saccharomyces cerevisae and the basidiomycete Moniliophthora perniciosa; and another report^[35] also indicated that phenolic content of CPH obtained through macerating the husk with ethanol (70%) or acetone:water (7:3) mixture demonstrated fungicidal activity against Fusarium oxysporum.

Table 5 shows the minimum inhibitory concentrations (MICs) of CPH ash aqueous and methanolic extracts determined by the micro-broth dilution assay. In this assay, both control agents (neomycin and acriflavine) demonstrated significantly greater antimicrobial activities against all the test organisms than both types of CPH ash extract (p<0.05). Also the MICs for the methanolic CPH ash extract were consistently lower (ranged from 2.60 to 10.42 mg/ml) than the MICs for the aqueous CPH ash extract (which ranged from 3.55 to 28.38 mg/ml) against the same organisms, respectively; indicating stronger antibacterial and antifungal activities of the methanolic extract than the aqueous CPH ash extract.

All subcultures made from the micro-titer plate wells (using a multi-inoculator) into fresh identical plates (with subsequent 24 or 48 h incubation following the MIC determination) for minimum bactericidal and fungicidal concentrations (MBC/MFC) determination (i.e. for recovery, possibly, of inhibited but yet viable cells), revealed exactly the same results as obtained previously for the MIC data. This indicated that the activities of CPH ash extract were bactericidal and fungicidal. The data (Table 5) furthermore clearly established a stronger bactericidal effect of the methanolic CPH ash extract (5.2 – 10.4 mg/ml MIC range) than that of the aqueous (14.2 – 28.4 mg/ml MIC range); with implication that the methanol solvent-derived extract should be preferred above the aqueous solvent-sourced extract when antimicrobial activity of CPH ash extract for therapeutic use is needed, in that the former extract has demonstrated significant capacity to deliver greater lethal activities against the test organisms.

Table 5: Minimum inhibitory concentrations of T. cacaopod husk (CPH) ash extract against selected organisms

Key:

*Deduced from micro broth dilution assay: The same values were determined for MBCs and MFCs, respectively.

** Positive control (antibacterial agent)

*** Positive control (antifungal agent)

‒ Testing was excluded for incongruity

Conclusion

The physicochemical and antimicrobial properties of methanolic and aqueous extracts of CPH ash have been determined in preliminary study toward prospective pharmaceutical applications. CPH is ordinarily an agricultural waste, and the ash resulting from its combustion is not normally considered pertinent to medicinal application.This study has, however, revealed that both the methanolic and aqueous CPH ash extract powders (contaminant-free and freely soluble in water) demonstrate significant antimicrobial activities against selected test microorganisms, comparable to activities of positive control antimicrobial agents.Their potential for pharmaceutical application (e.g. in topical pharmaceuticals) was thus established. Topical application of the ash extract would require moderation of its high pH property, which the use of citric acid aqueous solution effectively achieved.

Conflict of Interests

We have no conflicts of interest to disclose in this study.

Funding

This research work was funded by the researchers. No external funding was received.

Author Contributions

Akinrotohun H.O., Oyedele A.O. and Igbeneghu O.A. carried out the research;

Oyedele A.O. and Orafidiya L.O. wrote the article.

References

1. 1. Prabhakaran-Nair, K.P. The agronomy and economy of important tree crops of the developing world. (2010) Cocoa (Theobroma cacao L.) 131-180.

   Pubmed| Crossref| Others

2. 2. Chan, S.Y., Choo, W.S. Effect of extraction conditions on the yield and chemical properties of pectin from cocoa husks. (2013)Food Chem 141(4): 3752-3758.

   Pubmed| Crossref| Others

3. 3. Filho, P.C.C., Carvalho-Silva, R., Ferreira de Sousa, D. et al. Use of Theobroma cacao pod husk-derived biofertilizer is safe as it poses neither ecological nor human health risks. (2017) J Ferti lPestic 8(3): 1-7.

   Pubmed| Crossref| Others

4. 4. Makinde, O.J., Okunade, S.A., Opoola, E. et al. Exploration of Cocoa (Theobroma cacao) by-products as valuable potential resources in livestock feeds and feeding systems. (2019).

   Pubmed| Crossref| Others

5. 5. Rachmat, D., Mawarani, L.J., Risanti, D.D. Utilization of cacao pod husk (Theobroma cacao L.) as activated carbon and catalyst in biodiesel production process from waste cooking oil. (2018). Mater Sci Eng 299.

   Pubmed| Crossref| Others

6. 6. Tsai, W.T., Huang, P.C. Characterization of acid-leaching cocoa pod husk (CPH) and its resulting activated carbon. (2018) Biomass Convers Biorefinery 1–8.

   Pubmed| Crossref| Others

7. 7. Campos-Vega, R., Nieto-Figueroa, K.H., Oomah, B.D. Cocoa (Theobroma cacao L.) pod husk: Renewable source of bioactive compounds. (2018) Trends Food Sci Tech 81: 172–184.

   Pubmed| Crossref| Others

8. 8. Rhein-Knudsen, N., Ale, M.T., Rasmussen, S. et al. Alkaline extraction of seaweed carrageenan hydrocolloids using cocoa pod husk ash. (2018) Biomass Convers Bior 8(3): 577–583.

   Pubmed| Crossref| Others

9. 9. Ankrah, A., Donkor, A., Ankrah, D. Assessment of the effectiveness of cocoa pod-husk ash extract as alkaline in reducing the crude fiber levels in sorghum spent grain (pito mash). (2014). J Biol Agric Healthcare 4(9): 70-75.

   Pubmed| Crossref| Others

10. 10. Yaw, M.I., Asiedu, E., Yalley, P.P.K. et al. Feasibility of using cocoa pod husk ash (CPHA) as a stabilizer in the production of compressed earth bricks. (2015) Int J Eng Res Gen Sci 3(6): 514-524.

    Pubmed| Crossref| Others

11. 11. Jayeola, C.O., Adebowale, B.A., Yahaya, L.E. et al. Production of bioactive compounds from waste. (2018)Acad Press 317-340.

    Pubmed| Crossref| Others

12. 12. Pedroza-Perinan, D., Villalobos-Vasquez, M., Meza-Castellar, P. et al. Evaluation of Theobroma cacao pod husk extracts as corrosion inhibitor for carbon steel. (2016) Ciencia Tecnol Futura 6(3): 147-156.

    Pubmed| Crossref| Others

13. 13. Adi-Dako, O., Ofori-Kwakye, K., Manso, S.F. et al. Physicochemical and antimicrobial properties of cocoa pod husk pectin intended as a versatile pharmaceutical excipient and nutraceutical. (2016) J Pharma .

    Pubmed| Crossref| Others

14. 14. Association of Official Analytical Chemists (AOAC)

    Pubmed| Crossref| Others

15. 15. Gul, S., Safdar, M. Proximate Composition and Mineral Analysis of Cinnamon. (2009) Pak J Nutr 8(9): 1456-1460.

    Pubmed| Crossref| Others

16. 16. Nguyen, V.T. Mass proportion, proximate composition and effects of solvents and extraction parameters on pigment yield from cacao pod shell (Theobroma cacao L.). (2014) J Food Process Preserv 39: 1414-1420.

    Pubmed| Crossref| Others

17. 17. Association of Official Analytical Chemists (AOAC).

    Pubmed| Crossref| Others

18. 18. Magalhaes, L. M., Santos, M., Segundo, M. A. et al. Automatic method for determination of total antioxidant capacity using 2,2-diphenyl-1-picrylhydrazyl assay. (2006) Anal Chim Acta 558(1-2): 310-318.

    Pubmed| Crossref| Others

19. 19. Hanane, A., Ahmed, M., Samia, M. et al. Phytochemical screening, quantitative analysis and antioxidant activity of Lifagodielsiischweinj and muschl. (2014) IntJ Phytomed 6(2): 280-285.

    Pubmed| Crossref| Others

20. 20. Lin, J., Tang, C. Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation. (2007) Food Chem 101(1): 140-147.

    Pubmed| Crossref| Others

21. 21. Khasnabis, J., Rai, C., Roy, A. Determination of tannin content by titrimetric method from different types of tea. (2015) J Chem Pharm Res 7(6): 238-241.

    Pubmed| Crossref| Others

22. 22. Van-Eys, J.E. Anti-nutritional factors: - Trypsin inhibitors, In: Manual of Quality Analysis for Soybean Products in the Feed Industry. (2012) US Soybean Export Council.

    Pubmed| Crossref| Others

23. 23. Siddiqui, N., Rauf, A., Latif, A. et al. Spectrophotometric determination of the total phenolic content, spectral and fluorescence study of the herbal Unani drug Gul-e-Zoofa (NepetabracteataBenth) (2017). J Taibah Univ Med Sci 12(4): 360-363.

    Pubmed| Crossref| Others

24. 24. Organization for Economic Cooperation and Development (OECD). Guidelines for the Testing of Chemicals Section 1: Physical-Chemical Properties.

    Pubmed| Crossref| Others

25. 25. Michałowska‐Kaczmarczyk, A.M., Spórna‐Kucab, A., Michałowski, T. Principles of titrimetric analyses according to Generalized Approach to Electrolytic Systems (GATES). (2017). 135-137.

    Pubmed| Crossref| Others

26. 26. Medu, E.O., Idowu, T.O., Oyedele, A.O. et al. Antimicrobial activity of eleagnine isolated from the seed cotyledons of Chrysophyllumalbidum. (2016) Nig J Nat Prod Med 20: 27-34.

    Pubmed| Crossref| Others

27. 27. Shodehinde, S.A., Adamson, A. Tapping in to the good use of cocoa (Theobroma cacao) pod husks: towards finding alternative sources of nutrients for animals in Nigeria. (2017) J Food TechnolPres 1(1): 42-46.

    Pubmed| Crossref| Others

28. 28. Ozung, P.O., Kennedy-Oko, O.O. Agiang, E.A. Chemical composition of differently treated forms of cocoa pod husk meal. (2016) Asian J AgricSci 8(2): 5-9.

    Pubmed| Crossref| Others

29. 29. Nguyen, V.T., Nguyen, N.H. Proximate composition, extraction, and purification of theobromine from cacao pod husk (Theobroma cacao L.). (2017) Technologies 5(14): 1-10.

    Pubmed| Crossref| Others

30. 30. Bonvehí, J.S., Jordà, R.E. Constituents of Cocoa Husks. (1998) Z Naturforsch 53c: 785-792.

    Pubmed| Crossref| Others

31. 31Gupta,V.K., Kumria, R., Garg, M. et al. Recent updates on free radicals scavenging flavonoids: An Overview. (2010). Asian J Plant Sci 9(3): 108-117.

    Pubmed| Crossref| Others

32. 32. British Pharmacopoeia (BP). British Pharmacopoeia Commission Office. (2009). London

    Pubmed| Crossref| Others

33. 33Schmid-Wendtner, M. H.,Korting, H.C. pH and skin care. (2007) Berlin, Germany: 15-97.

    Pubmed| Crossref| Others

34. 34. Santos, R.X., Oliveira, D.A., Sodré, G.A. et al. Antimicrobial activity of fermented Theobroma cacao pod husk extract. (2014). Genet Mol Res 13(3): 7725-7735.

    Pubmed| Crossref| Others

35. 35. Rachmawaty, A.M., Hasri, H.P., Hartati, Z. M. Active compounds extraction of cocoa pod husk (Theobroma cacao L.) and potential as fungicides. (2018). J Phys: IOP Conf Ser.

    Pubmed| Crossref| Others