Proximate Composition, Extraction, and Purification of Theobromine

technologies

Article Proximate Composition, Extraction, and Puriﬁcation of Theobromine from Cacao Pod Husk (Theobroma Cacao L.)

Van Tang Nguyen 1,2,* and Nghia Huu Nguyen 3 1 School of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Ourimbah, NSW 2258, Australia 2 Department of Food Technology, Faculty of Food Technology, Nha Trang University, No. 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa 8458, Vietnam 3 Department of Thermal and Refrigeration Technology, Faculty of Mechanical Engineering, Nha Trang University, No. 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa 8458, Vietnam; [email protected] * Correspondence: [email protected]; Tel.: +61-4-3423-8842

Academic Editor: Manoj Gupta Received: 4 March 2017; Accepted: 30 March 2017; Published: 2 April 2017

Abstract: The aims of this study were to determine the proximate composition of cacao pod husk as well as the optimal conditions for extraction and purification of theobromine from cacao pod husk. The results indicated that cacao pod husk had high contents of moisture and carbohydrate (87.06% and 11.03% by fresh weight, respectively), but low contents of crude protein, crude lipid, and ash (0.31%, 0.12%, and 1.48% by fresh weight, respectively). The optimal conditions for extraction of theobromine from cacao pod husk were of 70% ethanol, with an extraction time of 90 min, and 1 as the number of extractions. A concentration of 10% by volume of 10% lead acetate solution was the best selection for purification of the crude extracts containing theobromine from cacao pod husk. Under these optimal conditions, theobromine content obtained from cacao pod husk was 6.79 mg/100 g dry weight. The finding from this study is a valuable contribution for obtaining theobromine from an abundant, inexpensive, renewable, and sustainable source for potential application in the nutraceutical, medical, and pharmaceutical industries.

Keywords: cacao pod husk; theobromine; proximate composition; extraction; puriﬁcation

1. Introduction Theobroma cacao L. has been known as “the food of the gods”. Cacao tree is wildly grown in tropical and subtropical areas around the world including in Africa, such as Côte d'Ivoire, Ghana, Nigeria, and Cameroon, in American countries, like Brazil, Ecuador, Columbia, and Mexico, in Caribbean and Southwestern Paciﬁc countries, such as the Dominican Republic and Papua New Guinea, and in Southeast Asian countries, like Indonesia, Malaysia, and Vietnam. Total production of cacao bean is estimated worldwide to be approximately 4.0 million tonnes in 2013, with a value about $12 billion [1]. Residues or wastes from the cacao processing industry consist of cacao pod shell, husk, pulp/mucilage, and hull, which account for a high proportion, approximately 85% by fresh weight of total cacao pod mass [2], in which the annual worldwide amount of cacao pod husk is estimated to be about 55 million tonnes, which is equal to 13 times the total amount of cacao bean. Therefore, cacao pod husk needs to be exploited to produce high-value-added products and this waste has been considered as an abundant, inexpensive, and renewable source of theobromine, a valuable compound, which exhibits stimulatory effects on the central nervous, gastrointestinal, cardiovascular, renal, and respiratory systems [3]. The chemical structure of theobromine is shown in Figure1.

Technologies 2017, 5, 14; doi:10.3390/technologies5020014 www.mdpi.com/journal/technologies Technologies 2017, 14, 14 2 of 10 cardiovascular, renal, and respiratory systems [3]. The chemical structure of theobromine is shown Technologiesin Figure 20171. , 5, 14 2 of 10

Figure 1. Chemical structure of theobromine [4]. Figure 1. Chemical structure of theobromine [4].

As of late, few studies on cacao residues have been performed. For example, Arlorio et al. [5] [5] reported that cacao bean hull, principal by-product from from cacao industry, was commonly used as a secondary source of theobromine (1.29 g/100 g g by by dry dry weight), weight), while Hammerstone and Chimel [6] [6] extracted theobrominetheobromine fromfrom cacao cacao solids solids using using various various solvents, solvents, including including isopropanol, isopropanol, methanol, methanol, and andethanol ethanol at different at different temperatures temperatures and and found found that th theat the highest highest theobromine theobromine content content was was obtained obtained by byethanol ethanol at 50 at◦ C.50 Peralta-Jim°C. Peralta-Jiménezénez and Cañizares-Macand Cañizares-Macíasías [7] also [7] used also an used ultrasound-assisted an ultrasound-assisted method withmethod heated with waterheated at water 80 ◦C at for80 the°C for extraction the extracti of theobromineon of theobromine and caffeine and caffeine from from cacao cacao seeds seeds and andchocolate chocolate products, products, and and indicated indicated that that the extractionthe extraction efficiency efficiency of theobromine of theobromine from from cacao cacao seeds seeds and andchocolate chocolate products products increased increased 43.6% 43.6% and 23%, and respectively,23%, respectively, when comparedwhen compared to conventional to conventional stirring stirringextraction extraction methods. methods. Munier Munier and Hartadi and Hartadi [8] reported [8] reported that fresh that cacao fresh podcacao husk pod contained husk contained 0.40% 0.40%theobromine theobromine and it was and greatly it was reducedgreatly reduced during fermentation during fermentation by A. oryzae by, andA. oryzae Adamafio, and [Adamafio9] mentioned [9] mentionedtheobromine theobromine toxicity, theobromine toxicity, theobromine catabolism, andcatabolism, de-theobromine and de-theobromine methods from methods cacao by-products. from cacao by-products.With regard to extraction and purification processes of bioactive compounds, water or organic solvent,With or regard a mixture to extraction of water andand organicpurification solvent, proc hasesses been of usedbioactive to extract compounds, xanthine water alkaloids, or organic such solvent,as caffeine or a and mixture theobromine of water [ 3and,10 ],organic while bleachingsolvent, has earth been and used activated to extract carbon xanthine are usuallyalkaloids, used such to asremove caffeine foreign and theobromine matter in water [3,10], and while oils [ 11bleachin,12]. However,g earth and the activated study on thecarbon proximate are usually composition used to removeof cacao foreign pod husk matter and optimalin water conditions and oils [11,12]. for extraction However, and the purification study on the oftheobromine proximate composition from cacao ofpod cacao husk pod has husk not been and optimal sufficiently conditions reported. for Therefore, extraction this and study purification aimed toof determinetheobromine the from proximate cacao podcomposition husk has ofnot cacao been podsufficiently husk, asreported. well as Theref optimalore, conditions this study foraimed extraction to determine and purificationthe proximate of compositiontheobromine of from cacao cacao pod pod husk, husk as as well an abundant, as optimal inexpensive, conditions renewable,for extraction and and sustainable purification source of theobromineusing a non-toxic, fromaccessible, cacao podand husk cheap as an solvent abundant, for potential inexpensive, application renewable, in the and nutraceutical, sustainable medical, source usingand pharmaceutical a non-toxic, accessible, industries. and cheap solvent for potential application in the nutraceutical, medical, and pharmaceutical industries. 2. Materials and Methods 2. Materials and Methods 2.1. Plant Material 2.1. PlantTwenty Material kilograms of cacao pods (Theobroma cacao trinitario) were purchased from the Western Highlands Agriculture and Forestry Science Institute, Buon Ma Thuot city, Dak Lak province, Vietnam Twenty kilograms◦ of cacao pods (Theobroma◦ cacao trinitario) were purchased from the Western Highlands(latitude 12.6667 AgricultureN, longtitude and Forestry 108.0500 ScienceE). After Institute, collection, Buon cacao Ma pods Thuot were city, packed Dak inLak jute province, bags and Vietnamimmediately (latitude taken 12.6667° to the laboratory N, longtitude at Nha 108.0500° Trang University E). After for collection, further treatments. cacao pods The were fresh packed samples in − ◦ werejute bags then and stored immediately in the freezer taken at to20 theC laboratory until used forat Nha further Trang experiments. University The for overall further experimental treatments. Theprocedure fresh samples is described were in then Figure stored2. in the freezer at –20 °C until used for further experiments. The overall experimental procedure is described in Figure 2.

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FigureFigure 2. 2. OverallOverall experimental experimental procedure procedure of of the the study. study.

2.2.2.2. Chemicals Chemicals AllAll chemicals usedused in in this this research research were were of analyticalof analytical grade. grade. Standard Standard theobromine theobromine (purity (purity 98.5%) 98.5%)was purchased was purchased from Sigma-Aldrich from Sigma-Aldrich Pte Ltd. (Nucleos,Pte Ltd. Singapore).(Nucleos, Singapore). Absolute ethanol, Absolute chloroform, ethanol, chloroform,acetonitrile, acetonitrile, phosphoric acid,phosphoric lead acetate, acid, lead and sodiumacetate, sulfateand sodium were purchasedsulfate were from purchased the Chemical from andthe ChemicalScientiﬁc Technologicaland Scientific Technological Join Stock Company Join Stock (Ho Company Chi Minh (Ho City, Chi Vietnam). Minh City, Vietnam).

2.3.2.3. Preparation Preparation of of Dried Dried Samples Samples FreshFresh cacao cacao pod pod husk husk was was separated separated from from cacao cacao po pods,ds, then then cut cut into into small small pieces, pieces, and and dried dried at at ◦ 4545 °CC to to constant constant weight weight using an infrared drying cabinet (SHN-L, Nha Nha Trang Trang University, University, Vietnam) Vietnam) basedbased on on the the formerly formerly reported reported method method of of Nguyen Nguyen et et al. al. [13]. [13]. The The dried dried samples samples were were packed packed in in sealed sealed plasticplastic bags and and stored stored in a a dry dry place place until until required required.. Prior Prior to to use use for for all all experiments, experiments, the the dried dried samples samples werewere ground to fine fine particles using a hammer mill [[14].14]. 2.4. Analysis of Proximate Composition of Cacao Pod Husk 2.4. Analysis of Proximate Composition of Cacao Pod Husk The moisture, crude protein, crude lipid and ash contents of cacao pod husk were analyzed The moisture, crude protein, crude lipid and ash contents of cacao pod husk were analyzed according to the Association of Official Analytical Chemists (AOAC) official method (1998) and based according to the Association of Official Analytical Chemists (AOAC) official method (1998) and based on the previously-described method by Nguyen [2]. Moisture content was determined using a hot-air on the previously-described method by Nguyen [2]. Moisture content was determined using a hot- oven at 100 ◦C overnight. Nitrogen content was measured using the Kjeldahl method and the crude air oven at 100 °C overnight. Nitrogen content was measured using the Kjeldahl method and the protein content was then calculated by multiplying nitrogen content by a factor of 6.25. Crude lipids crude protein content was then calculated by multiplying nitrogen content by a factor of 6.25. Crude were extracted using the Soxhlet method and the crude lipid content was determined after oven-drying lipids were extracted using the Soxhlet method and the crude lipid content was determined after the extract at 100 ◦C for 30 min. Ash content was measured by heating the cacao pod shells in a furnace oven-drying the extract at 100 °C for 30 min. Ash content was measured by heating the cacao pod at 600 ◦C for 5 h. Carbohydrates (include crude fiber) was calculated from Equations (1) and (2) [15] shells in a furnace at 600 °C for 5 h. Carbohydrates (include crude fiber) was calculated from as follows: Equations (1) and (2) [15] as follows:

CarbohydrateCarbohydrate (% by (% fresh by fresh weight) weight) = 100 = 100− −moisture moisture −− crudecrude protein protein − crude− crude lipid lipid − ash− ash(1) (1)

CarbohydrateCarbohydrate (% by(% dryby dry weight) weight) = 100= 100− − crudecrude protein −− crudecrude lipid lipid − ash− ash(2) (2)

2.5. Extraction of Theobromine from Cacao Pod Husk 2.5. Extraction of Theobromine from Cacao Pod Husk The extracts from cacao pod husk were prepared according to the prior methods of Nguyen [2] withThe some extracts minor from modiﬁcations. cacao pod Brieﬂy,husk were to select prepared the most according effective to the solvent, prior 10methods g of dried of Nguyen cacao pod [2] withhusk some was extractedminor modifications. with 270 mL Briefly, of various to select solvents the most for 30effective min at solvent, temperatures 10 g of ofdried 100 cacao◦C, 80 pod◦C, huskand 60was◦C extracted for water, with 70% 270 ethanol mL of and various chloroform, solvents respectively, for 30 min usingat temperatures a Memmert of WB29100 °C, thermostatic 80 °C, and 60bath °C (Memmertfor water, 70% GmbH ethanol + Co.KG, and chloroform, Schwabach, respectively, Germany), which using wasa Memmert closely coveredWB29 thermostatic by a lid to avoid bath (Memmertwater evaporation GmbH + during Co.KG, extraction. Schwabach, Different Germany), extraction which was times closely and the covered number by a of lid extractions to avoid water were evaporationthen applied during with selectedextraction. solvent Different to identify extraction optimal times extraction and the number time and of numberextractions of extractions.were then applied with selected solvent to identify optimal extraction time and number of extractions. After extraction, the extracts were immediately cooled to room temperature using an ice water bath, and

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Afterthen filtered extraction, through the extracts a Whatman were No. immediately 1 filter paper cooled to toobtain room the temperature crude extracts using for an further ice water analysis bath, and thenexperiments. filtered through a Whatman No. 1 filter paper to obtain the crude extracts for further analysis and experiments. 2.6. Purification of Crude Extracts from Cacao Pod Husk 2.6. Purification of Crude Extracts from Cacao Pod Husk Purification of the crude extracts from cacao pod husk containing theobromine is to remove foreignPurification matters (protein, of the crude lipid, extractspectin, tannins, from cacao pigm podents, husk etc.) containingwithin the crude theobromine extracts isfor to obtaining remove foreignthe pure matters extracts (protein, or theobromine-enriched lipid, pectin, tannins, extracts. pigments, Briefly, etc.) within10% lead the acetate crude extractssolution for at obtaining different theconcentrations pure extracts of or2.5%, theobromine-enriched 5.0%, 7.5%, 10.0%, and extracts. 12.5% Briefly,by volume 10% was lead added acetate to solution 40 mL of at the different crude concentrationsextracts. The mixture of 2.5%, was 5.0%, then 7.5%, left at 10.0%, room andtemper 12.5%ature by (32 volume ± 2 °C) was for added15 min. to After 40 mL treatment of the crude with extracts.10% lead The acetate mixture solution, was thenthe residual left at room lead temperatureacetate in the (32 extracts± 2 ◦C) was for completely 15 min. After removed treatment by using with 10%10% leadsodium acetate sulfate solution, solution the (based residual on leadformation acetate of inwhite the extractsprecipitate was named completely as lead removed sulfate) byand using then 10%filtered sodium through sulfate a Whatman solution(based No. 1 filter on formation paper to of obtain white the precipitate pure extracts. named These as lead pure sulfate) extracts and were then filteredthen measured through viscosity a Whatman using No. a 1 viscometer filter paper LVDV-E to obtain (Brookfield, the pure extracts. NY, USA) These at pure a speed extracts of 60 were rpmthen and measuredspindle S03, viscosity and absorbance using a viscometer at 436 nm LVDV-E using (Brookfield, a Cary 50 NY,UV-VIS USA) spectrophotometer at a speed of 60 rpm (Varian, and spindle Inc., S03,Manasquan, and absorbance NJ, USA). at The 436 nmresults using of viscosity a Cary 50 and UV-VIS absorbance spectrophotometer at 436 nm were (Varian, expressed Inc., as Manasquan, centipoise NJ,(cP) USA). and absorbance The results unit, of viscosity respectively. and absorbance The best pure at 436 extract nm were obtained expressed after aspurification centipoise was (cP) andalso absorbanceanalyzed theobromine unit, respectively. content to The evaluate best pure the extracteffectiveness obtained of the after purification purification process. was also analyzed theobromine content to evaluate the effectiveness of the purification process. 2.7. Analysis of Theobromine Content 2.7. Analysis of Theobromine Content Theobromine content in the extracts from cacao pod husk was analyzed using HPLC system (Shimadzu,Theobromine Kyoto, contentJapan) based in the on extracts the previously from cacao developed pod husk method was analyzed of Nguyen using et al. HPLC [13] systemBriefly, (Shimadzu,the extracts Kyoto,and standard Japan) theobromine based on the solution previously (100 developed µg/mL in methodmethanol) of were Nguyen filtered et al. through [13] Briefly, 0.45 theµm extractsnylon membranes, and standard and theobromine 20 µL were solution then (100individuallyµg/mL in injected methanol) by werean auto filtered injector through (Shimadzu, 0.45 µm nylonKyoto, membranes, Japan) onto a and column 20 µL C were18 (250 then × 4.6 individually mm 5 µm; Shimadzu, injected by Kyoto, an auto Japan), injector which (Shimadzu, was maintained Kyoto, ◦ Japan)at 35 °C onto by a columna column C18 oven(250 × (Shimadzu,4.6 mm 5 µ m;Kyoto, Shimadzu, Japan). Kyoto, The Japan),isocratic which elut wasion maintainedconsisted of at 0.05% 35 C byphosphoric a column acid oven in (Shimadzu, distilled water Kyoto, (A) Japan). and 100% The acetonitrile isocratic elution (B) (85:15). consisted Flow of rate 0.05% was phosphoric set at 1 mL/min. acid in distilledThe theobromine water (A) compound and 100% acetonitrile was detected (B) (85:15).at 272 nm Flow using rate wasa UV-VIS set at 1detector mL/min. (Shimadzu, The theobromine Kyoto, compoundJapan). Theobromine was detected content at 272 in nm the using extracts a UV-VIS from detector cacao pod (Shimadzu, husk was Kyoto, quantified Japan). Theobrominebased on the contentcalibration in the curve extracts of standard from cacao theobromine pod husk wasby comparing quantified basedretention on thetime calibration and peak curve area of standard theobrominetheobromine bywith comparing those in retentionthe extracts time from and peakcacao area pod of husk. standard The theobrominecalibration curve with thoseof standard in the extractstheobromine from cacao(concentration pod husk. ranged The calibration from 0 to curve 5 mg/L of) standard is indicated theobromine in Figure (concentration 3. Limit of detection ranged from(LOD) 0 toand 5 mg/L)limit of is quantification indicated in Figure (LOQ)3. Limitof standard of detection theobromine (LOD) and were limit found of quantification to be 0.10 and (LOQ) 0.31 ofmg/L, standard respectively. theobromine were found to be 0.10 and 0.31 mg/L, respectively.

Figure 3. Calibration curve of standard theobromine.

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2.8. Statistical Analysis All experiments were run in triplicate. The data were analyzed using SAS software (version 9.2, SAS Inst., Inc., Cary, NC, USA) and expressed as the mean ± standard deviation (n = 3). Statistical comparisons were made using analysis of variance (ANOVA) and t-tests (LSD). Differences were considered signiﬁcantly when p-values were below 0.05 (p < 0.05).

3. Results and Discussion

3.1. Proximate Composition of Cacao Pod Husk The proximate composition of cacao pod husk was calculated based on fresh weight and dry weight (Table1) to use for different purposes. The results showed that cacao pod husk had high contents of moisture and carbohydrates (87.06% and 11.03% by fresh weight, respectively), but low contents of crude proteins, crude lipids, and ash (0.31%, 0.12%, and 1.48% by fresh weight, respectively). Based on dry weight, the contents of crude protein, crude lipid, ash, and carbohydrate were of 2.42%, 0.93%, 11.44%, and 85.21% by dry weight, respectively, indicating that cacao pod husk contains high carbohydrate content which forms a ﬁrm structure for its husk. The moisture content in cacao pod husk found in this study was similar to that in cacao pod shell (87.0%), but the contents of crude protein, crude lipid, and ash in cacao pod husk were lower than those in cacao pod shell (1.26%, 0.16%, 2.30% by fresh weight and 9.69%, 1.23%, and 17.69% by dry weight, respectively), and carbohydrate content in cacao pod husk was higher than that in cacao pod shell (9.28% by fresh weight and 71.39% by dry weight, respectively) [2]. Marcel et al. [16] indicated that crude protein content in cacao pod shell, cacao pod husk, and cacao bean hull (16.0%, 9.14%, and 17.9% by dry weight, respectively) was much higher than that in cacao pod husk found in this study, whereas ash content in cacao pod shell, cacao pod husk and cacao bean hull (7.5%, 9.07%, and 9.3% by dry weight, respectively) was lower than that in cacao pod husk found in this study. Arlorio et al. [5] reported that moisture, the contents of crude lipid, crude protein and ash in pre-roasted cacao bean hull were of 10.12%, 6.81%, 18.12%, and 8.1% by dry weight, respectively. These results showed that proximate composition in different parts of cacao pod was greatly different that directly relates to structural characteristics of cacao pod, in that cacao pod husk is a main part against insects and harmful effects from the environment in terms of physical, chemical, and microbial agents.

Table 1. Proximate composition of cacao pod husk.

Proximate Composition Fresh Weight (%) Dry Weight (%) Moisture 87.06 ± 0.58 * - Crude protein (protein factor: 6.25) 0.31 ± 0.21 2.42 ± 0.37 Crude lipid 0.12 ± 0.12 0.93 ± 0.34 Ash 1.48 ± 0.31 11.44 ± 0.41 Carbohydrate (include crude ﬁber) 11.03 ± 0.21 85.21 ± 0.29 * Means and standard deviations were of triplicate.

3.2. Effect of Extraction Conditions on Theobromine Content from Cacao Pod Husk

3.2.1. Effect of Various Solvents on Theobromine Content Theobromine has been known as the most valuable compound in cacao and cacao products. Figure4 illustrates HPLC chromatograms of standard theobromine (Figure4A) and theobromine in the crude extract from cacao pod husk (Figure4B). Theobromine content from cacao pod husk extracted by various solvents is indicated in Figure5. Among three solvents tested, 70% ethanol obtained the greatest level of theobromine (2.23 mg/100 g dry weight) as compared to those extracted by water and chloroform (0.09 and 0.35 mg/100 g dry weight), revealing that theobromine had a limited dissolvability in water, which Technologies 2017, 5, 14 6 of 10 is considered as a “green” solvent. This greatly related to the intermediate polarity of 70% ethanol, which allows it to solvate theobromine with low molecular weight containing functional groups (–C=O and –CH3)[17]. Hu et al. [10] reported that 95% theobromine and 99% caffeine could be recovered by using a chloroform-water system, while Kasabe and Badhe [4] recovered theobromine in tea samples Technologies 2017, 14, 14 6 of 10 usingTechnologies various 2017 organic, 14, 14 solvents and aqueous mixtures and found that an increase of recovery6 of in 10 order by n-hexane,recovery ethylin order acetate, by n-hexane, methylene ethyl acetate, dichloride, methylene chloroform, dichloride, methanol, chloroform, water, methanol, 5% sulphuric water, 5% acid in recovery in order by n-hexane, ethyl acetate, methylene dichloride, chloroform, methanol, water, 5% water,sulphuric and 5% acid diethyl in water, amine and in 5% water, diethyl with amine a maximum in water, with level aof maximum theobromine level of was theobromine found to bewas 2.31% sulphuric acid in water, and 5% diethyl amine in water, with a maximum level of theobromine was by dryfound weight. to be This 2.31% data by illustrateddry weight. that This the data extractability illustrated that of theobrominethe extractability was of greatly theobromine affected was by the found to be 2.31% by dry weight. This data illustrated that the extractability of theobromine was charactergreatly of affected the solvent. by the character of the solvent. greatly affected by the character of the solvent.

Figure 4. HPLC chromatograms of standard theobromine (A) and theobromine in the crude extract FigureFigure 4. HPLC 4. HPLC chromatograms chromatograms of of standard standard theobrominetheobromine (A (A) )and and theobromine theobromine in the in thecrude crude extract extract from cacao pod husk (B). fromfrom cacao cacao pod pod husk husk (B). (B).

Figure 5. Effect of solvents on theobromine content from cacao pod husk. Different letters (a, b, c) Figure 5. Effect of solvents on theobromine content from cacao pod husk. Different letters (a, b, c) Figureindicated 5. Effect significant of solvents differences on theobromine between treatments content (p from < 0.05). cacao pod husk. Different letters (a, b, c) indicatedindicated signiﬁcant significant differences differences between between treatments treatments ((p < 0.05). 0.05). Of these, 70% ethanol was selected for further extraction of theobromine from cacao pod husk, Of these, 70% ethanol was selected for further extraction of theobromine from cacao pod husk, which is inexpensive, accessible, and friendly to humans and the environment. which is inexpensive, accessible, and friendly to humans and the environment.

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Of these, 70% ethanol was selected for further extraction of theobromine from cacao pod husk, whichTechnologies is inexpensive, 2017, 14, 14 accessible, and friendly to humans and the environment. 7 of 10

3.2.2. Effect of Extraction TimeTime andand NumberNumber ofof ExtractionsExtractions onon TheobromineTheobromine ContentContent Figure6 6 indicates indicates the the effecteffect ofof differentdifferent extraction extraction timestimes andand numbernumber ofof extractionsextractions onon theobrominetheobromine contentcontent from cacao cacao pod pod husk. husk. Of Of which, which, theobromine theobromine content content was was significantly significantly higher higher when when cacao cacao pod podhusk husk was wasextracted extracted by 70% by 70%ethanol ethanol at longer at longer times times (2.23%, (2.23%, 3.29%, 3.29%, and 5.98 and mg/100 5.98 mg/100 g dry weight g dry weight for 30, for60, 30,and 60, 90 and min, 90 respectively). min, respectively). However, However, theobr theobromineomine content content was not was sign notificantly significantly different different when whennumber number of extractions of extractions increased increased from 1 time from to 12 ti timemes to(5.98 2 times and 6.06 (5.98 mg/100 and 6.06 g dry mg/100 weight, g respectively). dry weight, respectively).This finding is Thissupported finding by is Nguyen supported and by Pham Nguyen [14] and PhamNguyen [14 [2]] and who Nguyen reported [2 ]that who the reported extract thatconcentration the extract from concentration different parts from of different artichoke parts plant of (leaf, artichoke stalk, root, plant and (leaf, flower) stalk, and root, pigment and flower) yield andfrom pigment cacao pod yield shell from attained cacao podan equilibrium shell attained at extraction an equilibrium times at of extraction 60 and 80times min, respectively, of 60 and 80 min,and respectively,Tan et al. [18] and also Tan found et al. [ 18that] also and found the thatphenolic and theextraction phenolic efficiency extraction from efficiency bitter from melon bitter was melon not wassignificantly not significantly different different after an after extraction an extraction time timeof 5of min 5 min and and one one extraction. extraction. Considering Considering thethe productionproduction costs,costs, longerlonger extractionextraction times and more numbernumber of extractionsextractions consume more energy for extraction andand remove remove solvent solvent after after extraction, extraction, leading leading to higher to higher production production costs, this costs, is an this important is an factorimportant and needsfactor toand be needs properly to be considered properly consider when selectinged when extraction selecting time extraction and number time and of extractions.number of Therefore,extractions. an Therefore, extraction an time extraction of 90 min time and of one 90 asmi then and number one as of the extractions number of were extractions suitable forwere extraction suitable offor theobromine extraction of from theobromine cacao pod from husk. cacao pod husk.

Figure 6.6. EffectEffect ofof differentdifferent extractionextraction timestimes andand numbernumber ofof extractionsextractions onon theobrominetheobromine contentcontent fromfrom cacaocacao podpod husk.husk. Different letters (a, b, c) indicatedindicated signiﬁcantsignificant differences between treatments (p < 0.05).

3.3. Effect of PurificationPurification onon PhysicochemicalPhysicochemical PropertiesProperties ofof ExtractsExtracts andand TheobromineTheobromine ContentContent fromfrom CacaoCacao PodPod HuskHusk InIn thisthis study, study, lead lead acetate acetate was was used used as an as agent an ag toent remove to remove foreign foreign matter matter within thewithin crude the extracts crude fromextracts cacao from pod cacao husk pod including husk including protein, lipid,protein, pectin, lipid, tannins, pectin, pigments, tannins, pigments, and so on and due so to bleachingon due to earthbleaching and earth activated and carbonactivated could carbon absorb could greatly absorb theobromine greatly theobromine in the extracts, in the extracts, leading leading to the loss to the of theobromine.loss of theobromine. The effect The of 10%effect lead of acetate10% lead solution acetate concentration solution concentration on the viscosity on andthe absorbanceviscosity and of theabsorbance extracts of from the cacaoextracts pod from husk cacao is shown pod husk in Figure is shown7. In in this Figure instance, 7. In this the viscosityinstance, ofthe the viscosity extracts of wasthe extracts significantly was significantly reduced at a reduced concentration at a concentration of 2.5% by volume of 2.5% of by 10% volume lead acetate of 10% solution lead acetate (4.77 tosolution 3.90 cP), (4.77 but to no3.90 significant cP), but no difference significant in differ the absorbanceence in the absorbance of the extracts of the was extracts observed was (1.87observed and 1.83(1.87 absorbance and 1.83 absorbance units). However, units). when However, the concentration when the concentration of 10% lead acetate of 10% solution lead acetate increased solution from increased from 2.5% to 5.0% by volume, both the viscosity and absorbance of the extracts were significantly decreased (3.90 to 3.63 cP and 1.83 to 0.37 absorbance unit, respectively), and then they were significantly reduced with an increase of the concentration of 10% lead acetate solution from 5.0% to 7.5% by volume (3.63 to 3.54 cP and 0.37 to 0.30 absorbance units, respectively). The

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2.5% to 5.0% by volume, both the viscosity and absorbance of the extracts were significantly decreased (3.90 to 3.63 cP and 1.83 to 0.37 absorbance unit, respectively), and then they were significantly reduced Technologieswith an increase 2017, 14, 14 of the concentration of 10% lead acetate solution from 5.0% to 7.5% by volume8 of 10 (3.63 to 3.54 cP and 0.37 to 0.30 absorbance units, respectively). The absorbance of the extracts was absorbancecontinuously of reduced the extracts when was the concentrationcontinuously ofredu 10%ced lead when acetate the solutionconcentration rose from of 10% 7.5% lead to 10.0% acetate by solutionvolume, rose whereas from the 7.5% viscosity to 10.0% of by the volume, extracts wherea was nots the significantly viscosity of different. the extracts These was resultsnot significantly indicated different.that the concentration These results ofindicated 10.0% by that volume the concen of 10%tration lead acetateof 10.0% solution by volume was theof best10% selectionlead acetate for solutionpurification was of the the best extracts selection from cacaofor purification pod husk. Underof thethe extracts extraction from conditions cacao pod of husk. 70% ethanol, Under onethe extraction conditions for 90 min, of and 70% purification ethanol, one with extraction a concentration for 90 min, of and 10.0% purification by volume with of 10% a concentration lead acetate ofsolution, 10.0% theobromineby volume of content 10% lead achieved acetate 6.79 solution mg/100, theobromine g dry weight, content which wasachieved higher 6.79 than mg/100 that without g dry weight,purifying which by 10% was lead higher acetate than solution that without (5.58 mg/100 purifying g dry by weight).10% lead This acetate is due solution to the effect(5.58 ofmg/100 foreign g drymatter weight). present This in theis due extracts, to the leadingeffect of to foreign a decrease matter in present the detectability in the extracts, of theobromine leading to in a thedecrease extracts in theby HPLCdetectability analysis. of theobromine Howlader et in al. the [19 extr] andacts Reddy by HPLC and analysis. Lakshmi Howlader [20] also used et al. 10% [19] leadand acetateReddy andsolution Lakshmi as an [20] agent also to used detect 10% the lead presence acetate of solution tannins inas thean agent methanolic to detect extract the presence from Diospyros of tannins blancoi in theleaf methanolic and phenolic extract compounds from Diospyros in the extract blancoi from leaf the and whole phenolic plant compounds of Oxalis corniculata in the extractL, based from on the wholeformation plant of of white Oxalis precipitate corniculata in L, the based end-products. on the formation of white precipitate in the end-products.

Figure 7. Effect of concentration of 10% lead acetate solutionsolution on viscosity and absorbance of the extracts fromfrom cacao cacao pod pod husk. husk. Different Different letters letters (a, (a, b, b, c, c, d, d, A, B,B, BC,BC, C)C) indicatedindicated signiﬁcantsignificant differencesdifferences between treatmentstreatments ( (pp << 0.05). 0.05).

By applying the conventional extraction techni techniqueque in in this this study, study, theobromine content obtained from cacao podpod huskhusk waswas lower lower than than that that found found in in cacao cacao bean bean hull hull (1.29 (1.29 g/100 g/100 g g dry dry weight) weight) [5 ][5] and and in intea tea samples samples (2.31 (2.31 g/100 g/100 g g dry dry weight) weight) [ [4].4]. ThisThis outcomeoutcome revealedrevealed that although theobromine content inin cacao pod huskhusk isis lowerlower thanthan thatthat inin somesome other other materials, materials, a a very very large large amount amount of of cacao cacao pod pod husk husk is isdiscarded discarded from from the the cacao cacao processing processing industry, industry, approximately approximately 5555 millionmillion tonnestonnes per year. Therefore, Therefore, cacao pod husk is an inexpensive, renewable, and sustainable source source for for extraction extraction of of theobromine, theobromine, with an estimation about 457.1 tonnes per year. To increase the extraction efficiency efﬁciency of theobromine and reduce extraction time, further research can be applied some advanced extraction techniques, techniques, such asas ultrasound-assistedultrasound-assisted extraction extraction [17 [17],], microwave-assisted microwave-assisted extraction extraction [21 ],[21], and supercriticaland supercritical ﬂuid fluidextraction extraction [22]. [22].

4. Conclusions This study was an exploratory and valuable assessment for further studies. The findings from this study indicated that cacao pod husk had high contents of moisture and carbohydrates, but low contents of crude protein, crude lipid, and ash. The extraction and purification processes greatly influenced on theobromine content from cacao pod husk, particularly solvent, extraction time, and concentration of 10% lead acetate solution. Therefore, the use of these extraction and purification conditions for effective exploitation of theobromine from cacao pod husk is a promising and

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4. Conclusions This study was an exploratory and valuable assessment for further studies. The findings from this study indicated that cacao pod husk had high contents of moisture and carbohydrates, but low contents of crude protein, crude lipid, and ash. The extraction and purification processes greatly influenced on theobromine content from cacao pod husk, particularly solvent, extraction time, and concentration of 10% lead acetate solution. Therefore, the use of these extraction and purification conditions for effective exploitation of theobromine from cacao pod husk is a promising and sustainable trend. In further studies, it is necessary to determine the biological variability of theobromine in cacao pod husk samples, as well as produce theobromine-enriched powder and evaluate its biological activity in various models for potential application in the medical and pharmaceutical industries.

Acknowledgments: We sincerely acknowledge Nha Trang University, Vietnam for financial and mechanical support. The authors also would like to kindly thank Huu Tuan Nguyen and Thi Be Phan for their experimental support. Author Contributions: Van Tang Nguyen conceived and designed the experiments, performed the experiments, analyzed the data, and wrote the paper. Nghia Huu Nguyen reviewed and suggested the paper. Conflicts of Interest: The authors declare no conflict of interest.

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