Journal of ISSN: 2377-4282JNMR

Nanomedicine Research
Research Article
Volume 4 Issue 1 - 2016
Pequiá-Based Nanoemulsion Highlights an Important Amazon Fruit (Caryocar villosum (Aubl.) Pers.)
Jacqueline da S Leal1, Jonatas L Duarte2,3, Jessica CE Vilhena3, Alexandro C Florentino4, Didier Bereau1, Jean-Charles Robinson1, José CT Carvalho2,3, Rodrigo AS Cruz2,3, Caio P Fernandes2,3*, Anna EMFM Oliveira2,3
1UMR Qualitrop, Université de Guyane, France
2Laboratorio de Nanobiotecnologia Fitofarmaceutica, Universidade Federal do Amapá, Brazil
3Laboratorio de Pesquisa em Fármacos, Universidade Federal do Amapá, Brazil
4Laboratorio de Absorção Atômica e Bioprospecção, Universidade Federal do Amapá, Brazil
Received: May 12, 2016 | Published: August 01, 2016
*Corresponding author: Caio Pinho Fernandes, Laboratório de Nanobiotecnologia Fitofarmacêutica- Colegiado de Farmácia, Universidade Federal do Amapá, Macapá, AP, Brazil, Tel: 055(96)4009-2927, Email:
Citation: Leal JDS, Duarte JL, Vilhena JCE, Florentino AC, Bereau D, et al. (2016) Pequiá-Based Nanoemulsion Highlights an Important Amazon Fruit (Caryocar villosum (Aubl.) Pers.). J Nanomed Res 4(1): 00079. DOI: 10.15406/jnmr.2016.04.00079


The fruits from pequiá (Caryocar villosum) are an important source of antioxidant substances. However, despite the great potential of this natural product for pharmaceutical and food industries, to our knowledge, no study was carried out in order to obtain novel nanoformulation with this raw material. Our results suggested that required Hydrophyle-Lipophile Balance (HLB) of pequiá oil is around 12.0 and nanoemulsion with low mean droplet size (191.3 ± 0.8 nm) and Polydispersity Index (0.290 ± 0.040) was obtained. Thus, our studies contribute to valorization of an Amazon fruit, providing for the first time valuable information about nanoemulsion formation using pequiá fruits oil.

Keywords: Fruits; HLB; Natural oil; Non-ionic surfactants


HLB: Hydrophyle-Lipophile Balance; RHLB: Required Hydrophile-Lipophile Balance; PI: Polydispersity Index; S: Surfactant


The genus Caryocar belongs to the family Caryocaracea and comprehends 16 arboreous species. Their fruits are used as food and medicine, being also considered potentially useful for cosmetic industry [1]. Caryocar villosum (Aubl.) Pers. is widely distributed in the North region of Brazil, being commonly known as pequiá [2], piqui [3] or piquiá [4]. Fruits from C. villosum are very characteristic and used as raw material for extraction of yellow oil that is used in culinary and has great biological potential. Several substances were identified on fruits of C. villosum, including oleic acid, steroids and volatile substances, such as β-bisabolene and (E)-nerolidol [5]. Several antioxidants substances were also reported for this fruits, including phenolic compounds and carotenoids [3-7]. They scavenge reactive oxygen and nitrogen species and therefore may be useful to promote human health [4-7]. Moreover, piquiá fruits developed a main role in gene expressions that are related to oxidative stress and generation of free radicals [8]. More polar substances such as saponins were also found in fruits [9] and stem barks [10] of C. villosum.

Nanotechnology is a growing multidisciplinary area that is considered promising for development of novel products for pharmaceutical industry, including nanoemulsions [11]. Nanoemulsions are dispersed systems constituted by two immiscible liquids and are often stabilized by surfactants [12,13] presenting droplets with mean diameter between 30-300 nm [14]. The small size of these droplets is associated to kinetic stability of nanoemulsions during storage, preventing gravitational separation or particle aggregation. They also have a typical translucent or transparent aspect with bluish reflect [15-17]. They are very promising to enhance water solubility of vegetal oils and improve chemical stability and bioavailability of bioactive substances. On this context, several studies aiming to obtain nanoemulsions were carried out with important oils from natural origin [18-23]. However, despite great biological potential of Caryocar villosum fruits, to our knowledge, no study was carried out aiming to obtain nanoemulsions using its oil. Therefore, the present study presents a novel pequiá oil-based nanoemulsion with great potential for cosmetics, nutraceuticals and phytopharmaceuticals.

Material and Methods


Surfactants were obtained from Praid (SP, Brazil) and solvent (analytical grade) were obtained from Vetec (RJ, Brazil).

Extraction of C. villosum fruits

Fresh pulp (30 g) of C. villosum was extracted with 150 ml of hexane using a Soxhlet apparatus during 4 hours. After this period, solvent was removed under reduced pressure using a rotary evaporator with water bath (40 oC). Pequiá extract was stored under controlled temperature (4 oC) and protected from light.

Emulsification method

The preparation of C. villosum emulsions was carried out according to a previous described method [21]. The oily phase was composed by C. villosum extract and surfactants (1:1) and was heated at 65 ± 5 °C. Aqueous phase, constituted by distilled water was individually heated to the same temperature of oily phase. Then, water was titrated through water phase using a burette under constant magnetic stirring (800 rpm), obtaining a primary emulsion. Further homogenization step was carried out using a high speed homogenizer Ultra-turrax T25 IKA during 5 min at 8000 rpm for droplet size reduction.

Determination of required Hydrophile-Lipophile Balance (RHLB) Of C. villosum extract

Series of emulsions was prepared by ranging sorbitan monooleate: polysorbate 80 ratios. Therefore, different HLB values were achieved from 4.3 (100% of sorbitan monooleate) to 15.0 (100% of polysorbate 80). All emulsions were prepared as follows: 5% of pequiá extract (w/w), 5% of surfactant (s) (w/w) and 90% of water (w/w).

Characterization of droplet size distribution

Emulsions were characterized by dynamic light scattering using a Zetasizer-nano (Malvern Instruments, UK). They were diluted using deionized water (1:25). Droplet size and Polydispersity Index are represented as mean ± standard deviation in Figure 1.

Figure 1: A: Day 1-mean droplet size: 276.6 ± 3.8 / pdi: 0.398 ± 0.000; B: Day 7-mean droplet size: 191.3 ± 0.8 / pdi: 0.290 ± 0.040.

Statistical analysis

The significance of the results was analyzed using One-way Anova with 95% of confidence interval) and Tukey´s test, using Software R (R Core Team, 2013). Differences among mean droplet diameters were considered significant when p < 0.05.

Results and Discussion

Table 1 shows mean droplet size and Polydispersity Index of emulsions prepared with pequiá extract at different HLB values. Most of them presented instable behavior, such as creaming and phase separation (HLB 4.3-10 and 13-15). Among this first set of emulsions, two of them (HLB 11 and 12) presented a remarkable fine aspect with bluish reflect, characteristic for nanoemulsions. Analysis of mean droplet size after 1 day of preparation confirmed sub-micrometer diameter (HLB 11: 239.7 ± 4, 7 and HLB 12: 276.6± 3.8), however, relative broad distribution was confirmed by Polydispersity Index (HLB 11: 0.438 ± 0.011; EHL 12: 0.398 ± 0.000). Additional emulsion within this range was prepared in order to refine the HLB value determination (10.75; 11.25; 11.5; 11.75 and 12.25). After 1 day, emulsions at HLB 10.75 and 11.5 presented mean droplet size above 300 nm and emulsions at HLB 11.25, 11.75 and 12.25 presented mean droplet size below 300 nm, being considered nanoemulsions. After 1 day of preparation, lowest mean droplet size was observed for nanoemulsion at HLB 12.25, considering all prepared emulsions (198.7 ± 3.729 nm), in addition to low Polydispersity Index. After 7 days, most of them had an augmentation in droplet size, which is not expected for high stable systems [24]. During storage, increase on droplet size around 30 to 85% was observed for carotenoid-rich emulsions [25]. This class of secondary metabolite is predominant on C. villosum and several carotenoids were identified on the fruits, such as lutein-like and carotene-like substances, antheraxanthin, zeaxanthin, neoxanthin and others [7]. Despite slight change was observed for nanoemulsions prepared with surfactants at HLB 11, higher Polydispersity Index was observed, suggesting a polimodal distribution that is not expected for stable nanoformulation. Lower Polydispersity indices are associated to narrow distribution and polimodal distribution may be an indicative of system instable behavior [26-28]. Considering all formulations, the nanoemulsion prepared at HLB 11.0 presented the smallest mean droplet size (191.3 ± 0.8 nm) after the storage (p < 0.001) and low Polydispersity Index. This last parameter reflects homogeneity of particle size distribution and values below 0.400 are associated to more stable systems [26]. A study carried out with carotenoid-based nanodispersions and using volatile organic solvent revealed a reduction in droplet size, which was associated to solvent diffusion from internal to external phase [29]. Considering the analyzed parameter and the fact that nanoemulsion prepared with surfactants at HLB 11.0 had a tendency after 7 days to achieve a monomodal distribution with mean droplet size below 200 nm, we suggest that this is the required hydrophilic lypophile of pequiá extract used in the present study.


Day 1

Day 7

Mean Droplet (nm ± SD)

Polydispersity Index

Mean Droplet (nm ± SD)

Polydispersity Index


307.2 ± 4.3

0.711 ± 0.031

350.1 ± 22.0

0.664 ± 0.129


239.7± 4.7

0.438 ± 0.011

237.6 ± 5.19

0.430 ± 0.017


216.7 ± 1.4

0.387 ± 0.025

231.3 ± 12.3

0.409 ± 0.009


329.9 ± 6.1

0.630 ± 0.009

344.1 ± 12.0

0.583 ± 0.099


278.3 ± 3.1

0.507 ± 0.099

287.9 ± 8.0

0.488 ± 0.068


276.6± 3.8

0.398 ± 0.000

191.3 ± 0.8

0.290 ± 0.040


198.7 ± 3.7

0.285 ± 0.026

233.3 ± 2.1

0.401 ± 0.002

Table 1: Particle size distribution of nanoemulsions prepared with C. villosum oil.

*Required Hydrophyle-Lipophile Balance of C. villosum oil
All emulsions were prepared with 5% of pequiá extract (w/w), 5% of surfactant (s) (w/w) and 90% of water (w/w).

HLB was primarily defined as a semi-empirical scale for selecting surfactants according to its hydrophilicity or lipophilicity [30]. Further studies were carried out by preparing a set of emulsions using a wide range of surfactant blend. Since each surfactant mixture has a typical HLB value, the concept of rHLB of oil could be stated. Among the set of emulsions prepared with surfactant (s) at different HLB values, the rHLB of oil can be determined considering that it should be the HLB of surfactant or surfactant mixture that allowed achievement of most stable emulsion [31]. Considering that small droplets, including in the range of nanoemulsions, are intrinsically associated to enhancement of physical stability, it is worth mentioning that rHLB determination is a useful tool to generate nanoemulsions. On this context, several studies with this approach obtained plant-based nanoemulsions using natural oils [18,20-23,32,33].


Oils from fruits are valuable source of bioactive compounds. Several of them were subjected to studies aiming to generate novel nanoformulation, such as nanoemulsions. However, studies regarding Amazon species are scare. On the present study, we successfully determined required HLB value of pequiá oil and obtained nanoemulsion with good physical properties. Thus, we provide valuable contribution to nanobiotechnology and valorization of an important species that can be sustainable used to generate novel nanoemulsions for industry.


  1. Ascari J, Takahashi JA, Boaventura MAD (2013) The Photochemistry and Biological Aspects of Caryocaraceae Family. Rev Bras Pl Med 15 (2): 293-308.
  2. Medeiros H, Amorim AMA (2016) “Caryocaraceae”, in Lista De Espécies Da Flora Do Brasil, Brazil.
  3. Godoy HT, Rodriguez-Amaya DB (1994) Occurrence of Cis-Isomers of provitamin A in Brazilian fruits. J Agric Food Chem 42(6): 1306-1313.
  4. Chisté RC, Benassi MT, Mercadante AZ (2014) Efficiency of Different Solvents on the Extraction of Bioactive Compounds from the Amazonian Fruit Caryocar villosum and the Effect on its Antioxidant and Colour Properties. Phytochem Anal 25(4): 364-372.
  5. Marx F, Andrade EHA, Maia JG (1997) Chemical composition of the fruit pulp of Caryocar villosum. Z Lebensm Unters Forsch A 204(6): 442-44.
  6. Chisté RC, Freitas M, Mercadante AZ, Fernandes E (2012) The potential of extracts of Caryocar villosum pulp to scavenge reactive oxygen and nitrogen species. Food Chem 135(3): 1740-1749.
  7. Chisté RN, Mercadante AZ (2012) Identification and Quantification, by HPLC-DAD-MS/MS, of Carotenoids and Phenolic Compounds from the Amazonian Fruit Caryocar villosum. J Agric Food Chem 60(23): 5884-5892.
  8. Almeida MR, Aissa AF, Darim JDC, Gomes TDUH, Chisté RC, et al. (2012) Free Radical Biology and Medicine 53: S82.
  9. Magid AA, Nazabadioko L, Harakat D, Pouny I, Caron C, et al. (2006) Triterpenoid Saponins from the Fruits of Caryocar villosum. J Nat Prod 69(6): 919-926.
  10. Magid AA, Voutquenne-Nazabadioko L, Renimel I, Harakat D, Moretti C, et al. (2006) Triterpenoid saponins from the stem bark of Caryocar villosum. Phytochemistry 67(19): 2096-2102.
  11. Sutradhar KB, Amin L (2013) Nanoemulsions: increasing possibilities in drug delivery. Eur J Nanomed 5(2): 97-110.
  12. McClements DJ (2011) Edible nanoemulsions: fabrication, properties, and functional performance. Soft Matter 7(6): 2297–2316.
  13. Fryd MM, Mason TG (2012) Advanced Nanoemulsions. Annu Rev Phys Chem 63: 493-518.
  14. Zhang Y, Gao JG, Zheng HT, Zhang R, Han YC (2011) The preparation of 3,5-dihydroxy-4-isopropylstilbene nanoemulsion and in vitro release. Int J Nanomedicine 6: 649-657.
  15. Tadros T, Izquierdo P, Esquena J, Solans C (2004) Formation and stability of nano-emulsions Adv Colloid Interface 108-109: 303-18.
  16. Ostertag F, Weiss J, McClements DJ (2012) Low-energy formation of edible nanoemulsions: factors influencing droplet size produced by emulsion phase inversion. J Colloid Interface Sci 388(1): 95-102.
  17. Solans C, Solé I (2012) Nano-emulsions: Formation by low-energy methods. Curr Opin Colloid Interface Sci 17: 246–54.
  18. Fernandes CP, Mascarenhas MP, Zibetti FM, Lima BG, Oliveira RPRF, et al. (2013) HLB value, an important parameter for the development of essential oil phytopharmaceuticals. Braz J Pharmacog 23(1): 108-114.
  19. Duarte JL, Amado JRR, Oliveira AEMFM, Cruz RAS, Ferreira AM, et al. (2015) Evaluation of larvicidal activity of a nanoemulsion of Rosmarinus officinalis essential oil Braz J Pharmacog 25: 189-92.
  20. Oliveira AEMFM, Duarte JL, Amado JRR, Cruz RAS, Rocha CF, et al. (2016) Development of a Larvicidal Nanoemulsion with Pterodon emarginatus Vogel Oil. Plos ONE 11: e0145835.
  21. Costa IC, Rodrigues RF, Almeida FB, Favacho HAS, Falcão DQ, et al. (2013) Development of Jojoba Oil (Simmondsia chinensis (Link) C.K. Schneid.) Based Nanoemulsions. Lat Am J Pharm 33(3): 459-463.
  22. Rodrigues ECR, Ferreira AM, Vilhena JCE, Almeida FB, Cruz RAS, et al. (2014) Development of a larvicidal nanoemulsion with Copaiba (Copaifera duckei) oleoresin. Braz J Pharmacog 24(6): 699-705.
  23. Rodrigues ECR, Ferreira AM, Vilhena JCE, Almeida FB, Cruz RAS, et al. (2015) Development of Babassu Oil Based Nanoemulsions. Acta Farm Bonaerense 34(2): 338-343.
  24. Guttoff M, Saberi AH, McClements DJ (2015) Formation of vitamin D nanoemulsion-based delivery systems by spontaneous emulsification: Factors affecting particle size and stability. Food Chem 171: 117-122.
  25. Liang R, Shoemaker AF, Yang X, Zhong F, Huang Q (2013) Stability and bioaccessibility of β-carotene in nanoemulsions stabilized by modified starches. J Agric Food Chem 61(6): 1249-1257.
  26. Tan TB, Yussof NS, Abas F, Mirhosseini H, Nehdi IA, et al. (2016) Forming a lutein nanodispersion via solvent displacement method: the effects of processing parameters and emulsifiers with different stabilizing mechanisms. Food Chem 194: 416-423.
  27. Cheong JN, Tan CP, Man YBC, Misran MJ (2008) α-Tocopherol nanodispersions: Preparation, characterization and stability evaluation. J Food Eng 89(2): 204-209.
  28. Leong WF, Lai OM, Long K, Che MYB, Misran M, et al. (2011) Preparation and characterisation of water-soluble phytosterol nanodispersions. Food Chem 129: 77-83.
  29. Silva HD, Cerqueira MA, Souza BWS, Ribeiro C, Avides MC, et al. (2011) Nanoemulsions of β-carotene using a high-energy emulsification-evaporation technique. J Food Eng 102: 130-135.
  30. Griffin WC (1949) Classification of surface-active agents by “HLB”. J Soc Cosmet Chem 1: 311-326.
  31. Orafidiya LO, Oladimeji FA (2002) Determination of the required HLB values of some essential oils. Int J Pharm 237(1-2): 241-249.
  32. Kourniatis LR, Spinelli LS, Mansur CRE, González G. (2010) Nanoemulsões óleo de laranja/água preparadas em homogeneizador de alta pressão. Quim Nova 33(2): 295-300.
  33. Silva CNS, Hyacienth DC, Ferreira AM, Vilhena JCE, Fernandes CP, et al. (2015) Development of nanoemulsions with tucumã (Astrocaryum vulgare) fruits oil. J Nanomed Res 2(2): 24.
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