- Email: [email protected]
Encapsulation of herbal aqueous extract through absorption with ca-alginate hydrogel beads
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Contents lists available at ScienceDirect
Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp
Encapsulation of herbal aqueous extract through absorption with ca-alginate hydrogel beads Eng-Seng Chan ∗, Zhi-Hui Yim, Soon-Hock Phan, Rachel Fran Mansa, Pogaku Ravindra Centre of Materials and Minerals, School of Engineering and Information Technology, Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia
a b s t r a c t Encapsulation of herbal aqueous extract through absorption with ca-alginate hydrogel beads was studied. A model herbal aqueous extract, Piper sarmentosum, was used in this study. The effect of process variables (i.e. alginate M/G ratio, alginate concentration, extract concentration, bead size and bead water content) on encapsulation efficiency and biochemical compounds stability were studied. The stability of biochemical compounds was evaluated by using mass balance analysis and FT-IR spectroscopy. The results show that the encapsulation efficiency was mainly affected by alginate M/G ratio and bead water content. In general, ca-alginate beads made of higher alginate M/G ratio or dried to a lower water content were found to absorb significantly more aqueous extract. However, the beads made of higher M/G ratio were less rigid after the absorption process. Besides, the mass balance analysis reveals that the encapsulation process and material did not degrade the bioactive compounds, as the total antioxidant content remained unchanged. This is well supported by the FT-IR analysis where the characteristic bands of chemical groups remained unaltered. Interestingly, the beads made of lower alginate M/G ratio were found to have higher antioxidant affinity. In conclusion, this study demonstrates the potential of using absorption process and hydrogel material for encapsulation of herbal aqueous extract. © 2009 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Encapsulation; Ca-alginate; Absorption; Herbal extract; Antioxidants; Piper sarmentosum
1.
Introduction
The herbal-related products take into account herbs used as food or food additives, food supplements, traditional medicines, etc. The World Bank has projected the global market for herbal products to grow from USD 200 billion in 2008 to USD 5 trillion in 2050. In Malaysia alone, the herbal industry was reported to be worth USD 2.5 billion and it is growing faster than the general economy at 10% a year (Kamarul, 2006). A key factor for the rapid growth of the market is the growing knowledge and confidence of consumers in natural products or medicines. Besides, some high profile reports on the potential cures from plant such as Pacific yew (Taxus brevifolia Nutt.) for breast cancer and the Bintangor (Calophyllum spp.) for AIDS and tuberculosis (clinical trials and in vitro findings) have created significant awareness amongst consumers (Patil et al., 1993; National Cancer Institute, 2005; Xu et al., 2004; Wang et al., 2006; WWF-UK, 2006). In addition, bioactive
∗
compounds of herbal plants such as antioxidants have shown to have multiple functional and remedial properties that include anti-radical, anti-carcinogenic, reduction of oxidative stress, anti-inflammatory and cardio-protection (Owen et al., 2000; Kris-Etherton et al., 2004). In recent decades, some of the popular herbal plants in Malaysia have also gained increasing attention from both industrial and academic sectors. These include Eurycoma longifolia, Piper sarmentosum, Labisia pumila, Andrographis paniculata, Orthosiphon stamineus and Centella asiatica. Encapsulation is defined as a process of confining active compounds within a matrix or membrane in particulate form to achieve one or more desirable effects (Chan et al., 2009). From the standpoint of herbal products, encapsulation could achieve a number of desirable effects that includes controlled-delivery, extending shelf-life, separating incompatible compounds and improving final product qualities (Chan and Zhang, 2002, 2005; Shu et al., 2006; Kosaraju et al., 2006;
Corresponding author. Tel.: +60 88 320 000; fax: +60 88 320 348. E-mail addresses: [email protected], [email protected] (E.-S. Chan). Received 6 February 2009; Received in revised form 24 July 2009; Accepted 30 September 2009 0960-3085/$ – see front matter © 2009 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2009.09.005
196
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Deladino et al., 2008). For example, controlled-delivery could enhance bioavailability of an active compound by customising the release mechanism or rate in gastro-intestinal tract. In fact, a delivery system may be mandatory if direct consumption of a herbal active compound may cause interferences with the human body. In addition, encapsulation may promote better product stability by isolating active compounds from the detrimental effects of oxygen, moisture or incompatible compounds. Therefore, encapsulation could be a useful technological tool for the commercial sector to develop valueadded products or to create product differentiation from competitors. Hydrogel is a commonly used material for encapsulation due to its capability to absorb large amount of water or biological fluids. Among many materials, calcium-alginate hydrogel is the most widely used due to several advantageous features such as non-toxicity, biocompatible, easily produced; thermally and chemically stable (Chan et al., 2009). Since liquid herbal concentrates are normally prepared through an aqueous extraction process, encapsulation could be performed through absorption by using pre-prepared ca-alginate hydrogel beads. The absence of published work on the encapsulation of herbal aqueous extract by using this approach and the possible interaction between the matrix materials and herbal active compounds have led to the motivation to carry out this work. In this study, an aqueous extract of P. sarmentosum was used as the model herbal fluid. P. sarmentosum is one of the most abundant and widely used traditional herbal medicines in this region. The effect of process variables (i.e. alginate M/G ratio, alginate concentration, extract concentration, bead size and bead water content) on encapsulation efficiency was studied. The effect of encapsulation on antioxidant and chemical stability was evaluated by using mass balance and Fourier transform infrared analysis respectively.
2.
Materials and methods
2.1.
Materials
Sodium alginates of high guluronic acid content (Manugel GHB), denoted as high-G and high mannuronic content (Manugel DH), denoted as high-M were obtained from ISP Technologies Inc., UK. P. sarmentosum aqueous extract was prepared and supplied by Furley, Malaysia.
Fig. 1 – Experimental setup of this study: (1) syringe containing P. sarmentosum aqueous extract; (2) sieve; (3) cylinder; (4) blank ca-alginate beads; (5) cap; (6) connected to an air compressor; (7) encapsulated P. sarmentosum; (8) universal bottle; (9) residual extract.
2.3.
The experimental setup of this study is shown in Fig. 1. Blank ca-alginate beads were first prepared by using an extrusion method described by Chan et al. (2009). The beads were then briefly dried to remove the free water. One gram of beads was loaded into a cylinder followed by injection of 2 g of aqueous extract. The cylinder was gently tapped until a uniform packing was achieved. The beads were immersed in the aqueous extract and they were left in a dark cabinet for 1 h. Subsequently, the residual extract was withdrawn with the help of an air compressor. The overall encapsulation efficiency was calculated based on the mass of extract absorbed (g) by 1 g of blank ca-alginate beads, as shown in Eq. (1): overall encapsulation efficiency
g extract g beads
=
mEb mb
(1)
where mEb is the mass of extract absorbed by beads (g) and mb is the mass of beads (g).
2.4. 2.2.
Encapsulation of herbal aqueous extract
Analysis of antioxidant content
Preparation of blank beads and aqueous extract
The experimental design of this study is shown in Table 1. Sodium alginate solution was prepared to the desired concentration with deionised distilled water. The solution was extruded through a flat tip and allowed to drip into a gelation bath containing calcium chloride 1.5% (w/v) (Mallinckrodt, USA). The ca-alginate beads were then hardened for 4 h. Different sizes of beads were produced by varying the tip diameter. The beads were air-dried to obtain lower water content, if required. The herbal aqueous extract was kept at 4 ◦ C upon receipt and it was used directly, unless specified. Different concentrations of aqueous extract were prepared by diluting the original extract (1×) with deionised distilled water on a mass basis. For instance, an aqueous extract of 0.75× concentration was prepared by diluting 3 g of the extract with 1 g of deionised distilled water.
Antioxidant content of herbal extract in ca-alginate beads and residual extract was determined by using the 2,2-diphenyl-1picryl-hydrazyl (DPPH) colorimetric method as described by Zhou et al. (2004). Ascorbic acid (Sigma, USA) was used as the reference. Antioxidant stability was determined by using a mass balance analysis based on the antioxidant content, as shown in Eq. (2): sum of antioxidant mass fractions =
m
AOXb
mAOXo
m AOXr +
mAOXo (2)
where mAOXb is the mass of antioxidant in beads (mg), mAOXo is the original mass of antioxidant (mg) and mAOXr is the mass of antioxidant in residual extract (mg). If the sum of the two fractions is about one, it indicates no significant degradation of antioxidant.
197
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Table 1 – Experimental design of this study. No
Alginate type
Alginate concentration (%, w/v)
1 High-G High-M
Bead diameter (mm)
2 3 4 5
Bead water content (%)
Extract to bead ratio (w/w)
Extract strengtha
2.2
100
2
1×
100
2
1×
2
1×
2 High-G High-M
2
1.9 2.2 3.3
High-G High-M
2
2.2
High-G High-M
2
2.2
3
25 50 75 100
4
a
100
2
0.25× 0.50× 0.75× 1×
1× designates original concentration of P. sarmentosum aqueous extract.
2.5.
Analysis of chemical stability
Fourier transform infrared (FT-IR) spectra of aqueous extract, encapsulated extract and extract released from alginate beads were generated by using the Nicolet 5700 FT-IR spectrometer (Thermo Electron Corporation, USA). The samples were first mixed with potassium bromide powder (Sigma, USA) at 5% (w/w) and the mixture was then laminated by using a pellet mold. The wave number used was in the range of 4000–600 cm−1 .
2.6.
Statistical analysis
The experiments were repeated at least twice and the mean values were calculated by using Microsoft Office Excel 2007. The standard deviations of the mean values were also presented.
3.
Results and discussion
3.1.
Overall encapsulation efficiency
Fig. 2a–d shows the effect of process variables on the overall encapsulation efficiency of ca-alginate beads. In general, the high-M beads showed higher encapsulation efficiency than that of the high-G beads (0.78 ± 0.18 and 0.26 ± 0.06 g/g, respectively). The alginate concentration, the extract concentration and the bead size did not show clear effect on the overall encapsulation efficiency. On the other hand, the beads with lower water content were found to have higher encapsulation efficiency for both types of alginates. The encapsulation efficiency of the beads improved 2- to 4-fold when they were dried to a quarter of their original mass. In this study, the physical and chemical properties of the beads were found to have major influence on the encapsulation efficiency. The effect was evident when the beads made of two different types of alginates, were compared. It was found that the size of the high-G alginate beads remained unchanged after the absorption process (Table 2). On the other hand, the high-M alginate beads were found to swell to a larger volume and thus resulted in a higher encapsulation efficiency.
The swelling capability of the high-M beads could be caused by two factors. It is generally known that beads made of high-M alginate are elastic and they are susceptible to chelating agent that could destabilise the gel network. Since a herbal extract contains numerous biochemical compounds, it is speculated that some could act as chelating agents. This speculation is supported by the fact that the high-M beads became more fragile after encapsulation. In addition, the residual extract was found to be more viscous than the original extract. This indicates partial dissolution of gel network. In comparison, the high-G beads did not show significant swelling and they remained rigid after encapsulation. Another important factor that influenced the overall encapsulation efficiency was the bead water content. It is known that a hydrogel contains a large amount of water. In this case, the removal of water from the hydrogel matrix was found to promote the absorption of extract by the beads. On the other hand, drying was found to have different effects on the beads made from the two types of alginates (Fig. 3). The high-M beads shrunk to a greater extent than that of the highG beads when dried to a same water content. In addition, the swelling capability of the high-M beads was clearly reduced at a drier state. However, an opposite effect was observed for the high-G beads. This explains the improved encapsulation efficiency of the beads.
3.2.
Antioxidant and chemical stability
In this study, the antioxidant stability was determined through a mass balance method based on the antioxidant content in ca-alginate beads and residual extract, compared to that of original extract. In general, the total antioxidant content remained unchanged as the sum of the mass fractions was about one (Fig. 4). The mass fractions of antioxidant found in the high-M and high-G beads were about 0.3–0.4 and 0.2–0.3 respectively and the fractions were not affected by the alginate concentration, the extract concentration and the bead size. It was also found that the high-G beads with a lower water content contained higher antioxidant mass fractions (up to 0.45) whereas the high-M beads did not show significant changes. In addition, the chemical stability of herbal extract was analysed by using a Fourier transform infrared spectroscopy.
198
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Fig. 2 – Effect of (a) alginate concentration, (b) bead water content, (c) extract concentration and (d) bead diameter on encapsulation efficiency.
Fig. 3 – Effect of drying and encapsulation on bead diameter.
The characteristic bands of extract encapsulated within the ca-alginate beads (Fig. 5a) and that released from the beads (Fig. 5b) were compared to the original extract. In general, the bands remained unaltered and they can be assigned as follows (ALS Infrared Beamlines, 2009; Bhatt and Ray, 1998): conjugated C C or C O stretching vibrations at 1610 cm−1 , aromatic ring vibrations at 1500–1600 cm−1 , methyl group vibrations at 1380 cm−1 , C O C vibrations of esters at 1240 cm−1 , C OH stretching vibrations of secondary cyclic alcohols at 1070 cm−1 and CH out-of-plane bending vibrations at 760 and 625 cm−1 . Nevertheless, the hydrogel material could have minor effects on the signal strength. For example, the absorbances of encapsulated extract at 918 and 1237 cm−1 are absent or weaker when compared to the original extract (Fig. 5a). However, the signals reappear in the spectra of the extract released from the hydrogel matrix (Fig. 5b). This indicates that the signals could have been shielded by the hydrogel matrix. On the other hand, it is noteworthy that several new bands appear at about 813, 900, 1028 cm−1 in the spectra of the encapsulated extract. These could be the characteristic bands of blank caalginate hydrogel beads since it has been reported that the
Table 2 – Effect of encapsulation on the physical properties of beads and aqueous extract. High-G beads Swelling Rigidity Viscosity of residual extract
No obvious swelling Remained rigid No obvious change
High-M beads Swelled to more than 6% in diameter Less rigid Became more viscous
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
199
Fig. 4 – Mass fractions of antioxidant in beads and residual extract.
characteristic bands of alginate that correspond to the mannuronic and guluronic acid blocks are in the range between 780 and 1100 cm−1 (Pereira et al., 2003; Leal et al., 2008). Plant extracts normally contain numerous biochemical compounds such as alkaloids, amides, prophenylphenols, steroids, hydrocinnamic acid and oxalic acid (Masuda et al., 1991). These phytochemical compounds could be an important source of natural antioxidants and their efficacy could only be conferred when they are consumed together (Liu, 2004). Therefore, the activity and content of these compounds should be preserved during processing. In general, this study indicates that the encapsulation process through absorption with ca-alginate beads did not change the biochemical profile of P. sarmentosum extract. Although some chemical interac-
tions (i.e. chelation) could have occurred between the herbal extract and the hydrogel, the reaction did not affect the biochemical compounds of the extract. This is clearly shown by the unaltered FT-IR spectra of the encapsulated herbal extract. The chemical interactions may be caused by other compounds (i.e. metal ions) that were present in trace amounts since the extract was provided in a crude form. The finding is in good agreement with a previous work where no chemical interaction was found between a plant-derived essence (i.e. Azadirachta indica A. Juss.) and an alginate-based hydrogel matrix (Kulkarni et al., 2000). Therefore, it can be deduced that ca-alginate hydrogel matrix is a compatible material for encapsulating biochemical active compounds extracted from plants.
200
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Table 3 – Antioxidant affinity of ca-alginate beads. High-G beads
Mean
± s.d.
Alginate concentration (%, w/v) 2 2.54 0.52 3 2.12 0.55 4 1.50 0.34 5 1.78 0.41
0.82 0.94 0.89 0.76
0.09 0.04 0.15 0.04
Extract or strength 0.25× 1.99 0.50× 1.98 0.75× 1.85 1.0× 2.54
0.11 0.34 0.35 0.52
1.25 1.05 0.95 0.82
0.06 0.06 0.12 0.09
Bead diameter (mm) 1.9 2.79 2.2 2.54 3.3 2.36
0.41 0.52 0.39
0.93 0.82 1.36
0.02 0.09 0.06
Bead water content (%) 25 0.82 50 1.23 75 1.56 100 2.54
0.02 0.17 0.34 0.52
0.40 0.50 0.52 0.82
0.03 0.04 0.02 0.09
Mean
± s.d.
High-M beads
work is required to ascertain the mechanism for antioxidant affinity of ca-alginate hydrogel.
3.4.
Fig. 5 – FT-IR spectra of P. sarmentosum extract (a) encapsulated in beads and (b) released from beads.
3.3.
Antioxidant affinity
In this study, the ratio of antioxidant content in the beads to the theoretical amount (i.e. based on the original antioxidant content in the extract absorbed by the beads) was calculated (see Table 3). It was found that the antioxidant in the high-G beads did not correspond to the theoretical amount since the beads contained 1.5- to 3-fold more antioxidant than expected. However, the antioxidant found in the high-M beads was close to the theoretical amount. The ratio was also found to decrease with lower water content regardless of the alginate type. In brief, the ratio indicates the capability of ca-alginate hydrogel to attract and to concentrate antioxidant of a herbal aqueous extract. This capability could be termed as ‘antioxidant affinity’. Similar to the encapsulation efficiency, the antioxidant affinity was also found to be influenced by the chemical and physical properties of ca-alginate beads. The results indicate that the guluronic acid content of ca-alginate beads could play a role in accumulating antioxidant within the hydrogel matrix. In addition, it was found that water is a requirement to achieve high antioxidant affinity as it could change the physicochemical properties of hydrogel beads. It was reported that alginate-based materials (e.g. ca-alginate beads and algal biomass) had varying biosorption capacities for different metallic cations and the mechanisms involved could be ion exchange, chelation and reduction reactions (Papageorgiou et al., 2006; Raize et al., 2004). However, further
Comparison with other encapsulation methods
Previous studies have shown that the spray-drying method could be used to encapsulate herbal compounds such as lycopene, olive leaf extract, Amaranthus betacyanin extracts, carotene, d-Limonene and procyanidins (Desobry et al., 1997; Cai and Corke, 2000; Kosaraju et al., 2006; Shu et al., 2006). This method is popular because it is economical and it can achieve high productivity. However, the use of high processing temperature was found to cause degradation of active compounds (Desobry et al., 1997; Cai and Corke, 2000; Kosaraju et al., 2006; Shu et al., 2006). Therefore, this method may not be suitable for encapsulation of heat-sensitive compounds. Encapsulation of herbal compounds within hydrogel beads has also been performed by using the classical directextrusion method. A polymeric solution containing an active compound is extruded through an orifice and the droplets formed are allowed to fall into a gelling solution. Although the method is simple, many studies have reported low encapsulation efficiency of water soluble compounds due to diffusion of the compounds to the gelling solution (Moses et al., 2000; Kulkarni et al., 2000). We have subsequently conducted an experiment to verify this findings. By using the same herbal extract and encapsulation materials, it was found that the maximum encapsulation efficiency of the direct-extrusion method was about 0.14 g/g. The result is in good agreement with the finding of Kulkarni et al. (2000) where the encapsulation efficiency of liquid pesticide was found to be in the range of 0.074–0.267 g/g. In comparison, the absorption method gave 2-6 times higher encapsulation efficiency than the directextrusion method.
4.
Conclusion
Encapsulation of a model herbal aqueous extract, P. sarmentosum, through absorption with ca-alginate beads has been demonstrated. Depending on the encapsulation objective, a
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
suitable encapsulation process and material should take into consideration the encapsulation efficiency, stability of biochemical compounds and the final particle qualities. In this study, it was found that these criteria were closely related to the physicochemical properties of ca-alginate beads. In addition, the encapsulation method and material were found to have no interaction with the biochemical compounds of the extract. Further analysis shows that the high-G beads had higher antioxidant affinity than the high-M beads. It is envisaged that this simple and effective process can be easily adopted for industrial production of encapsulated herbal products. Nomenclature mEb mass of extract absorbed by beads (g) mass of beads used (g) mb mass of antioxidant in beads (mg) mAOXb original mass of antioxidant (mg) mAOXo mAOXr mass of antioxidant in the residual extract (mg)
Acknowledgements The authors thank the Ministry of Science, Technology and Innovation, Malaysia for the financial support through the EScience Project (SCF0038-IND2007). The authors also thank the colleagues and collaborators who have contributed to the development of this work.
References ALS Infrared Beamlines., (2009). Useful FTIR Info & Links—Characteristic IR Band Positions. http://infrared.als.lbl.gov/content/web-links/60-ir-bandpositions (access: 14 July, 2009) Bhatt, V.D. and Ray, A., 1998, Synthesis, characterization and electrical conductivity of polyesters, polyamides and doped polymers. Synthetic Metals, 92(2): 115–120. Cai, Y.Z. and Corke, H., 2000, Production and properties of spray-dried Amaranthus betacyanin pigments. Journal of Food Science, 65(6): 1248–1252. Chan, E.-S. and Zhang, Z., 2002, Encapsulation of probiotic bacteria Lactobacillus acidophilus by direct compression. Food and Bioproducts Processing, 80: 78–82. Chan, E.-S. and Zhang, Z., 2005, Bioencapsulation by compression coating of probiotic bacteria for their protection in an acidic medium. Process Biochemistry, 40(10): 3346–3351. Chan, E.-S., Lee, B.-B., Ravindra, P. and Poncelet, D., 2009, Prediction models for shape and size of ca-alginate macrobeads produced through extrusion-dripping method. Journal of Colloid and Interface Science, 338(1): 63–72. Deladino, L., Anbinder, P.S., Navarro, A.S. and Martino, M.N., 2008, Encapsulation of natural antioxidants extracted from Ilex paraguariensis. Carbohydrate Polymer, 71: 126–134. Desobry, S.S., Netto, F.M. and Labuza, T.P., 1997, Comparison of spray-drying, drum-drying and freeze-drying for -carotene encapsulation and preservation. Journal of Food Science, 62(6): 1158–1162. Kamarul, Y., (2006). Research Vital in Promoting Herbal Products Industry. (New Straits Times). Kosaraju, S.L., D’ath, L. and Lawrence, A., 2006, Preparation and characterisation of chitosan microspheres for antioxidant delivery. Carbohydrate Polymers, 64: 163–167.
201
Kris-Etherton, P.M., Lefevre, M., Beecher, G.R., Gross, M.D., Keen, C.L. and Etherton, T.D., 2004, Bioactive compounds in nutrition and health-research methodologies for establishing biological function: the antioxidant and anti-inflammatory effects of flavonoids on atherosclerosis. Annual Review of Nutrition, 24: 511–538. Kulkarni, A.R., Soppimath, K.S., Aminabhavi, T.M., Dave, A.M. and Mehta, M.H., 2000, Glutaraldehyde crosslinked sodium alginate beads containing liquid pesticide for soil application. Journal of Controlled Release, 63: 97–105. Leal, D., Matsuhiro, B., Rossi, M. and Caruso, F., 2008, FT-IR spectra of alginic acid block fractions in three species of brown seaweeds. Carbohydrate Research, 343(2): 308–316. Liu, R.H., 2004, Potential synergy of phytochemicals in cancer prevention: mechanism of action. International Research Conference on Food, Nutrition, and Cancer, 3479S–3485S. Masuda, T., Inazumi, A., Yamada, Y., Padolina, W.G., Kikuzaki, H. and Nakatani, N., 1991, Antimicrobial phenylpropanoids from Piper sarmentosum. Phytochemistry, 30(10): 3227–3228. Moses, L.R., Dileep, K.J. and Sharma, C.P., 2000, Beta cyclodextrin–insulin-encapsulated chitosan/alginate matrix: oral delivery system. Journal of Applied Polymer Science, 75: 1089–1096. National Cancer Institute., (2005). Weekly Paclitaxel (Taxol®) Before Any Other Treatment Improves Outcomes in Early-stage Breast Cancer. http://www.cancer.gov/clinicaltrials/results/weeklypaclitaxel0905 (access: 22 June, 2008) Owen, R.W., Giacosa, A., Hull, W.E., Haubner, R., Spiegelhalder, B. and Bartsch, H., 2000, The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. European Journal of Cancer, 36: 1235–1247. Papageorgiou, S.K., Katsaros, F.K., Kouvelos, E.P., Nolan, J.W., Deit, H.L. and Kanellopoulos, N.K., 2006, Heavy metal sorption by calcium alginate beads from Laminaria digitata. Journal of Hazardous Materials B, 137: 1765–1772. Patil, A.D., Freyer, A.J., Eggleston, D.S., Haltiwanger, R.C., Bean, M.F., Taylor, P.B., Caranfa, M.J., Breen, A.L., Bartus, H.R., Johnson, R.K., Hertzberg, R.P. and Westley, J.W., 1993, The inophyllums, novel inhibitors of HIV-1 reverse transcriptase isolated from the Malaysian tree, Calophyllum inophyllum Linn. Journal of Medicinal Chemistry, 36(26): 4131–4138. Pereira, L., Sousa, A., Coelho, H., Amado, A.M. and Ribeiro-Claro, P.J.A., 2003, Use of FTIR, FT-Raman and 13 C-NMR spectroscopy for identification of some seaweed phycocolloids. Biomolecular Engineering, 20: 223–228. Raize, O., Argaman, Y. and Yannai, S., 2004, Mechanisms of biosorption of different heavy metals by brown marine macroalgae. Biotechnology and Bioengineering, 87(4): 451–458. Shu, B., Yu, W., Zhao, Y. and Liu, X., 2006, Study on microencapsulation of lycopene by spray-drying. Journal of Food Engineering, 76: 664–669. Wang, L., Ma, T. and Liu, G., 2006, Recent progress in Calophyllum coumarins as potent anti-HIV agents, in Medicinal Chemistry of Bioactive Natural Products, Liang, X.T. and Fang, W.S., Fang, W.S. (eds) (John Wiley and Sons Inc., New York, USA), pp. 325–351. WWF-UK., (2006). Heart of Borneo: Medical Treasure-trove at Risk. http://www.wwf.org.uk/news/n 0000002549.asp (access: 22 June, 2008) Xu, Z.Q., Barrow, W.W., Suling, W.J., Westbrook, L., Barrow, E., Lina, Y.M. and Flavina, M.T., 2004, Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis. Bioorganic and Medicinal Chemistry, 12: 1199–1207. Zhou, K., Laux, J. and Yu, L., 2004, Comparison of Swiss red wheat grain and fractions for their antioxidant properties. Journal of Agricultural and Food Chemistry, 52: 1118–1123.
Contents lists available at ScienceDirect
Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp
Encapsulation of herbal aqueous extract through absorption with ca-alginate hydrogel beads Eng-Seng Chan ∗, Zhi-Hui Yim, Soon-Hock Phan, Rachel Fran Mansa, Pogaku Ravindra Centre of Materials and Minerals, School of Engineering and Information Technology, Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia
a b s t r a c t Encapsulation of herbal aqueous extract through absorption with ca-alginate hydrogel beads was studied. A model herbal aqueous extract, Piper sarmentosum, was used in this study. The effect of process variables (i.e. alginate M/G ratio, alginate concentration, extract concentration, bead size and bead water content) on encapsulation efficiency and biochemical compounds stability were studied. The stability of biochemical compounds was evaluated by using mass balance analysis and FT-IR spectroscopy. The results show that the encapsulation efficiency was mainly affected by alginate M/G ratio and bead water content. In general, ca-alginate beads made of higher alginate M/G ratio or dried to a lower water content were found to absorb significantly more aqueous extract. However, the beads made of higher M/G ratio were less rigid after the absorption process. Besides, the mass balance analysis reveals that the encapsulation process and material did not degrade the bioactive compounds, as the total antioxidant content remained unchanged. This is well supported by the FT-IR analysis where the characteristic bands of chemical groups remained unaltered. Interestingly, the beads made of lower alginate M/G ratio were found to have higher antioxidant affinity. In conclusion, this study demonstrates the potential of using absorption process and hydrogel material for encapsulation of herbal aqueous extract. © 2009 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Encapsulation; Ca-alginate; Absorption; Herbal extract; Antioxidants; Piper sarmentosum
1.
Introduction
The herbal-related products take into account herbs used as food or food additives, food supplements, traditional medicines, etc. The World Bank has projected the global market for herbal products to grow from USD 200 billion in 2008 to USD 5 trillion in 2050. In Malaysia alone, the herbal industry was reported to be worth USD 2.5 billion and it is growing faster than the general economy at 10% a year (Kamarul, 2006). A key factor for the rapid growth of the market is the growing knowledge and confidence of consumers in natural products or medicines. Besides, some high profile reports on the potential cures from plant such as Pacific yew (Taxus brevifolia Nutt.) for breast cancer and the Bintangor (Calophyllum spp.) for AIDS and tuberculosis (clinical trials and in vitro findings) have created significant awareness amongst consumers (Patil et al., 1993; National Cancer Institute, 2005; Xu et al., 2004; Wang et al., 2006; WWF-UK, 2006). In addition, bioactive
∗
compounds of herbal plants such as antioxidants have shown to have multiple functional and remedial properties that include anti-radical, anti-carcinogenic, reduction of oxidative stress, anti-inflammatory and cardio-protection (Owen et al., 2000; Kris-Etherton et al., 2004). In recent decades, some of the popular herbal plants in Malaysia have also gained increasing attention from both industrial and academic sectors. These include Eurycoma longifolia, Piper sarmentosum, Labisia pumila, Andrographis paniculata, Orthosiphon stamineus and Centella asiatica. Encapsulation is defined as a process of confining active compounds within a matrix or membrane in particulate form to achieve one or more desirable effects (Chan et al., 2009). From the standpoint of herbal products, encapsulation could achieve a number of desirable effects that includes controlled-delivery, extending shelf-life, separating incompatible compounds and improving final product qualities (Chan and Zhang, 2002, 2005; Shu et al., 2006; Kosaraju et al., 2006;
Corresponding author. Tel.: +60 88 320 000; fax: +60 88 320 348. E-mail addresses: [email protected], [email protected] (E.-S. Chan). Received 6 February 2009; Received in revised form 24 July 2009; Accepted 30 September 2009 0960-3085/$ – see front matter © 2009 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2009.09.005
196
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Deladino et al., 2008). For example, controlled-delivery could enhance bioavailability of an active compound by customising the release mechanism or rate in gastro-intestinal tract. In fact, a delivery system may be mandatory if direct consumption of a herbal active compound may cause interferences with the human body. In addition, encapsulation may promote better product stability by isolating active compounds from the detrimental effects of oxygen, moisture or incompatible compounds. Therefore, encapsulation could be a useful technological tool for the commercial sector to develop valueadded products or to create product differentiation from competitors. Hydrogel is a commonly used material for encapsulation due to its capability to absorb large amount of water or biological fluids. Among many materials, calcium-alginate hydrogel is the most widely used due to several advantageous features such as non-toxicity, biocompatible, easily produced; thermally and chemically stable (Chan et al., 2009). Since liquid herbal concentrates are normally prepared through an aqueous extraction process, encapsulation could be performed through absorption by using pre-prepared ca-alginate hydrogel beads. The absence of published work on the encapsulation of herbal aqueous extract by using this approach and the possible interaction between the matrix materials and herbal active compounds have led to the motivation to carry out this work. In this study, an aqueous extract of P. sarmentosum was used as the model herbal fluid. P. sarmentosum is one of the most abundant and widely used traditional herbal medicines in this region. The effect of process variables (i.e. alginate M/G ratio, alginate concentration, extract concentration, bead size and bead water content) on encapsulation efficiency was studied. The effect of encapsulation on antioxidant and chemical stability was evaluated by using mass balance and Fourier transform infrared analysis respectively.
2.
Materials and methods
2.1.
Materials
Sodium alginates of high guluronic acid content (Manugel GHB), denoted as high-G and high mannuronic content (Manugel DH), denoted as high-M were obtained from ISP Technologies Inc., UK. P. sarmentosum aqueous extract was prepared and supplied by Furley, Malaysia.
Fig. 1 – Experimental setup of this study: (1) syringe containing P. sarmentosum aqueous extract; (2) sieve; (3) cylinder; (4) blank ca-alginate beads; (5) cap; (6) connected to an air compressor; (7) encapsulated P. sarmentosum; (8) universal bottle; (9) residual extract.
2.3.
The experimental setup of this study is shown in Fig. 1. Blank ca-alginate beads were first prepared by using an extrusion method described by Chan et al. (2009). The beads were then briefly dried to remove the free water. One gram of beads was loaded into a cylinder followed by injection of 2 g of aqueous extract. The cylinder was gently tapped until a uniform packing was achieved. The beads were immersed in the aqueous extract and they were left in a dark cabinet for 1 h. Subsequently, the residual extract was withdrawn with the help of an air compressor. The overall encapsulation efficiency was calculated based on the mass of extract absorbed (g) by 1 g of blank ca-alginate beads, as shown in Eq. (1): overall encapsulation efficiency
g extract g beads
=
mEb mb
(1)
where mEb is the mass of extract absorbed by beads (g) and mb is the mass of beads (g).
2.4. 2.2.
Encapsulation of herbal aqueous extract
Analysis of antioxidant content
Preparation of blank beads and aqueous extract
The experimental design of this study is shown in Table 1. Sodium alginate solution was prepared to the desired concentration with deionised distilled water. The solution was extruded through a flat tip and allowed to drip into a gelation bath containing calcium chloride 1.5% (w/v) (Mallinckrodt, USA). The ca-alginate beads were then hardened for 4 h. Different sizes of beads were produced by varying the tip diameter. The beads were air-dried to obtain lower water content, if required. The herbal aqueous extract was kept at 4 ◦ C upon receipt and it was used directly, unless specified. Different concentrations of aqueous extract were prepared by diluting the original extract (1×) with deionised distilled water on a mass basis. For instance, an aqueous extract of 0.75× concentration was prepared by diluting 3 g of the extract with 1 g of deionised distilled water.
Antioxidant content of herbal extract in ca-alginate beads and residual extract was determined by using the 2,2-diphenyl-1picryl-hydrazyl (DPPH) colorimetric method as described by Zhou et al. (2004). Ascorbic acid (Sigma, USA) was used as the reference. Antioxidant stability was determined by using a mass balance analysis based on the antioxidant content, as shown in Eq. (2): sum of antioxidant mass fractions =
m
AOXb
mAOXo
m AOXr +
mAOXo (2)
where mAOXb is the mass of antioxidant in beads (mg), mAOXo is the original mass of antioxidant (mg) and mAOXr is the mass of antioxidant in residual extract (mg). If the sum of the two fractions is about one, it indicates no significant degradation of antioxidant.
197
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Table 1 – Experimental design of this study. No
Alginate type
Alginate concentration (%, w/v)
1 High-G High-M
Bead diameter (mm)
2 3 4 5
Bead water content (%)
Extract to bead ratio (w/w)
Extract strengtha
2.2
100
2
1×
100
2
1×
2
1×
2 High-G High-M
2
1.9 2.2 3.3
High-G High-M
2
2.2
High-G High-M
2
2.2
3
25 50 75 100
4
a
100
2
0.25× 0.50× 0.75× 1×
1× designates original concentration of P. sarmentosum aqueous extract.
2.5.
Analysis of chemical stability
Fourier transform infrared (FT-IR) spectra of aqueous extract, encapsulated extract and extract released from alginate beads were generated by using the Nicolet 5700 FT-IR spectrometer (Thermo Electron Corporation, USA). The samples were first mixed with potassium bromide powder (Sigma, USA) at 5% (w/w) and the mixture was then laminated by using a pellet mold. The wave number used was in the range of 4000–600 cm−1 .
2.6.
Statistical analysis
The experiments were repeated at least twice and the mean values were calculated by using Microsoft Office Excel 2007. The standard deviations of the mean values were also presented.
3.
Results and discussion
3.1.
Overall encapsulation efficiency
Fig. 2a–d shows the effect of process variables on the overall encapsulation efficiency of ca-alginate beads. In general, the high-M beads showed higher encapsulation efficiency than that of the high-G beads (0.78 ± 0.18 and 0.26 ± 0.06 g/g, respectively). The alginate concentration, the extract concentration and the bead size did not show clear effect on the overall encapsulation efficiency. On the other hand, the beads with lower water content were found to have higher encapsulation efficiency for both types of alginates. The encapsulation efficiency of the beads improved 2- to 4-fold when they were dried to a quarter of their original mass. In this study, the physical and chemical properties of the beads were found to have major influence on the encapsulation efficiency. The effect was evident when the beads made of two different types of alginates, were compared. It was found that the size of the high-G alginate beads remained unchanged after the absorption process (Table 2). On the other hand, the high-M alginate beads were found to swell to a larger volume and thus resulted in a higher encapsulation efficiency.
The swelling capability of the high-M beads could be caused by two factors. It is generally known that beads made of high-M alginate are elastic and they are susceptible to chelating agent that could destabilise the gel network. Since a herbal extract contains numerous biochemical compounds, it is speculated that some could act as chelating agents. This speculation is supported by the fact that the high-M beads became more fragile after encapsulation. In addition, the residual extract was found to be more viscous than the original extract. This indicates partial dissolution of gel network. In comparison, the high-G beads did not show significant swelling and they remained rigid after encapsulation. Another important factor that influenced the overall encapsulation efficiency was the bead water content. It is known that a hydrogel contains a large amount of water. In this case, the removal of water from the hydrogel matrix was found to promote the absorption of extract by the beads. On the other hand, drying was found to have different effects on the beads made from the two types of alginates (Fig. 3). The high-M beads shrunk to a greater extent than that of the highG beads when dried to a same water content. In addition, the swelling capability of the high-M beads was clearly reduced at a drier state. However, an opposite effect was observed for the high-G beads. This explains the improved encapsulation efficiency of the beads.
3.2.
Antioxidant and chemical stability
In this study, the antioxidant stability was determined through a mass balance method based on the antioxidant content in ca-alginate beads and residual extract, compared to that of original extract. In general, the total antioxidant content remained unchanged as the sum of the mass fractions was about one (Fig. 4). The mass fractions of antioxidant found in the high-M and high-G beads were about 0.3–0.4 and 0.2–0.3 respectively and the fractions were not affected by the alginate concentration, the extract concentration and the bead size. It was also found that the high-G beads with a lower water content contained higher antioxidant mass fractions (up to 0.45) whereas the high-M beads did not show significant changes. In addition, the chemical stability of herbal extract was analysed by using a Fourier transform infrared spectroscopy.
198
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Fig. 2 – Effect of (a) alginate concentration, (b) bead water content, (c) extract concentration and (d) bead diameter on encapsulation efficiency.
Fig. 3 – Effect of drying and encapsulation on bead diameter.
The characteristic bands of extract encapsulated within the ca-alginate beads (Fig. 5a) and that released from the beads (Fig. 5b) were compared to the original extract. In general, the bands remained unaltered and they can be assigned as follows (ALS Infrared Beamlines, 2009; Bhatt and Ray, 1998): conjugated C C or C O stretching vibrations at 1610 cm−1 , aromatic ring vibrations at 1500–1600 cm−1 , methyl group vibrations at 1380 cm−1 , C O C vibrations of esters at 1240 cm−1 , C OH stretching vibrations of secondary cyclic alcohols at 1070 cm−1 and CH out-of-plane bending vibrations at 760 and 625 cm−1 . Nevertheless, the hydrogel material could have minor effects on the signal strength. For example, the absorbances of encapsulated extract at 918 and 1237 cm−1 are absent or weaker when compared to the original extract (Fig. 5a). However, the signals reappear in the spectra of the extract released from the hydrogel matrix (Fig. 5b). This indicates that the signals could have been shielded by the hydrogel matrix. On the other hand, it is noteworthy that several new bands appear at about 813, 900, 1028 cm−1 in the spectra of the encapsulated extract. These could be the characteristic bands of blank caalginate hydrogel beads since it has been reported that the
Table 2 – Effect of encapsulation on the physical properties of beads and aqueous extract. High-G beads Swelling Rigidity Viscosity of residual extract
No obvious swelling Remained rigid No obvious change
High-M beads Swelled to more than 6% in diameter Less rigid Became more viscous
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
199
Fig. 4 – Mass fractions of antioxidant in beads and residual extract.
characteristic bands of alginate that correspond to the mannuronic and guluronic acid blocks are in the range between 780 and 1100 cm−1 (Pereira et al., 2003; Leal et al., 2008). Plant extracts normally contain numerous biochemical compounds such as alkaloids, amides, prophenylphenols, steroids, hydrocinnamic acid and oxalic acid (Masuda et al., 1991). These phytochemical compounds could be an important source of natural antioxidants and their efficacy could only be conferred when they are consumed together (Liu, 2004). Therefore, the activity and content of these compounds should be preserved during processing. In general, this study indicates that the encapsulation process through absorption with ca-alginate beads did not change the biochemical profile of P. sarmentosum extract. Although some chemical interac-
tions (i.e. chelation) could have occurred between the herbal extract and the hydrogel, the reaction did not affect the biochemical compounds of the extract. This is clearly shown by the unaltered FT-IR spectra of the encapsulated herbal extract. The chemical interactions may be caused by other compounds (i.e. metal ions) that were present in trace amounts since the extract was provided in a crude form. The finding is in good agreement with a previous work where no chemical interaction was found between a plant-derived essence (i.e. Azadirachta indica A. Juss.) and an alginate-based hydrogel matrix (Kulkarni et al., 2000). Therefore, it can be deduced that ca-alginate hydrogel matrix is a compatible material for encapsulating biochemical active compounds extracted from plants.
200
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
Table 3 – Antioxidant affinity of ca-alginate beads. High-G beads
Mean
± s.d.
Alginate concentration (%, w/v) 2 2.54 0.52 3 2.12 0.55 4 1.50 0.34 5 1.78 0.41
0.82 0.94 0.89 0.76
0.09 0.04 0.15 0.04
Extract or strength 0.25× 1.99 0.50× 1.98 0.75× 1.85 1.0× 2.54
0.11 0.34 0.35 0.52
1.25 1.05 0.95 0.82
0.06 0.06 0.12 0.09
Bead diameter (mm) 1.9 2.79 2.2 2.54 3.3 2.36
0.41 0.52 0.39
0.93 0.82 1.36
0.02 0.09 0.06
Bead water content (%) 25 0.82 50 1.23 75 1.56 100 2.54
0.02 0.17 0.34 0.52
0.40 0.50 0.52 0.82
0.03 0.04 0.02 0.09
Mean
± s.d.
High-M beads
work is required to ascertain the mechanism for antioxidant affinity of ca-alginate hydrogel.
3.4.
Fig. 5 – FT-IR spectra of P. sarmentosum extract (a) encapsulated in beads and (b) released from beads.
3.3.
Antioxidant affinity
In this study, the ratio of antioxidant content in the beads to the theoretical amount (i.e. based on the original antioxidant content in the extract absorbed by the beads) was calculated (see Table 3). It was found that the antioxidant in the high-G beads did not correspond to the theoretical amount since the beads contained 1.5- to 3-fold more antioxidant than expected. However, the antioxidant found in the high-M beads was close to the theoretical amount. The ratio was also found to decrease with lower water content regardless of the alginate type. In brief, the ratio indicates the capability of ca-alginate hydrogel to attract and to concentrate antioxidant of a herbal aqueous extract. This capability could be termed as ‘antioxidant affinity’. Similar to the encapsulation efficiency, the antioxidant affinity was also found to be influenced by the chemical and physical properties of ca-alginate beads. The results indicate that the guluronic acid content of ca-alginate beads could play a role in accumulating antioxidant within the hydrogel matrix. In addition, it was found that water is a requirement to achieve high antioxidant affinity as it could change the physicochemical properties of hydrogel beads. It was reported that alginate-based materials (e.g. ca-alginate beads and algal biomass) had varying biosorption capacities for different metallic cations and the mechanisms involved could be ion exchange, chelation and reduction reactions (Papageorgiou et al., 2006; Raize et al., 2004). However, further
Comparison with other encapsulation methods
Previous studies have shown that the spray-drying method could be used to encapsulate herbal compounds such as lycopene, olive leaf extract, Amaranthus betacyanin extracts, carotene, d-Limonene and procyanidins (Desobry et al., 1997; Cai and Corke, 2000; Kosaraju et al., 2006; Shu et al., 2006). This method is popular because it is economical and it can achieve high productivity. However, the use of high processing temperature was found to cause degradation of active compounds (Desobry et al., 1997; Cai and Corke, 2000; Kosaraju et al., 2006; Shu et al., 2006). Therefore, this method may not be suitable for encapsulation of heat-sensitive compounds. Encapsulation of herbal compounds within hydrogel beads has also been performed by using the classical directextrusion method. A polymeric solution containing an active compound is extruded through an orifice and the droplets formed are allowed to fall into a gelling solution. Although the method is simple, many studies have reported low encapsulation efficiency of water soluble compounds due to diffusion of the compounds to the gelling solution (Moses et al., 2000; Kulkarni et al., 2000). We have subsequently conducted an experiment to verify this findings. By using the same herbal extract and encapsulation materials, it was found that the maximum encapsulation efficiency of the direct-extrusion method was about 0.14 g/g. The result is in good agreement with the finding of Kulkarni et al. (2000) where the encapsulation efficiency of liquid pesticide was found to be in the range of 0.074–0.267 g/g. In comparison, the absorption method gave 2-6 times higher encapsulation efficiency than the directextrusion method.
4.
Conclusion
Encapsulation of a model herbal aqueous extract, P. sarmentosum, through absorption with ca-alginate beads has been demonstrated. Depending on the encapsulation objective, a
food and bioproducts processing 8 8 ( 2 0 1 0 ) 195–201
suitable encapsulation process and material should take into consideration the encapsulation efficiency, stability of biochemical compounds and the final particle qualities. In this study, it was found that these criteria were closely related to the physicochemical properties of ca-alginate beads. In addition, the encapsulation method and material were found to have no interaction with the biochemical compounds of the extract. Further analysis shows that the high-G beads had higher antioxidant affinity than the high-M beads. It is envisaged that this simple and effective process can be easily adopted for industrial production of encapsulated herbal products. Nomenclature mEb mass of extract absorbed by beads (g) mass of beads used (g) mb mass of antioxidant in beads (mg) mAOXb original mass of antioxidant (mg) mAOXo mAOXr mass of antioxidant in the residual extract (mg)
Acknowledgements The authors thank the Ministry of Science, Technology and Innovation, Malaysia for the financial support through the EScience Project (SCF0038-IND2007). The authors also thank the colleagues and collaborators who have contributed to the development of this work.
References ALS Infrared Beamlines., (2009). Useful FTIR Info & Links—Characteristic IR Band Positions. http://infrared.als.lbl.gov/content/web-links/60-ir-bandpositions (access: 14 July, 2009) Bhatt, V.D. and Ray, A., 1998, Synthesis, characterization and electrical conductivity of polyesters, polyamides and doped polymers. Synthetic Metals, 92(2): 115–120. Cai, Y.Z. and Corke, H., 2000, Production and properties of spray-dried Amaranthus betacyanin pigments. Journal of Food Science, 65(6): 1248–1252. Chan, E.-S. and Zhang, Z., 2002, Encapsulation of probiotic bacteria Lactobacillus acidophilus by direct compression. Food and Bioproducts Processing, 80: 78–82. Chan, E.-S. and Zhang, Z., 2005, Bioencapsulation by compression coating of probiotic bacteria for their protection in an acidic medium. Process Biochemistry, 40(10): 3346–3351. Chan, E.-S., Lee, B.-B., Ravindra, P. and Poncelet, D., 2009, Prediction models for shape and size of ca-alginate macrobeads produced through extrusion-dripping method. Journal of Colloid and Interface Science, 338(1): 63–72. Deladino, L., Anbinder, P.S., Navarro, A.S. and Martino, M.N., 2008, Encapsulation of natural antioxidants extracted from Ilex paraguariensis. Carbohydrate Polymer, 71: 126–134. Desobry, S.S., Netto, F.M. and Labuza, T.P., 1997, Comparison of spray-drying, drum-drying and freeze-drying for -carotene encapsulation and preservation. Journal of Food Science, 62(6): 1158–1162. Kamarul, Y., (2006). Research Vital in Promoting Herbal Products Industry. (New Straits Times). Kosaraju, S.L., D’ath, L. and Lawrence, A., 2006, Preparation and characterisation of chitosan microspheres for antioxidant delivery. Carbohydrate Polymers, 64: 163–167.
201
Kris-Etherton, P.M., Lefevre, M., Beecher, G.R., Gross, M.D., Keen, C.L. and Etherton, T.D., 2004, Bioactive compounds in nutrition and health-research methodologies for establishing biological function: the antioxidant and anti-inflammatory effects of flavonoids on atherosclerosis. Annual Review of Nutrition, 24: 511–538. Kulkarni, A.R., Soppimath, K.S., Aminabhavi, T.M., Dave, A.M. and Mehta, M.H., 2000, Glutaraldehyde crosslinked sodium alginate beads containing liquid pesticide for soil application. Journal of Controlled Release, 63: 97–105. Leal, D., Matsuhiro, B., Rossi, M. and Caruso, F., 2008, FT-IR spectra of alginic acid block fractions in three species of brown seaweeds. Carbohydrate Research, 343(2): 308–316. Liu, R.H., 2004, Potential synergy of phytochemicals in cancer prevention: mechanism of action. International Research Conference on Food, Nutrition, and Cancer, 3479S–3485S. Masuda, T., Inazumi, A., Yamada, Y., Padolina, W.G., Kikuzaki, H. and Nakatani, N., 1991, Antimicrobial phenylpropanoids from Piper sarmentosum. Phytochemistry, 30(10): 3227–3228. Moses, L.R., Dileep, K.J. and Sharma, C.P., 2000, Beta cyclodextrin–insulin-encapsulated chitosan/alginate matrix: oral delivery system. Journal of Applied Polymer Science, 75: 1089–1096. National Cancer Institute., (2005). Weekly Paclitaxel (Taxol®) Before Any Other Treatment Improves Outcomes in Early-stage Breast Cancer. http://www.cancer.gov/clinicaltrials/results/weeklypaclitaxel0905 (access: 22 June, 2008) Owen, R.W., Giacosa, A., Hull, W.E., Haubner, R., Spiegelhalder, B. and Bartsch, H., 2000, The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. European Journal of Cancer, 36: 1235–1247. Papageorgiou, S.K., Katsaros, F.K., Kouvelos, E.P., Nolan, J.W., Deit, H.L. and Kanellopoulos, N.K., 2006, Heavy metal sorption by calcium alginate beads from Laminaria digitata. Journal of Hazardous Materials B, 137: 1765–1772. Patil, A.D., Freyer, A.J., Eggleston, D.S., Haltiwanger, R.C., Bean, M.F., Taylor, P.B., Caranfa, M.J., Breen, A.L., Bartus, H.R., Johnson, R.K., Hertzberg, R.P. and Westley, J.W., 1993, The inophyllums, novel inhibitors of HIV-1 reverse transcriptase isolated from the Malaysian tree, Calophyllum inophyllum Linn. Journal of Medicinal Chemistry, 36(26): 4131–4138. Pereira, L., Sousa, A., Coelho, H., Amado, A.M. and Ribeiro-Claro, P.J.A., 2003, Use of FTIR, FT-Raman and 13 C-NMR spectroscopy for identification of some seaweed phycocolloids. Biomolecular Engineering, 20: 223–228. Raize, O., Argaman, Y. and Yannai, S., 2004, Mechanisms of biosorption of different heavy metals by brown marine macroalgae. Biotechnology and Bioengineering, 87(4): 451–458. Shu, B., Yu, W., Zhao, Y. and Liu, X., 2006, Study on microencapsulation of lycopene by spray-drying. Journal of Food Engineering, 76: 664–669. Wang, L., Ma, T. and Liu, G., 2006, Recent progress in Calophyllum coumarins as potent anti-HIV agents, in Medicinal Chemistry of Bioactive Natural Products, Liang, X.T. and Fang, W.S., Fang, W.S. (eds) (John Wiley and Sons Inc., New York, USA), pp. 325–351. WWF-UK., (2006). Heart of Borneo: Medical Treasure-trove at Risk. http://www.wwf.org.uk/news/n 0000002549.asp (access: 22 June, 2008) Xu, Z.Q., Barrow, W.W., Suling, W.J., Westbrook, L., Barrow, E., Lina, Y.M. and Flavina, M.T., 2004, Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis. Bioorganic and Medicinal Chemistry, 12: 1199–1207. Zhou, K., Laux, J. and Yu, L., 2004, Comparison of Swiss red wheat grain and fractions for their antioxidant properties. Journal of Agricultural and Food Chemistry, 52: 1118–1123.