An Initial Investigation into the Anti-inflammatory Activity and Antioxidant Capacity of...

Volume 9, Issue 1 2012 Article 25

Journal of Complementary andIntegrative Medicine

An Initial Investigation into the Anti-inflammatory Activity and Antioxidant

Capacity of alpha-Cyclodextrin-ComplexedManuka Honey

Lynne M. Chepulis, Waiariki Institute of Technology,Rotorua, New Zealand

Evelyn Francis, Waiariki Institute of Technology, Rotorua,New Zealand

Recommended Citation:Chepulis, Lynne M. and Francis, Evelyn (2012) "An Initial Investigation into the Anti-inflammatory Activity and Antioxidant Capacity of alpha-Cyclodextrin-Complexed ManukaHoney," Journal of Complementary and Integrative Medicine: Vol. 9: Iss. 1, Article 25.DOI: 10.1515/1553-3840.1646

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An Initial Investigation into the Anti-inflammatory Activity and Antioxidant

Capacity of alpha-Cyclodextrin-ComplexedManuka Honey

Lynne M. Chepulis and Evelyn Francis

AbstractThe bioactive properties of Manuka honey are now well recognised, but the nature of honey

(a sticky, viscous fluid) can make it hard to use as a health remedy. A new technology usingencapsulation of Manuka honey with alpha-cyclodextrin molecules has been developed, creatinga free-flowing powder that can easily be added to foods and beverages, or tableted / made intocapsules for use in health. In this study, we investigated for antioxidant and anti-inflammatoryactivity. Antioxidant capacity of raw Manuka honeys and matched complexes was measured usingthe CUPRAC method. Results showed that the antioxidant activity of honey decreased whencomplexed, this being directly related to dilution of the final product with alpha-cyclodextrin. Anti-inflammatory activity was assessed by measuring inhibition of neutrophil TNF-alpha secretion.Contradictory results were produced, with both stimulation and inhibition of TNF-alpha beingobserved. Data from this study indicate that the formation of cyclodextrin-based complexes ofManuka honey may potentiate the anti-inflammatory activity of honey, but this may differdepending on methylglyoxal content and the presence of other factors.

KEYWORDS: manuka honey, cyclopower, alpha-cyclodextrin, antioxidant capacity, anti-inflammatory activity

Author Notes: This study was funded in its entirety by Manuka Health New Zealand Ltd. We thankTrinity Bioactives (Wellington, New Zealand) for their assistance with this study.



INTRODUCTION The health-promoting benefits of honey have long been recognised, and texts as far back as 1900-1250 BC have reported the use of honey in medicines and prescriptions (Stomfay-Stitz, 1960, Traynor, 2011 (with a foreword from Peter Molan; Lee, Sinno and Khachemoune, 2011)). More recently, honey has gained popularity as both an internal and external remedy (Beck and Smedley, 1997; Molan, 2006; Al-Waili et al., 2011) and researchers are now beginning to realise that honey inherently possesses a number of health-related benefits. These include a potent antibacterial effect (reviewed by Molan, 1992; Al-Waili et al., 2011) positive effects on weight, blood glucose and cholesterol levels (Chepulis, 2007; Chepulis and Starkey, 2008), a prebiotic effect (Ustunol and Ghandi, 2001; Kajiwara et al., 2002; Sanz et al., 2005), anti-inflammatory (Molan, 2006; Al-Waili, 2005) and immunostimulatory effects (Al-Waili and Haq, 2005; Tonks et al., 2003, 2007) and wound-healing capabilities (reviewed by Molan, 2006).

More recently, much honey research has focused on the bioactive properties of New Zealand Manuka (Leptospernum scoparium) honey as its properties have often been shown to be more potent and stable than those of other honeys. It has been shown to have a unique antimicrobial profile due to the often high levels of heat- and light-stable methylgyoxal (Mavric et al., 2008). Manuka honey has also been studied extensively for use in wound care, and it appears to demonstrate a number of wound-healing properties (reviewed by Al-Waili et al., 2011). This can include a potent anti-inflammatory and antibacterial action on the wound as well as the ability to stimulate epithelialisation, tissue growth and wound debridement.

Inflammation is an important process during wound healing, but it also occurs during many other disease states including arthritis, cancer and inflammatory bowel disease (Wan et al., 1989). Systemic inflammation has also been shown to initiate or exacerbate autoimmune diseases, pre-senile dementia and atherosclerosis (reviewed by Brod, 2000), and to possibly be involved in the development of obesity (Cancello and Clement, 2006). Several studies have reported that honey can have an anti-inflammatory activity (Lee, Sinno and Khachemoune, 2011; Malik et al., 2010; Leong et al. 2012). Both in vitro and in vivo studies have reported that honey applied to wounds reduces localised swelling, heat and pain associated with inflammation (Burlando, 1798; Efem, 1993; Subrahmanyam 1993, 1998) and the effect has been widely observed in clinical settings (reviewed by Molan, 2006). It has been suggested that some of the anti-inflammatory properties of honey may be due to its ability to reduce prostaglandin production (Al-Waili, 2005), and limited evidence suggests that it may be related to the high antioxidant levels of the honey (Medhi et al., 2008). Indeed, studies have demonstrated that Manuka honey is able to modulate production and quenching of free radicals (Henriques et al., 2006), this being an

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Chepulis and Francis: Alpha-Cyclodextin complexed Manuka Honey

Published by De Gruyter, 2012



important consequence of inflammation (Flohe et al., 1985). Furthermore, honey with a high phenolic content has been shown to induce apoptosis in some cancer cells, effectively inhibiting cell proliferation (as evidenced in both in vivo and in vitro studies). It is suggested that this mechanism of action is involved with the mitochondrial and ROS-mediated apoptotic mechanisms (Jaganathan and Mandal, 2009; Jaganathan et al, 2010; Samarghandian et al., 2011).

Cyclodextrins are naturally-occurring cyclical oligosaccharides produced from starch by enzymatic conversion (Martin Del Valle, 2003). They have been used in the food, pharmaceutical and chemical industries for many years, and they have been shown to have a range of applications because of their ability to form host-guest complexes with hydrophobic molecules (Eastburn and Tao, 1994). A range of Manuka honey-based cyclodextrin complexes (Manuka Honey with Cyclopower™) have been developed in New Zealand (patent pending WO 2010/044042) with the intention of improving the bioactive properties of the raw materials. Thus, this study was designed to evaluate whether the creation of cyclodextrin-based Manuka honey complexes would have any effect on the antioxidant capacity and the anti-inflammatory properties of Manuka honey.

METHODS

Samples

Samples of raw Manuka Honey with high levels of methylglyoxal (MGO) (MGO250™ and MGO400™) were sourced from beekeepers in the Waiarapa region of New Zealand and supplied by Manuka Health New Zealand Ltd.

Preparation of Cyclodextrin-based Cyclopower™ Complexes

Manuka Honey/cyclodextrin complexes (Manuka Honey with Cyclopower™) were prepared by CycloChemBio, Japan. To make the complex, samples of honey (MGO250™ and MGO400™) were mixed with aqueous solutions of -cyclodextrin (-CD; Cavamax, Wacker Biosolutions) (final ratio 45% honey / 55% -CD) and the water content of each was adjusted to 30% w/w by addition of deionised water (the initial moisture contents of the honey and -CD were determined using a refratometer (As ONE Corporation, APAL-J) and a halogen moisture analyzer (Mettler-Toldedo International, HB43); respectively). The solutions were mixed at 40oC for 10 minutes before freeze-drying (Tokyo Rikakikai Co. Ltd., FDU-2200) until dry.

Methylgyoxal levels were determined in the raw Manuka honeys and in the complexes using RP-HPLC with UV detection as the corresponding quinoxaline after pre-column derivatization with o-phenylenediamine as outlined in Mavric et al. (2008). This analysis was carried out in duplicate for each sample.

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Journal of Complementary and Integrative Medicine, Vol. 9 [2012], Iss. 1, Art. 25



Assays

Antioxidant Capacity

Total antioxidant capacity was measured using the CUPRAC method as described by Apak et al., (2005) and Ozyurek et al., (2008). In brief a sodium urate (Sigma) standard was prepared by making a 2.5 mM (475.48 ug/ml) sodium urate solution in deionised water, then diluting quantitatively 1:24 with phosphate buffered saline (PBS) to give 0.1 mM. Standard and samples (200 µL) were pipetted into a 96-well microtitre plate in triplicate. Aliquots (100 l) of 15% ethanol / Hank’s balanced salt solution (HBSS; Gibco) were pipetted into test sample wells, whilst 100 l PBS was pipetted into standard wells. Samples and standards were sequentially diluted (1:2) across the rows of the microtitre plate. The final concentration of all test samples was 0.78 µM (0.078 MGO/ml). Neocuproine hydrochloride hydrate (DMP; Sigma) solution (2 mM, 50 µL) was then pipetted into each well, except for blank wells. Copper chloride solution (1 mM, 50 µL; Aldrich) was then added to all wells (except blank wells) and the plate was incubated at room temperature for two hours. The absorbance of each well was measured at 450 nm using a Versamax 96 well plate reading, and final readings were determined after subtraction of background absorbance values. All reagents were of analytical grade.

Anti-inflammatory Activity

Anti-inflammatory activity was determined by measuring inhibition of Neutrophil TNF- secretion, as reported in Yoshimura et al (1994). Samples were dissolved in 15% ethanol/HBSS buffer (Gibco) and working stock solutions of each at 4 MGO/ml and 1 MGO/ml were prepared. Chloroquine (Sigma) was prepared at a stock concentration of 1 mM (0.52 MGO/ml) in 15% ethanol/HBSS and further diluted to 0.1 mM and 0.05 mM. These working stock solutions gave final concentrations of 10 µM and 5 μM in the cell culture.

The isolation of neutrophil cells and the determination of anti-inflammatory activity were based on the Standard Operating Procedure (BIG SOP XNSO 3.0).

Isolation of Polymorphonuclear Granulocytes from Whole Blood

Four Lewis rats were anaesthetised with ketamine/xylazine and ~28ml of blood was drawn and collected in EDTA tubes. The blood was stored on ice or at 4C until use. Approximately 2.5 ml of Polymorphprep™ (Axis-Shield PoC, Norway) was added to each of four 12 ml glass centrifuge tubes. Aliquots (7.0 ml) of anticoagulated whole blood were carefully layered over the 2.5 ml of Polymorphprep™. The tubes were centrifuged at 500 x g for 30 min at 20ºC in a

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Beckman J-6 centrifuge using a JS-5.2/4.0 rotor. After centrifugation, two leukocyte bands were visible, and the lower band was harvested into a 15 ml plastic centrifuge tube using a Pasteur pipette. The polymorphonuclear fraction was brought up to a total of 15 ml using HBSS and centrifuged at 1200 rpm (JS-5.2/4.0 rotor, Beckman J-6 centrifuge) for 5 min at 4ºC. The supernatant was discarded and the cell pellet was resuspended and re-washed by adding 15 ml of washing buffer HBSS and centrifuged at 1200 rpm for 5 min at 4ºC. Again, the supernatant was discarded and the cell pellet was re-suspended in 10 ml of HBSS. The cell number was counted and the cell density adjusted to 1.2-2.0 x 106/ml with HBSS. The cell suspension was kept on ice until needed.

Culturing Neutrophils for TNF-α and IL-8 ELISA

Assays were performed using a 96-well microplate. Aliquots of cell suspension (190 l) were added to all wells (except blanks which contained medium only). Samples (25 µl of each) were added into the appropriate wells and 25 µl of either 0.1 mM or 0.05 mM Chloroquine added. Sample-free and cell-only controls were also included on the plate.

The plate was incubated in a humidified incubator at 37ºC in 95% air/5% carbon dioxide for 20 minutes. Next, 10 l of 250 μg/ml LPS (Sigma) and 25 l of foetal bovine serum (Sigma) was added to each well. The plate was incubated at 37ºC for 24 hours. The following day, the plate was centrifuged at 500 rpm for 15 min. Aliquots (120 l/well) of the supernatant/medium from each test sample triplicate wells and blanks were pooled and duplicate aliquots (100 µl) were transferred to two sets of new plates and stored at -20C until used.

Each test sample (at both concentrations, 400 and 100 µg/ml) was assayed in duplicate by ELISA for the determination of the concentrations of TNF- α (R and D Systems, RTA00) and IL-8 (Cusabio Viotech Co Ltd; CSB-E07273r) following the recommendations in the manuals provided by the manufacturer.

Statistics

The percentage standard error of the mean (SEM) was assessed and when SEM was greater than 20% they were regarded as outliers and were removed. Preliminary statistical significance was assessed with an independent Student t-test at < 0.05 (with and without outliers).

RESULTS

Antioxidant Capacity

The total antioxidant capacity (TAC) values of the various samples are shown in Table 1. Eight different concentrations of each sample were assessed on the

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microtitre place, and those falling within the range of sodium urate standards were used to calculate the TAC values (all samples demonstrated a linear dilution response). The TAC values are expressed as the equivalent reference sodium urate units per ml of sample.

Table 1: Antioxidant capacity of raw Manuka honey compared with -cyclodextrin-complexed Cyclopower™ samples (reported as sodium urate equivalents (g/ml))

Raw Honey Cyclopower™Complex (as received)

Percent antioxidant activity retained in the Cyclopower™ complex when adjusted for honey concentration*

MGO250™ 11.72 5.08 96.4% MGO 400™ 9.84 4.40 99.4%

* Manuka Cyclopower complex is 45% honey and 55% a-cyclodextrin

All Manuka honey samples had TAC values below 12.0 sodium urate equivalents (g/ml). The comparable Manuka honey with Cyclopower™ complex TAC values were ~55% less compared with the raw honey samples and correlated with the fact that these complexes are 45% honey and 55% -cyclodextrin. When adjusted to an equivalent weight per weight of honey basis, more than 96% of the antioxidant activity was retained in the Manuka Cyclopower™ products.

Anti-inflammatory Activity

The total anti-inflammatory activities of the various samples (measured as inhibition of TNF-) are shown in Figure 1. Both the MGO250™ and the MGO400™ Manuka honey samples demonstrated inhibition of TNF- when measured at 400 g/ml (29.9% and 19.5%; both p < 0.05 vs control cells (0% inhibition, not shown)); however, the MGO250™ Manuka honey sample showed stimulation of TNF- when measured at 100 g/ml (+ 30.2%; p = 0.02 vs control). Both honeys also demonstrated a reduction in TNF- activity when they were complexed with -cyclodextrin. The MGO250™ Manuka honey complex sample showed a 50% reduction in inhibition of TNF-when compared with the raw Manuka honey at 400 g/ml (15.5% vs 29.9%; p < 0.05). At 100 µg/ml the effect of MGO250™ Manuka honey-cyclodextrin complex was slightly greater - it showed a 23.8% inhibition of TNF-(p < 0.001 vs control cells). The MGO400™ Manuka honey-cyclodextrin sample was a stronger anti-inflammatory modulator. At 400 g/ml it inhibited the production of TNF- by 48.7% (p < 0.0001 vs control cells; p < 0.01 vs raw honey (19.45% inhibition)). Inhibition of TNF- was similar for both complex and raw samples when measured at 100 g/ml with the MGO400™ Manuka honey sample (12.9% vs 15.3%).

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Figure 1: Effects of raw Manuka honey compared with -cyclodextrin-complexed Cyclopower™ samples on TNF- inhibition from activated neutrophils after 24 hours of incubation at 37oC for 24 hours.

Methylglyoxyl levels in the MGO250™ and MGO400™ Manuka honeys were shown to be 82.3% and 82.7% of raw honey levels, respectively, after complexing with -cyclodextrin.

No IL-8 was detectable in any of the cultures, including controls.

DISCUSSION Manuka honey has been recognised for many years now as having a suite of bioactive properties, and much of this has been attributed to the compound methylglyoxal (particularly antimicrobial activity). However, despite the fact that Manuka honey, and honey in general, has been demonstrated to exhibit anti-inflammatory properties (Molan, 2006), researchers have still not fully elucidated the compound(s) responsible. Various ideas have been postulated in the literature, including reduction of prostaglandin synthesis in the presence of honey (Al Waili, 2005), but one commonly accepted theory is that it likely involves the antioxidant content of the honey. Reactive oxygen species are a serious and common complication of excessive inflammation as they can lead to the amplification of the inflammatory response via oxidative activation of the transcription factor NF-B that promotes the production of pro-inflammatory cytokines (Flohe et al., 1985). Thus, it has been suggested that the antioxidant content of honey may contribute to its anti-inflammatory activity as the activation of NF-B can be

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0

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MGO250™ Manuka Honey

MGO250™ Manuka Honey Complex

MGO 400™ Manuka Honey

MGO400™ Manuka Honey Complex

% In

hibition of TN

F‐Production

100 mg/ml

400 mg/ml

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prevented by the presence of antioxidants (Gribble, 1994) and direct application of antioxidants to burns has been demonstrated to reduce inflammation (Subramanyam 1991, 1996; Malik et al., 2010; Lee et al., 2011). Further, other food-based antioxidants have also been shown to demonstrate a clinical anti-inflammatory effect (reviewed by Katiyar and Elmets (2001) and Geronikaki et al. (2006)).

A small number of studies have recently evaluated the antioxidant capacities of honey (Rodríguez et al., 2012; Sant’ana et al., 2012), and results generally agree with work published earlier (Frankel et al., 1998; Chen et al., 2000; Gheldof and Engeseth, 2002) that the antioxidant capacity can differ dramatically between different honey samples (both within and between floral sources), with higher levels tending to occur in the darker coloured honeys. Common theories by these authors suggest that the antioxidant capacity of honey is due to its phenolic content, and two authors have demonstrated a linear correlation between the phenolic profile of the honey and the antioxidant (ORAC) activity of the honeys (Gheldof et al., 2002; Jaganathan and Mandal, 2009).

However, despite the work that has been done with other honeys, there is still little data available describing the antioxidant content or the anti-inflammatory properties of Manuka honeys. Limited animal studies have reported that oral administration of Manuka honey at 5 and 10 g/kg significantly reduces induced colonic inflammation, and improves the antioxidant defense system in vivo (Medhi et al., 2008b; Prakash et al., 2008) but further studies are required in this area to better elucidate and understand the potential anti-inflammatory properties of Manuka honey.

As mentioned earlier, Manuka honey is becoming recognised for its health-promoting capabilities (including treatment of burns, diabetic foot ulcers and other wounds, gastro-intestinal illness including gastroenteritis and peptic ulcer and its extensive anti-bacterial properties), and today there are a number of medical grade Manuka honey products available on the market. However, there are issues associated with consuming raw honey for health. These include the amount of sugar that is ingested, and the fact that it can be difficult to consume the quantity of honey needed to ensure an effective bioactive effect in vivo. Further, honey, when consumed orally, is quickly diluted with saliva and/or gastric and intestinal juices and the ingredients are rapidly absorbed or degraded. Thus, any bioactive effects in the mouth or gut are usually limited to the upper gastrointestinal tract and only for a short initial period of time. The ability to capture the bioactive components of Manuka honey into a complex that allows for better delivery therefore holds great potential. Formation of a Manuka honey/cyclodextrin complex allows the sticky, sweet consistency of honey to be transformed into a powder than can easily and readily be mixed into food and drink with relatively little sweetness and taste. Further it allows the bioactive

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components of Manuka honey to be incorporated into capsules, lozenges and inhalers, something which cannot be achieved with raw honey. However, if such a product is to become commercially viable in a medical or health and wellness capacity then it is important that honey researchers and consumers alike understand what effects this complexing may have on the bioactive properties of Manuka honey.

As the results of this small study demonstrate, the two samples of raw Manuka honey tested (MGO250™ and MGO400™) did display relatively high levels of antioxidant activity (11.72 and 9.84 sodium urate equivalents), and more than 95% of the total antioxidant activity of the raw honey was retained in the Manuka Cyclopower complex when compared on a weight per weight of honey basis. Although only a small number of samples were tested, the results were similar and indicated that there was no synergistic antioxidant activity of Manuka honey with cyclodextrin.

However, there does not appear to be any direct correlation with the total antioxidant levels and the anti-inflammatory results observed in this study. The MGO400™ Manuka honey, for example, showed a marked increase in anti-inflammatory activity when complexed and tested at 400 g/ml but this was not observed when tested at the lower concentration of 100 g/ml. Further, the lower methylglyoxal honey (MGO250™) actually showed a 30% stimulation of TNF-a when measured at 100 g/ml but not when tested at 400 g/ml. The validity of the anti-inflammatory assay was not in question as both positive and negative controls reacted appropriately, suggesting that other factors in the assay or the honey are leading to the contrasting results. Tonks et al., (2003; 2007) have reported that honey can stimulates inflammatory cytokine production (including TNF- release) from monocyte cells (involved in the generation of an immune response), therefore there may well be conflicting processes occurring in Manuka honey samples. Honeys are recognised as being complex in composition, containing acids, phenolic compounds, enzymes, sugars and many, as yet, unidentified substances.

These limited results suggest that Manuka honey does have anti-inflammatory activities (at least when tested under certain conditions) and these may be potentiated by formation of a cyclodextrin-based complex in some samples. Clearly, further research with more samples (both raw and complexed honey) is needed to clarify this situation and to elucidate what pathways or compounds are involved in this anti-inflammatory process. Thus, whilst we have an exciting new technology that may offer advantages (including delayed release, delivery to the large intestine, improved taste and new delivery systems (including nasal and bronchial)) we need to further evaluate these products and their potential clinical applications before drawing more definite conclusions about their use.

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