Instituto Tecnológico y de Estudios Superiores de Monterrey
Campus Monterrey
School of Engineering and Sciences
Characterization and anti-inflammatory effects evaluation of chili oleoresin SNEDDS containing capsaicinoids
A thesis presented by
Ana Emilia Nava Ochoa
Submitted to the
School of Engineering and Sciences
in partial fulfillment of the requirements for the degree of
Master of Science In
Biotechnology
Monterrey Nuevo León, June 3rd, 2021
iv Dedication
To my mom who has been a role model for me as a person but also as a professional. To the one I have always seen working and studying and motivates me to keep going and keep learning. You have taught me how to be responsible and balanced.
Most importantly, you motivate me to keep being better and thanks to you I’m pursuing this degree.
To my dad who has taught me how to balance my studies and professional life with recreational activities that have taught me how to grow personally and be more disciplined and constant. You have taught me that at the end, everything gets solved and that nothing is ever good or bad, it just is. Thanks to you I have learned to let things go and you made me realize that I’m “Better than Perfect”.
To both of you, I owe everything I have and am. I’ll never cease to thank you all the effort it has taken you to get me where I am today. Thanks for always being there for me, encouraging me and giving me your unconditional love.
To my sisters, who always make me realize I don’t have to take everything serious and are always taking me out of my comfort zone. I love you lady girls.
To my grandparents (Nany, Lulu & Joaquin), uncles and aunts who have been my biggest supporters since day one and are always there for me. It’s amazing to have a family as united as mine.
To my grandfather Ruben, who is in heaven. I always wanted to include him in my thesis dedication just as he thanked me on his.
To Carlos, who has given me unconditional support these last years and has encouraged me to keep moving forward. Thanks for always being there for me and for making my life more fun. You have been a role model of hard work and persistency.
Thank you for teaching me that distance means nothing and that sometimes you have to risk things in order to become a better person.
v
Acknowledgements
First of all, I would like to acknowledge all the support given by my advisors Dr.
Daniel Guajardo and Dr. Marilena Antunes during these hard years. Thank you for supporting me in difficult times and for sharing all your knowledge with me. I appreciate your guide, advice and patience with me. Thanks for risking your personal time on helping me when I needed it and for being there for me not only as advisors but also as personal support. I also want to thank Dr. Mariana Martinez for always helping me and being there for me when I got stuck on something. Thanks for your patience and your help.
I want to thank my friends Antonio Jimenez and Kathya Huesca who have helped me through all this time. Thank you for helping me on all my lab activities and writing process. But most importantly, thank you for being my friends during this hard time. Yours is a friendship I’ll cherish all my life.
To my greatest support for 10 years now, Alejandra Figueroa. You’re my right hand and my third sister. You are the best, intelligent, careless henchman on this ambitious road. Thanks for always keeping my feet off the ground and make me realize that there’s no need to stress that much on everything.
A big thank you my sister Kathya Ximena who helped me do all the Figures on this and previous works.
Lastly, I would like to thank to the Consejo Nacional de Ciencia y Tecnología (CONACYT) for scholarship (CVU: 1006991) and the Tecnológico de Monterrey for the academic scholarship and to NutriOmics focus group for their economic support.
Also, I acknowledge the donation of oleoresin by Intalmesa and Applied Biotech.
vi
Characterization and anti-inflammatory effects evaluation of chili oleoresin SNEDDS containing capsaicinoids
By
Ana Emilia Nava Ochoa
Abstract
Capsaicinoids are the compounds found in chili plants (Capsicum genus) that confer the pungency to the plant. These have been evaluated due to their anti- inflammatory and analgesic effects as they act directly by binding to Transient receptor potential vanilloid 1 (TRPV1), responsible for the perception and sensation of pain.
Transdermal application of these compounds has its side effects as these compounds produce irritation in skin and burning sensation. Therefore, this work is focused on implementing these compounds into self-nanoemulsifying drug delivery systems (SNEDDS) for their potential of enhancing these compounds’ anti-inflammatory properties while reducing its side effects by changing its particle size and its properties.
Oleoresins evaluated were obtained from Guajillo chili, Arbol chili and a synthesized oleoresin. Characterization and quantification of capsaicinoids on oleoresins were done by high performance liquid chromatography coupled with diode array detectors (HPLC- DAD) and they were reported as capsaicin equivalents. SNEDDS formulations stability was evaluated after 45 days and they were characterized by their particle size, zeta potential and polydispersity index (PDI) on a Zetasizer. SNEDDS entrapment efficiency (%) was also evaluated by HPLC-DAD. Lastly, in vitro tests were done to indicate anti- inflammatory activity. RAW 264.7 cells were used to determine nitric oxide (NO) inhibition, human dermal fibroblasts (HDFa) were used to evaluate cellular uptake and a fluorescent activity assay kit was used to evaluate COX-2 inhibition. As a result, the major capsaicinoids found were capsaicin and dihydrocapsaicin on Arbol oleoresin and four capsaicin analogues on the synthetic oleoresin. No capsaicinoids were found on Guajillo sample. Average entrapment efficiency was of 91.03%. Formulation stability was determined by PDI values which were lower than 0.500 on all samples and by zeta potential which showed an average of -18.98 mV, indicating formulation stability for both parameters. For cellular assays, RAW 264.7 cells showed no cytotoxicity from these compounds. Higher NO inhibition (%) was shown on SNEDDS formulation with synthetic oleoresin compared to normal oleoresins with an inhibition of 83.1 ± 1.99%. Synthetic oleoresin also showed the highest COX-2 inhibition with an activity of 79.19 ± 1.07%.
Despite cellular uptake being higher on oleoresins, nanoemulsions showed a high cellular uptake of 21.18 ± 0.07% on synthetic SNEDDS formulations. This study showed the capability of oleoresins containing capsaicinoids incorporated into SNEDDS as anti- inflammatory agents. This type of formulations has the potential to be applied transdermally in order to be used on treatments for inflammation.
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List of Figures
Figure 1. Capsaicinoids chemical structure. ... 5 Figure 2. Capsaicinoids elution pattern on chromatogram. (A) nordihyrocapsaicin (B) capsaicin (C) dihydrocapsaicin (D) homocapsaicin (E) homodihydrocapsaicin. ... 7 Figure 3. Effects of capsaicin binding to TRPV1 channel that cause analgesic and anti- inflammatory effects. Abbreviations: Transient receptor potential vanilloid 1 (TRPV1), Phosphatidylinositol 4,5-biphosphate (PIP2), Prostaglandin E2 (PGE2), Interleukin-1 Beta (IL-1B), Interleukin 8 (IL-8), Interleukin 10 (IL-10), Nuclear factor kappa-light-chain- enhancer of activated B cells (NF-kB), inducible nitric oxide synthase (iNOS),
Cyclooxygenase 2 (COX-2). ... 13 Figure 4. Comparison of skin permeation between conventional particles and nano- sized carriers. Nano-sized carriers penetrate the three layers of skin and hair follicles allowing the compound to get to its desired site of action. ... 16 Figure 5. Polydispersity index (PDI) representation. Left: monodispersed solution with a PDI<0.5. Right: polydisperse solution of a PDI>0.5. ... 20 Figure 6. SNEDDS Chemical structure. ... 22 Figure 7. SNEDDS Control 1 (left) and SNEDDS Control 2 (right) visual differences after 10 days. ... 29 Figure 8. Visual differences of SNEDDS control with oleoresins. (A) SNEDDS 1 at 4ºC.
(B) SNEDDS 1 at 25ºC. (C) SNEDDS 2 at 4ºC. (D) SNEDDS 2 at 25ºC. ... 30 Figure 9. Chromatograms of different samples. (A) ABX oleoresin (B) Capsaicin
standard (C) Arbol (D) Guajillo. ... 32 Figure 10. Capsaicinoids wavelength at the UV spectrum. ... 34 Figure 11. ABX chemical structure compared to capsaicin chemical structure. ... 36 Figure 12. Nitric oxide inhibition (%) of Arbol, Guajillo and ABX samples caused by oleoresins (0.10%) and nanoemulsion formulations (0.075% and 0.10%). Different letters represent a statistical difference (p<0.05). ... 39 Figure 13. Cell viability (%) of RAW 264.7 cells after oleoresins and formulations
application. Different letters represent a statistical difference (p<0.05). ... 41 Figure 14. Nanoemulsions and oleoresins ABX and Arbol cellular uptake (%) on HDFa cells after 24 hours. Different letters represent a statistical difference (p<0.05). ... 44 Figure 15. COX-2 inhibition (%) of arbol and ABX oleoresins and nanoemulsion at 0.10%. Different letters represent a statistical difference (p<0.05). ... 47 Figure 16. Sensory evaluation of Arbol. Pictures were taken at time 0, 30 min, 60 min and 120 min after application. 30 min picture was enhanced to show visible skin
irritation. ... 50 Figure 17. Sensory evaluation of ABX. Pictures were taken at time 0, 30 min, 60 min and 130 min after application. ABX also showed irritation after 24 hours. ... 50
viii
List of Tables
Table 1. Levels of pungency based on SHU (Gonzalez-Zamora et al. 2013) ... 7 Table 2. Properties and benefits of nano-sized carriers. ... 17 Table 3. SNEDDS formulations: oil, surfactant, co-surfactant and oleoresin composition ... 24 Table 4. Identification and quantification of most abundant capsaicinoids associated with oleoresins. ... 33 Table 5. Formulations' characterization based on capsaicin equivalents content, particle size, Polydispersity index (PDI), Z Potential and entrapment efficiency. ... 35 Table 6. Formulations’ characterization study after 45 days based on particle size, polydispersity index and zeta potential. ... 37
ix Contents
Chapter 1 1
Hypothesis and Objectives 4
a) Hypothesis 4
b) General objectives 4
c) Specific objectives 4
1. Theoretical framework 5
1.1. Capsaicinoids 5
1.1.1. Functional properties 5
1.1.1. Techniques of capsaicin characterization 6
1.1.2. Capsaicinoids topical application 8
1.1.2. Previous work on capsaicin effects on inflammation and analgesia 9
1.2. Inflammation/analgesia 11
1.2.1. Inflammation and analgesic mechanisms 11
1.2.2. Capsaicinoids effect in inflammation and analgesia 12
1.2.3. Inflammation assessment techniques 13
1.2.3.1. In vitro 13
1.3. Limitations 14
1.4. Nanotechnologies 15
1.4.1. Benefits on using nanotechnologies 15
1.4.2. Nanotechnologies’ properties 15
1.4.3. Nanocapsules characterization and quantification techniques 19
1.4.4. Delivery systems 20
1.4.5. SNEDDS 21
Chapter 2 23
2. Materials and methods 23
2.1. Chemicals and reagents 23
2.2. Biological material 23
2.3. Development of SNEDDS formulation using capsaicin as standard 23
2.4. Macroscopic SNEDDS formulations aspects 24
2.5. Capsaicinoids oleoresin obtaining 25
2.5.1. Capsicum annum v. de Arbol 25
2.5.2. Capsicum annum v. Guajillo 25
2.5.3. Synthetic oleoresin 25
2.6. Capsaicinoid characterization by liquid chromatography coupled to diode array
(HPLC-DAD) 26
x
2.7. Preparation and characterization of SNEDDS 26
2.7.1. Capsaicinoids SNEDDS preparation 26
2.7.2. Formulations’ characterization: Size and zeta potential 26 2.7.3. Quantification of entrapped capsaicinoids in formulations by liquid
chromatography coupled to diode array (HPLC-DAD) 27
2.8. In vitro studies 27
2.8.1. Cell culture 27
2.8.2. Nitrite assay to determine inflammation using RAW 264.7 as model 27
2.8.3. Cellular uptake assay 28
2.8.4. COX-2 inhibition as inflammation marker 28
2.9. Statistical analysis 28
Chapter 3 29
3. Results and discussion 29
3.1. Analysis of the formulations’ stability 29
3.2. Capsaicinoid characterization 31
3.3. Capsaicin formulation quantification and characterization 34 3.4. Effect of formulations and oleoresins in LPS-induced nitric oxide (NO) inhibition
in RAW 264.7 cells 38
3.5. Cellular uptake in HDFa cells 42
3.6. Measurement of COX-2 inhibition in RAW 264.7 cells 45
4. Conclusions and future work 47
5. Appendix A 49
5.1.1. Sensory irritation evaluation 49
Abbreviations 51
6. References 52
7. Published papers 64
8. Curriculum Vitae 65
1
Chapter 1
Introduction
Among cultivars in Mexico, chili plants are one of the most important given that it’s cultivated in 25 of its states and each contains a wide genetic variety of this cultivar.
Capsicum, the genus of this plant, belongs to the Solanaceae family. Among the domesticated species of this plant there is Capsicum frutescens, Capsicum chinense and Campsicum annuum (Basharat et al., 2021). The most common varieties grown in Mexico are Ancho, De arbol, Guajillo, Habanero, Jalapeño, Pasilla, Piquin and Serrano (González-Zamora et al. 2013). These plants contain compounds such as phenolics, flavonoids, alkaloids, carotenoids and capsaicinoids. The aforementioned are responsible for the pungency of the plants. These compounds are known to be produced by chili plants to protect themselves against predators (Hayman & Kam, 2008). These have been investigated for their application in cosmetics, food and therapeutic industry. They have been used mainly by their effects on pain relief, weight reduction, anti-cancer properties, cardiovascular effects, dermatological conditions, among others (Sharma et al., 2013).
Nowadays, emphasis has been made in investigating these compounds in treatments that reduce pain caused by several conditions given their analgesic and anti-inflammatory properties.
Capsaicinoids have been proved to treat topically several types of neuropathic pain caused by chronic diseases (Mason et al., 2004). The majority of these conditions provoke painful symptoms such as burning, tingling skin or shooting (electric shock-like feelings). Treatment of these conditions often includes drugs that fail to lower the pain sensation caused by them. The need of implementing compounds that reduce these symptoms comes from this failed attempt to treat them with common analgesic and anti- inflammatory drugs. Commonly used analgesics such as paracetamol, ibuprofen and diclofenac have a failure rate of 66% in patients with acute pain (Moore, et al., 2013). The implementation of special analgesic agents has also been widely studied in the treatment for pain. These agents include tricyclic antidepressants, serotonin noradrenaline reuptake inhibitors, anticonvulsants, opioids, cannabinoids, among others. The efficacy of these
2
has been proved in clinical trials for some neuropathic conditions that cause several types of pain (Reyes-Escogido et al., 2011). Still, the implementation of them has adverse secondary effects such as drowsiness, arrhythmia, hypertension, nausea, ataxia, seizures, hypotension, among many others (Moulin et al., 2007). These secondary adverse effects make it necessary to find alternative analgesic agents. For this, nutraceuticals have been studied as an alternative to treat conditions that fail to be treated with common drugs.
Nutraceuticals are defined as compounds extracted from food that can provide health benefits by preventing or treating several conditions (Chen & Hu, 2019).
Capsaicinoids are examples of nutraceuticals that have been added to commercial products to treat pain and inflammation. Although these contain beneficial effects, there are only few commercially available products given the limitations in their physicochemical properties and their secondary effects. These compounds have low solubility, are susceptible to oxidation, have low selectivity and high toxicity depending on concentration(Saffarionpour, 2019; Luo et al., 2011). These negative effects cause their therapeutic efficacy to be low (Kumar et al., 2019). Also, their secondary effects consist of skin irritation, erythema and the sensation of stinging and burning (Raza et al., 2014).
Alternatives of delivering these compounds have to be found in order to reduce these effects and to stabilize these compounds without reducing their potential analgesic and anti-inflammatory properties. Nano structures have the potential to act as delivery systems of nutraceuticals such as capsaicinoids. By doing so, these compounds can be delivered into the desired site of action and their stability, bioaccessibility and bioavailability can be enhanced (Lu et al., 2017).
Nanoparticles behave differently than conventional size particles. By their characteristics, they can have improved bioavailability and enhance bioactivity of compounds (Ingle et al., 2020). Other characteristics that are enhanced by reducing compounds to a nanoscale are: their preservation during process, their interaction with other compounds (like the ability of being dispersed in water-based systems), their release can be easily controlled, and they are harder to degrade in certain pH conditions when encapsulated (Singh et al., 2019). This can also help reduce the toxicity of the drug and lower intake dose as some drugs’ efficiency is related to its particle size (Rizvi &
3
Saleh, 2018). The incorporation of compounds into nano-sized delivery systems can enhance their penetration through skin because their size allows them to pass through tissue and cell barriers (Agrawal et al., 2015; Chen & Hu, 2019). A nano-sized delivery system can help penetrate skin layers and allows the compound to get to the desired site of action beneath the epidermis or through hair follicles. This is given by its ability of crossing some molecular barriers due to their small size and large surface area (Rizvi &
Saleh, 2018).
The implementation of capsaicinoids into a nanostructured delivery system is thought to be an effective solution in reducing its adverse effects while keeping their anti- inflammatory and analgesic effects (Wang et al., 2017). The use of nanotechnologies has been emerging in the last years and the outcomes of implementing them as therapeutic agents have given positive results. Few studies exist that implement capsaicinoids into nanostructured delivery systems like nanoemulsions, nanoliposomes, nanostructured lipid carriers, among others (Kim et al., 2014; Giri et al., 2017; Agrawal et al., 2015). The results of all of them conclude that implementing these compounds into nano-scaled delivery systems has fewer secondary effects and similar analgesic and anti-inflammatory properties compared to the direct implementation of the capsaicinoids. SNEDDS are a type of nano-sized delivery system that have arisen interest in applying it to treat several conditions given their physicochemical properties. These are easily solubilized and have improved dissolution and absorption rates which makes them attractive to use. Also, these systems allow industrial production at low cost, high stability and reproducibility (Rahmann et al., 2020).
The following study was based on the incorporation of oleoresin containing capsaicinoids into SNEDDS to evaluate their effect as anti-inflammatory and analgesic agents for their potential use on treating different types of pain. The purpose of this project was to develop and characterize a low-dose oleoresin nanoemulsion to evaluate its stability, particle size, zeta potential, entrapment efficiency and in vitro effects on inflammation markers.
4 Hypothesis and Objectives
a) Hypothesis
The incorporation of capsaicinoids into SNEDDS has anti-inflammatory properties with reduced skin irritation compared to regular capsaicinoid formulations.
b) General objectives
To develop SNEDDS that integrate oleoresin containing capsaicinoids and evaluate its physicochemical properties and its anti-inflammatory potential applied topically.
c) Specific objectives
i. Develop a SNEDDS formulation using capsaicin as standard to establish its oil-water ratio in order to make it physically stable.
ii. Obtain oleoresins from two different chili matrixes (Capsicum annum v.
Guajillo, Capsicum annum v. Arbol) and a synthesized oleoresin to compare their capsaicinoid profile and concentration.
iii. Characterize samples in terms of capsaicinoids identification and
quantification through High Performance Liquid Chromatography coupled to Diode Arrays Detector (HPLC-DAD) to see their profile and concentration.
iv. Formulate capsaicinoids into SNEDDS in different proved effective low-dose concentrations (0.025%, 0.050%, 0.075% and 0.10%).
v. Evaluate physicochemical properties from capsaicinoid SNEDDS formulations (physical stability through 45 days, drop size, PDI and zeta potential on
Zetasizer and entrapment efficiency on HPLC-DAD).
vi. Evaluate cellular uptake of chili oleoresins vs SNEDDS using Human Dermal Fibroblasts (HDFa) as model.
vii. Evaluate chili oleoresins vs SNEDDS inflammatory effect on inflammatory biomarker NO using macrophages from from mus musculus (RAW 264.7) as model.
viii. Evaluate chilli oleoresins vs SNEDDS inflammatory effect on inflammatory biomarker COX-2 using a fluorescent activity assay kit.
5 1. Theoretical framework
1.1. Capsaicinoids
1.1.1. Functional properties
Chili oleoresins are composed mainly by lipids, carotenoids and capsaicinoids (de Aguiar et al., 2013). Capsaicinoids refer to a variety of compounds such as capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin and homodihydrocapsaicin.
Capsaicin and dihydrocapsaicin represent around 90% of the total capsaicinoids in chili peppers (Lu et al., 2017). Different capsaicinoids differ from each other in their lateral chain carbons and by the presence/absence of unsaturations (Figure 1). They are naturally synthesized in the placenta of chili fruits by the addition of a branched fatty acid to vanillylamine (Sharma et al., 2013). Capsaicin and dihydrocapsaicin are non-polar, volatile, hydrophobic, colorless and odorless compounds that have a molecular weight of 305.4 g/mol and 307.43 g/mol, respectively, and are fat, alcohol and oil soluble (Reyes- Escogido et al., 2011; Sharma et al., 2013).
Figure 1. Capsaicinoids chemical structure.
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Capsaicinoids are made of a nonpolar phenolic structure which means they can’t be solubilized in water. Therefore, nonpolar solvents are used to extract these compounds in order to maintain their properties. Topical absorption of these compounds has been reported up to 94% given its chemical structure (Basharat et al., 2021).
Biosynthesis of capsaicinoids is done in two ways: via the phenylpropanoid pathway or the fatty acid metabolism. The first pathway will determine the structure of the phenolic compound, the second one determines the molecule’s fatty acids. The concentration of these compounds is increased during the development of the fruit where it reaches its maximum levels at 40-50 days of fruit maturity. Afterwards, they degrade due to peroxidase action. Also, it has been proved that hydric stress can increase the compounds levels via the phenylpropanoid pathway (Reyes-Escogido et al., 2011).
1.1.1. Techniques of capsaicin characterization
There exist several key methods to characterize compounds such as capsaicin.
Most common methods include spectrophotometry UV-VIS and chromatography. Each of these exist with different modifications depending on the sensitivity of the method and in order to have faster characterization. For chromatography, analytical separation, quantitation and identification of capsaicinoids can be achieved. For this, methods such as reverse-phase HPLC coupled with mass spectrometry, capillary electrophoresis and magnetic resonance have been applied (Gonzalez-Zamora et al., 2013). HPLC is a widely used method to characterize capsaicinoids. HPLC-DAD allows quantification of these compounds even at low levels (Reyes-Escogido et al., 2011). Capsaicinoids have already been characterized on different chili matrixes (Gonzalez-Zamora et al., 2013) (Figure 2). Retention time varies depending on the method and equipment, but the elution pattern always stays similar.
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Figure 2. Capsaicinoids elution pattern on chromatogram. (A) nordihyrocapsaicin (B) capsaicin (C) dihydrocapsaicin (D) homocapsaicin (E) homodihydrocapsaicin.
These methods can also help identify these compounds, separate them and quantify them. Thanks to capsaicinoid concentration, relative pungency can be calculated based on Scoville heat units (SHU) as 15 SHU per 1ppm of capsaicinoids. There exist different levels of pungency based on SHU for different types of chili plants (Table1) (Gonzalez-Zamora et al., 2013).
Table 1. Levels of pungency based on SHU (Gonzalez-Zamora et al. 2013)
Level SHU*
NON-PUNGENT 0-700
MILDLY PUNGENT 700-3,000
MODERATELY PUNGENT 3,000-25,000
HIGHLY PUNGENT 25,000-70,000
VERY HIGHLY PUNGENT >80,000
*SHU = Scoville Heat Units
8 1.1.2. Capsaicinoids topical application
Capsaicin is related to treating several types of neuropathic pain which can affect the nervous system. There are several types of neuropathic pain such as postherpetic neuralgia, post-stroke pain, diabetic neuropathy, chronic post-operative pain or arthritic disorders (Baron, R. et al., 2010; Mason, L. et al., 2004). Topical capsaicinoids have the ability to act directly in the skin in order to reduce pain by a process called defunctionalization of nociceptor fibers. This is achieved by multiple applications of these compounds which can lead to loss of the ability to respond against external stimuli (Wang et al., 2017). The dose of application is very important, and the effects of these compounds are said to depend on it along with the time of exposure. Exposure to doses over 100 mg of capsaicin per kg body weight can cause ulcers and accelerate the development of certain cancers. On the other hand, low concentrations are related to analgesic properties and can be found in few commercial products (Rollyson et al., 2014).
Capsaicin commercial products have been studied for their absorption rates and metabolism. Metabolism of capsaicin on the skin is slow, it has been reported to last about 20 hours in the skin. Also, its transport through skin depends on the dose administered and, on the vehicle used to administer it.
Currently, there are two different types of formulation commercially available based on their capsaicinoid concentration. High-dose capsaicin is normally referred to application of 8% of the compound while low-dose capsaicin is referred to 0.025%- 0.075% (Webster et al., 2010). High doses are the most commonly evaluated and there’s a lack of studies on the effect of low-dose application. One important difference of these formulations is that high-doses require a single application while low-doses require several applications. Products with both formulations contain undesirable effects. Data regarding low doses is limited and studies have shown different conclusions on their effectiveness (Smith et al., 2014). Still, there are some results that show them to have a potential as a therapeutic option for patients that have failed to find pain relief using other types of formulations. Topically applied capsaicin has shown positive effects on pruritus on low-dose concentrations (Basharat et al., 2021). These doses have also shown effectiveness in patients with severe fibromyalgia; thus, it can be hypothesized that these doses are effective depending on the type/severity of the disease (Smith et al., 2014).
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1.1.2. Previous work on capsaicin effects on inflammation and analgesia In vivo studies have shown some positive outcomes of topically applied capsaicin in different conditions. The effect of topical application of a capsaicin-containing cream on rats was evaluated to check for inflammation and thermal nociception (Yoshimura et al., 2000). The cream was applied once a day and concentration of capsaicin was 0.10%, 0.30% and 1% in different rats. Results showed a stimulation of pain receptors on the capsaicin area. Also, after one day of application a block on neurogenic inflammation was visible. This study concluded that continuous application of capsaicin has a therapeutic effect on noxious pain. Another study investigated the effect of capsaicin in a rat model of inflammatory muscle pain (Fang et al., 2020). A solution with 0.30% of capsaicin, along with other irritants, was applied to rats and it was evaluated using electrophysiological methods. Their results confirmed that capsaicin relieved inflammatory muscle pain and an inhibition of nociceptive neurons was visible.
There are few clinical trials that have proved the efficacy of Capsaicinoids when administering it topically. Some of them consist of only capsaicin administered alone while others evaluate its effect along with other drugs. The topical administration of capsaicin in patients with post-herpetic neuralgia, diabetic neuropathy and chronic musculoskeletal pain has also been evaluated (Anand & Bley, 2011). Another studyshowed the analgesic effects of capsaicin by administering it to patients along with doxepin in a placebo- controlled, double-blind study (McCleane, 2001). The combination of doxepin with 0.025% of capsaicin showed the reduction of shooting pain in patients and also reduction of patients’ perception of sensitivity after application. In patients with HIV-Distal Symmetric Polyneuropathy associated pain, a commercial patch was applied directly to skin and results showed no increase in skin irritation and prolonged pain reduction associated with their disease (Simpson et al., 2013). These patients had a decrease on the Neuropathic Pain Rating Scale (NPRS) of 25.8% after 12 weeks of treatment. A review of clinical trials of topical capsaicin as treatment for pain (Baron et al., 2010) suggested a 50% reduction of pain in patients with different conditions. These results were obtained by analysis of data of the different clinical trials which showed a statistical difference of results of capsaicin and placebo patients. Along with the positive effects
10
found in these studies, several secondary effects after administering capsaicin are also reported.
Several secondary effects of capsaicin have been found when administered topically. In the previously mentioned review of clinical trials, it was found that around 54% of the patients administered with capsaicin had adverse effects after its application (Mason et al., 2004). It has also been reported that capsaicin treatments applied to patients increased burning pain sensations after applications, but these were difficult to identify as effects of the compound or their own pain (McCleane, 2001). Also, capsaicin administered alone has shown low potential penetrating human skin showing reduction of transdermal delivery in skin (Anand & Bley, 2011). This study also reported that the low doses that are able to penetrate skin, have short exposure in the active site. It is believed by these authors that if capsaicin is delivered faster, its topical application could be more tolerated due to its potential rapid metabolism after penetrating skin and its possible low interaction with superficial skin layers.
For commercial products, a dermal patch containing capsaicin is available in the US and was approved by the Food and Drug administration. This patch is used for pain relief and has a concentration of 8% capsaicin (Simpson et al., 2013). In a clinical trial from this product, patients showed reduction of their disease-related pain and this effect lasted for about 12 weeks. Still, the effects of this patch show the same characteristics as applying capsaicin directly into skin: redness, irritation, burn sensation, among others (McCleane, 2001). There are some creams that contain low doses of capsaicin (0.075%) to treat postherpetic neuralgia and diabetic neuropathy and others that treat osteoarthritis and rheumatoid arthritis (0.025%). Both of these need an application of 3-4 times daily for a specific period (Mason et al., 2004). Other registered formulations of capsaicin exist such as dry powder, liquid capsaicin and liquid spray (Sharma et al., 2013). All of these are formulated to find the proper amount to be administered in order to treat pain via inflammation and analgesic mechanisms but none of them prevent the undesirable side effects of capsaicin.
11 1.2. Inflammation/analgesia
1.2.1. Inflammation and analgesic mechanisms
Inflammation is a cascade of events that are made by certain stimuli such as pathogens or noxious agents. This cascade is presented by some characteristics such as heat pain and swelling. The body creates this process as a way to repair and defend itself from stimuli. Prolonged inflammation can lead to a dysfunction (Fujiwara et al., 2005).
When the process of inflammation is activated, several compounds are released from cells or tissues such as cytokines, prostaglandins, inflammatory molecules, among others. Among the most characteristic compounds in the process of inflammation there are interleukins, which regulate inflammatory and anti-inflammatory responses.
Interleukin-10 (IL-10) is an anti-inflammatory interleukin produced by macrophages, fibroblasts and T lymphocytes. This interleukin has the ability to inhibit production of pro- inflammatory interleukins and Tumor Necrosis Factor (TNF). This interleukin can inhibit prostaglandins such as Prostaglandin E2 (PGE2) by down regulating COX2. It also induces production of anti-inflammatory molecules (Fujiwara et al., 2005). COX2 is an enzyme that is induced in response to stimulants on the site of inflammation. This is induced in macrophages and endothelial cells by proinflammatory cytokines. These are said to be responsible for edemas and vasodilatation in the process of inflammation. Inhibition of mediators produced by this enzyme is related to preventing some inflammatory diseases.
The most important product by this enzyme is PGE2 (Hseu et al., 2005). Inducible nitric oxide synthase (iNOS) is another enzyme expressed from cells (microglial cells, keratinocytes and epithelial cells) under some inflammatory conditions. When there are pro-inflammatory stimuli, iNOS produces nitric oxide which is related to DNA damage (Murakami et al., 2007). Also, TNF-α is related to the stimulation of hyperalgesic cytokines Interleukin-8 (IL-8) and Interleukin-1 (IL-1) (Ferreira et al., 1993).
All of the previously mentioned compounds are related to the inflammation process and are targeted directly by several drugs. By the inhibition or overproduction of these mediators, the inflammation process can be interrupted or enhanced. If this cascade of events is targeted by some drugs, there would be an inflammation reduction and it has been proved that capsaicin has a direct mechanism of action in these (Kim et al., 2003).
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1.2.2. Capsaicinoids effect in inflammation and analgesia
Capsaicin has been proved to reduce inflammation and pain caused by it due to its mechanism of action. For the reduction of pain, it can bond to the Transient receptor potential vanilloid 1 (TRPV1); a receptor expressed in sensory neurons which is a cationic channel located in nociceptive neurons, responsible for the perception or sensation of pain(Reyes-Escogido et al., 2011). Capsaicin, as an agonist of TRPV1, can activate it and make it go into a refractory state which makes it resistant to several stimuli such as pain and proinflammatory agents. When TRPV1 is activated by capsaicin, its refractory state makes it resistant to exogenous pain and leads to changes in extracellular calcium in the receptor protein which closes TRPV1 (Sharma et al., 2013). The intracellular calcium activates calmodulin and calcineurin which dephosphorylate and inhibit the receptor, respectively, and degrades PIP2 (Phosphatidylinositol 4,5-bisphosphate)which helps recruit proteins to the plasmatic membrane for its activation and signal transduction (Chung & Campbell, 2016). When this happens, membrane potential is lost, and sensorial neuropeptides are liberated, and their restoration is blocked by substance P and somatostatin. Therefore, it is said that intracellular calcium override leads to the neuron’s defunctionalization due to its membrane potential loss and causes reversible retraction of terminal nervous fibers in dermis and epidermis (Luo et al., 2011). Because TRPV1 is responsible for the release of transmitters, its blocking inhibits the transmission of signals from nociceptive sensory neurons (Winter et al., 1995).
Capsaicin can also have anti-inflammatory properties as it has been known to reduce the production of PGE2 by the inhibition of COX-2 activity and the expression of iNOS. Also, capsaicin helps express IL-10 via nuclear factor kappa-light-chain-enhancer of activated B cells (NKkB), which is an anti-inflammatory cytokine and suppress Interleukin 1 beta (IL-1ß) and interleukin 8 (IL-8) which are pro-inflammatory cytokines (Lu et al., 2020) (Figure 3).
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Figure 3. Effects of capsaicin binding to TRPV1 channel that cause analgesic and anti- inflammatory effects. Abbreviations: Transient receptor potential vanilloid 1 (TRPV1),
Phosphatidylinositol 4,5-biphosphate (PIP2), Prostaglandin E2 (PGE2), Interleukin-1 Beta (IL-1B), Interleukin 8 (IL-8), Interleukin 10 (IL-10), Nuclear factor kappa-light-chain-
enhancer of activated B cells (NF-kB), inducible nitric oxide synthase (iNOS), Cyclooxygenase 2 (COX-2).
1.2.3. Inflammation assessment techniques 1.2.3.1. In vitro
Inflammation mediators are released as a reaction of the inflammation process.
Main cells responsible in this process are macrophages. These have three main objectives: present antigens, phagocytosis and immunomodulation. On the latter, proinflammatory cytokines and TNF-α are activated. This activation is due to the exposure of any inflammatory stimuli. Most commonly, bacterial lipopolysaccharides(LPS) are implemented, which leads to the activation of macrophages via the immunomodulation process (Fujiwara et al., 2005). Other inflammatory mediators are NO produced by iNOS
14
under pathological conditions. Inhibition of these can be done by inhibiting COX-2. COX- 2 protein is expressed in tissue as a response of stimuli such as UV light exposure or LPS as mentioned before. COX-2 has been a target for analgesic and antiinflammatory remedies (Murakami et al., 2007). In order to assess inflammation of compounds such as capsaicinoids, studies have been made by measuring the previously mentioned markers.
Therefore, quantification of NO, COX-2 and interleukins provide an indirect measurement on the impact of compounds on the inflammation process.
1.3. Limitations
The most common treatments for neuropathic pain, analgesia and inflammation include anti-epileptics and anti-depressants for the strongest type of pain, which are considered first-line treatments. All of these have reported side effects such as nausea, impairment, dizziness among others and their use is restricted to only certain types of pain (Hall et al., 2020). Another type of treatment is the use of opioids which has been reported effective to treat these conditions, but they are extremely controlled as they can cause addiction (Pham & Kim, 2020). Topical analgesic and patches come second for in- site treatment of pain and have proved effects in the treatment of pain with reduced side effects compared to the first-line treatments.
As it has been mentioned, capsaicin can be an effective treatment for several types of pain because it works directly on inflammation and has analgesic effects. However, the topical administration of capsaicin has shown several side effects such as pain, burning, erythema in the application site and the need of repeated applications for it to stop the feeling of burning in the skin. Capsaicin shows physiological effects: the stimulation of the nervous system and induction of intense pain in tissues and its antinociceptive and anti- inflammatory effects (Kim et al., 2014). Given these contradictory effects, implementation into therapeutic drugs has been controversial. Also, these have shown limitations when applying them into drugs given their low solubility, susceptibility to oxidation, low selectivity and high toxicity (Saffarionpour, 2019; Luo et al., 2011). As an alternative to take advantage of its effects and with the purpose of avoiding the contradictory effects, nanostructured compounds are proposed to act as delivery systems in order to deliver the compounds in an encapsulated way in order to protect the compounds and the user.
15 1.4. Nanotechnologies
1.4.1. Benefits on using nanotechnologies
Conventional drugs are not able to have a controlled drug release and sometimes they cannot cross the skin barrier due to their properties. When compounds are reduced to a nano-size they can have enhanced localized effects because they can cross the skin barrier and hair follicles and sometimes, they can present lower toxicity. Active ingredients have enhanced skin penetration due to large surface area, low surface tension and low interfacial tension (Kim et al., 2014). With this, the compound can be delivered directly to the desired site of action, preventing secondary effects that can arise when the compound is normally delivered. Also, this type of technology is said to protect the compound and it can be retained longer in the target side. All of this is achieved given that modification of compounds leads to a modification on their surface charge, size, nanoparticle shape and stealth (Koppa et al., 2020).
1.4.2. Nanotechnologies’ properties
Nano-sized drug delivery systems can enhance the therapeutic properties of their compounds. This type of system has been proved to cross biological barriers which normally retain foreign materials to protect themselves, but small specific molecules are able to cross them (Pham and Kim, 2020). Because the stratum corneum (found in the epidermis, the highest layer of the skin) is lipophilic, high-molecular weight and hydrophilic compounds can’t penetrate this layer. On the contrary, the next layer (epidermis) is hydrophilic which makes it a limitation for any compound to penetrate either one of these layers (Kim et al., 2014). The stratum corneum presents a big challenge for topical drug delivery because it is a limiting step in drug skin transport given its barrier property that impedes the transport of drug through skin, mostly lipophilic compounds. The most important limitation is the molecular weight of compounds and the affinity for lipophilic or hydrophilic compounds. Drug delivery through skin can achieve controlled release of compounds on specific site of action (Mota et al., 2017).
Nano-sized drug delivery systems improve their drug specificity into the target because it is proved that they avoid non-specific drug availability in unwanted sites.
Furthermore, it allows for compounds to have reduced doses as they have the same
16
activity as normal-sized compounds, and they can achieve high concentration that can reach a therapeutic effect. Compounds’ solubility is enhanced because these delivery systems form stable droplets where the compound is encapsulated or emulsified (Beg et al., 2020). By their characteristics, they can have improved bioavailability and enhance bioactivity of compounds (Ingle et al., 2020). Other characteristics can also be enhanced by reducing compounds to a nanoscale such as their preservation during process, their interaction with other compounds (like the ability of being dispersed in water-based systems), their release can be easily controlled and they are harder to degrade in certain pH conditions when encapsulated (Singh et al., 2019). It can also help reduce the toxicity of the drug and lower the intake dose for patients as some drugs’ efficiency is related to its particle size (Rizvi & Saleh, 2018). The incorporation of compounds into nano-sized delivery systems can enhance their penetration through skin (Agrawal et al., 2015) (Figure 4). Their size allows them to pass through tissue and cell barriers (Chen & Hu, 2019). A nano-sized delivery system can help penetrate all skin layers and allow the compound to get to the desired site of action beneath the epidermis given its ability of crossing some molecular barriers due to their small size and large surface area (Rizvi &
Saleh, 2018).
Figure 4. Comparison of skin permeation between conventional particles and nano- sized carriers. Nano-sized carriers penetrate the three layers of skin and hair follicles
allowing the compound to get to its desired site of action.
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The most common nano-sized delivery systems are nanoemulsions, nano- liposomes, nanocapsules, solid-lipid nanoparticles and nanostructured lipid carriers. The type of vehicle to choose for the active compound is important given the nature of it.
Parameters that influence this are particle size, thickness of layers, chemical interaction between its phases (Surassmo et al., 2010) and the characteristics of the compound like its polarity, solubility, desired site of action among others. Some of the benefits that have been acquired to these structures are that they can control the release of the active compound, protect it against external factors, improve bioaccessibility and bioavailability, enhance skin permeability and maintain the compound stable until it reaches the desirable site of action (Table 2).
Table 2. Properties and benefits of nano-sized carriers.
Nano-sized carrier
Structure Properties Benefits
Nanoemulsion ● Homogenized two
immiscible liquids
● Lipid based.
● Suitable for
hydrophobic/lipophilic compounds
● Protect active compounds against oxidative degradation
● Controlled release
● Promote bioaccessibility
● Enhance permeability
● Stable against separation
● Can help disperse water in oil or oil in water.
Nanoliposome ● Compound
encapsulated by lipid bilayers.
● Made of phospholipids and cholesterol.
● Has hydrophilic and lipophilic groups in the same molecule.
● Enhance solubility, stability and bioavailability
● Compounds’ encapsulation, delivery and liberation are facilitated
● Easy and rapid lipid exchange between lipophilic compound and cell membrane
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● Controlled release
Nanocapsule ● Core-shell morphology
● Oil or aqueous cavity depending on
compound
● Compounds
entrapped by polymer membrane
● Good for lipophilic compounds
● High encapsulation efficiency
● Improves compound’s stability
● Controlled release
● Controlled permeation in skin
Solid lipid nanoparticle
● Oil phase is in solid state
● Active compounds can be in the core or in the surface
● Higher stability
● Higher biocompatibility
● Better delivery and controlled release than other nano-sized carriers
● Protects active compound from degradation
● Easy to scale-up Nanostructured
lipid carrier
● Solid lipid dispersed in liquid lipid in an
aqueous medium
● Suitable for lipophilic compounds
● Enhanced stability
● Higher dispersibility of active compound in aqueous medium
● Better permeation of skin
● Enhanced drug localization
● Easy to scale-up
The properties and benefits of these nano-sized carriers have a direct effect on the performance of the active compound from the moment it has contact with the skin, as it
19
improves absorption, to the point where it gets to the desired site of action. There is proof that incorporating some compounds into a nano-sized carrier helps improve their activity.
1.4.3. Nanocapsules characterization and quantification techniques
Key methods to characterize nanocapsules include assessment of morphology, size, surface properties and physicochemical properties which affect the stability and the function of the compounds. Particle size can affect appearance, stability and release properties. Among the methods to obtain this are static light scattering, gravity sedimentation, dynamic light scattering, among others. For this, size distribution range is usually measured as the polydispersity index (PDI) of a solution. PDI indicates the particle homogeneity of a solution. These values range from 0-1, being 1 highly heterogeneous and homogeneous (Figure 5). Other factors that affect stability are electrical factors. The most important one is the Zeta Potential, which is used to measure strength of attraction between particles. The charge of dispersed particles increases Zeta potential (Chen &
Hu, 2019). In order of a zeta potential to be considered good, the solution must be monodispersed and with a concentration that can effectively scatter light. Also, low salt concentration is required for its effect in conductivity and solution must be well dispersed in a polar dispersant. Zeta potential can determine the surface charge of nanoparticles.
This charge attracts ions to the nanoparticle’s surface. This value has a relationship with the solution’s stability. Desired values range from ±30 indicating higher stability (Kumar
& Chandra, 2017).
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Figure 5. Polydispersity index (PDI) representation. Left: monodispersed solution with a PDI<0.5. Right: polydisperse solution of a PDI>0.5.
1.4.4. Delivery systems
Delivery systems are made to protect bioactive compounds such as nutraceuticals from degrading in processing and storage but also, they are important because they help to control their release (Benshitrit et al., 2012). These delivery systems can be classified depending on their size, production methods, their intended site of delivery or their composition (lipid, protein or carbohydrate based). Because of the nature of certain compounds, there is a limitation in implementing them into drugs such as their low solubility, permeability and bioavailability. The use of drug delivery systems can help improve the compound’s solubility, stability and their limitation by encapsulating them and reducing their drop size. Stratum corneum can be penetrated by nano-sized drug delivery systems given their physicochemical properties such as size, surface polarity and concentration of nanoparticles. The size of these compounds helps increase solubility and stability of bioactive compounds (Mota et al., 2017).
For the production of nano-sized drug delivery systems, the appropriate method needs to be chosen by its scaling-up capacity and the size of the nanoparticle that is aimed. By establishing this, appropriate formulation conditions should be considered such as stirring rate, temperature, surfactants and co-surfactants. Because nanotechnology has been evolving so fast, there are nowadays three nanomedicines approved for clinical use to treat pain. This has opened the possibility to produce different types of nano-sized drug delivery systems. These have shown significant effects in reducing pain because of
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their ability of a sustained release and by enhanced target of glial cells (Pham & Kim, 2020). It has been proved that these delivery systems can help tolerate the effects of capsaicinoids by their improved permeation, sustained release and enhanced skin retention (Wang et al., 2017).
Some benefits of delivering these systems topically are the improvement of compounds’ physical stability, biocompatibility, safety for human health and site-specific prolonged release. Also, there’s lower toxicity provided by the dose reduction given that it can have the same effects with less dose. This mechanism avoids the metabolism of compounds through some organs. Also, therapeutic efficacy of low activity compounds can be enhanced by applying them directly through skin. Local application of compounds into skin can also reduce side effects (Harwansh et al., 2019).
1.4.5. SNEDDS
Lipid-based drug delivery systems can incorporate agents into a lipidic system in order to enhance the bioavailability of compounds with low solubility. Nanoemulsions consist of oil droplets dispersed in water or otherwise. These are stabilized with surfactants and co-surfactants and their normal size ranges from 500 nm and under.
Nanoemulsions are said to show superior transdermal efficiency in comparison to other nanoparticles due to their capacity of solubilizing lipophilic compounds and their skin affinity. Nanoemulsions that fail to enter across the epidermis have been proved to enter through hair follicles. These is common in nanoemulsions with an average of 200 nm (Su et al., 2017).
SNEDDS are lipid-based drug delivery systems that are easy to process and manufacture and can carry compounds with therapeutic activity. These compounds are formed by surfactant, co-surfactant, the oil matrix and the desired compound to entrap (Figure 6). Adequate selection of compatible surfactant, co-surfactant, oil and compound is crucial for the elaboration of these complexes. This type of delivery system has increased drug loading capacity compared to normal emulsions, liposomes, solid lipid nanoparticles and other lipid-based delivery systems (Beg et al., 2020). These are formed with gentle and continuous agitation. Their easy solubilization and improved dissolution
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and absorption rates make this type of formulation more attractive (Rahmann et al., 2020).
Also, this system allows industrial production at low cost, high stability and reproducibility.
As described before, there are reports that show that capsaicin nanoemulsions have higher analgesic activities, anti-inflammatory properties and lower side effects than conventional application of capsaicin (Hall et al., 2020). This project’s purpose is to implement capsaicinoids into SNEDDS. By implementing these compounds into SNEDDS by a simple formulation process, it is believed that capsaicinoids can conserve the same benefits while reducing their undesirable side effects.
Surfactant
Co-surfactant
Active compound
Oil matrix
WATER PHASE
Figure 6. SNEDDS Chemical structure.
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Chapter 2
2. Materials and methods 2.1. Chemicals and reagents
Lauroglycol FCC, Gelucire 44/14 and Labrafil M1944CS were obtained from LYONTEC Químicos (Mexico City, Mexico). Methanol was obtained from DEQ (Monterrey, N.L.). Acetonitrile was obtained from J.T. Chemical Co. (Phillipsburg, NJ, USA). HPLC-grade water and methanol were obtained from VWR international LLC (West Chester, PA, USA). Lipopolysaccharides (LPS) from Salmonella enterica serotype typhimurium L7261 were obtained from Sigma Aldrich (St. Louis, MO, USA).
Dulbecco’s Modified Medium: Nutrient Mixture F-12 (DMEM-F12), ampicillin/streptomycin and trypsin were obtained from Gibco Invitrogen (Carlsbad, CA, USA). Celltiter96®AQueous One Solution Cell Proliferation Assays from Promega (Madison, WI, USA). Carbon dioxide (CO2) was obtained from AOC (Nuevo Leon, Mexico).
2.2. Biological material
Guajillo oleoresin obtained by residues was kindly donated by Intalmesa (Chipilo, PUE, Mexico), lipase-catalyzed synthetic capsaicin analogues oleoresin was donated by Applied Biotec (Cuernavaca, MOR, Mexico). Residues from chile de árbol were bought at a local market in Monterrey, Mexico. Primary Dermal Fibroblasts, human, adult (HDFa) and macrophages from mus musculus (RAW 264.7) cell lines were obtained from ATCC (ATCC, Manassas, VA). Fetal bovine serum (FBS) and antibiotics (Penicilin- Streptomycin-Amphotericin) were obtained from Gibco Laboratories (Grand Island, NY, USA). Lipopolysaccharides (LPS) from Salmonella enterica serotype typhimurium L7261 were obtained from Sigma Aldrich (St. Louis, MO, USA).
2.3. Development of SNEDDS formulation using capsaicin as standard
Two pre-formulations were developed based on previous results shown by Muñoz- Correa et al. (2020). These formulations were made to establish oil-water ratio in order to choose the most physically stable (Table 3). For these formulations, Lauroglycol FCC
24
was used as the oil phase, Gelucire 44/14 as the surfactant and Labrafil M1944CS as the co-surfactant.
Gelucire 44/14 was previously heated at 70ºC for 10 minutes before incorporation.
The three components (oil, surfactant and co-surfactant) were incorporated under magnetic stirring and heat at 70ºC. For the control, formulations without capsaicin were made by just incorporating the oil phase. For standard formulations, capsaicin was added at 0.1% v/v in order to evaluate formulations’ stability with and without compound. After incorporation of the oil phase, 100ml of water was slowly added to each oil phase under 700rpm at 70ºC for 20 minutes to create the SNEDDS. Afterwards, SNEDDS were sonicated (QSonica Sonicators, Newtown, CT) at 40% amplitude for 5 minutes each to reduce their particle size.
Table 3. SNEDDS formulations: oil, surfactant, co-surfactant and oleoresin composition
Oil (Lauroglycol
FCC) (mg)
Surfactant (Gelucire
44/14) (mg)
Co- surfactant
(Labrafil M1944CS)
(mg)
Oleoresin (mg)
Total content
(mg)
Water (mL)
SNEDDS1
CONTROL 350 500 150 0 1000 100
SNEDDS2
CONTROL 150 600 250 0 1000 100
SNEDDS1 350 500 150 100 1100 100
SNEDDS2 150 600 250 100 1100 100
2.4. Macroscopic SNEDDS formulations aspects
Formulations were observed for physical changes such as separation, change of color, creaming or coalescence. Evaluation was carried out for 10 days on two different
25
conditions: room temperature (25ºC) and refrigeration (4ºC). For visual evaluation, formulations were evaluated in front of a white screen under illumination. The formulation that showed no phase separation or physical changes either in the control and in the formulation with the sample was chosen to move further into the addition of compound at different concentrations.
2.5. Capsaicinoids oleoresin obtaining 2.5.1. Capsicum annum v. de Arbol
Capsicum Annum v. arbol was used in supercritical fluid extraction to obtain oleoresin. This was done using a supercritical carbon dioxide extractor (Thar 100 F, Thar technologies, Inc, Pittsburgh, PA). First, 200g of arbol sample was placed into an extraction vessel of 1L. Extraction vessel was tightly sealed, and conditions were set at 450 bar, temperature of 65ºC for 45 minutes. CO2 flow was set to 60g/min. Ethanol as co-solvent was set at 5% flow rate. Extraction lasted 40 minutes and the sample was collected after vessel depressurization.
2.5.2. Capsicum annum v. Guajillo
Guajillo oleoresin was also donated by Intalmesa. It was obtained from supercritical fluid extraction at 240-260 bar at 45ºC. Temperature and pressure at the collector were 50ºC and 62 bar, respectively. Sample was extracted from Guajillo chili with a particle size of 2-3 mm.
2.5.3. Synthetic oleoresin
Synthetic oleoresin was provided by AB Solutions (Cuernavaca, MOR, Mexico) and it was obtained by synthesizing pungent capsaicin analogues (Castillo et al., 2007). Resulting sample was an oleoresin that contained chili extract and synthetic capsaicinoids, among them, the most abundant denominated
“capsaicinoid ABX”.
26
2.6. Capsaicinoid characterization by liquid chromatography coupled to diode array (HPLC-DAD)
Quantification of capsaicinoids in oleoresin samples was done by HPLC-DAD, Agilent Technologies, Santa Clara, CA). Samples were diluted with HPLC-grade methanol (1:10 v/v) and were analyzed by HPLC. Separation was carried in a reverse- phase Zorbax Eclipse Plus C18 column 4.6x100mm (Agilent technologies, Santa Clara, CA). Solvents used for this method were Acetonitrile and HPLC-grade water acidified with .1% formic acid (1:1) at a flow rate of .8 ml/min. Detection was set at 280 nm. Injection volume was 5ul and the analysis was done at 40ºC with a pressure of 120 bar. A capsaicin standard was used to quantify Capsaicin Equivalents contained in the sample as there were no capsaicinoids standards available. Results were expressed as mg of capsaicin equivalents on g of oleoresin (mg CapEq./g oleoresin).
2.7. Preparation and characterization of SNEDDS 2.7.1. Capsaicinoids SNEDDS preparation
SNEDDS formulations with capsaicinoids were done at four different concentrations: 0.025%, 0.050%, 0.075% and 0.10%. The procedure was the same as the first formulation elaboration. Each oleoresin was weighed and incorporated to the oil phase under magnetic agitation and at 70ºC. Afterwards, 100 ml of water was slowly added to the oil phase in agitation of 700rpm for 20 minutes. Solutions were sonicated for 5 minutes at 40% amplitude (QSonica Sonicators, Newtown, CT).
2.7.2. Formulations’ characterization: Size and zeta potential
Particle size and zeta potential were measured with a dynamic light scattering instrument (Zetasizer Nano ZSP (Malvern Instruments Ltd., Malvern, Worcestershire, UK). Samples were diluted 1:8 v/v in MiliQ-grade water. Because stability is measured in the particle’s surface, the equipment was configured at a refractive index of 1.465 based on Gelucire’s properties (Jannin, 2009) and a 0.025 absorbance. The dispersant was set to water at a 25ºC temperature. Samples were read in disposable folded capillary cells suited for the equipment.
27
2.7.3. Quantification of entrapped capsaicinoids in formulations by liquid chromatography coupled to diode array (HPLC-DAD)
Quantification of capsaicinoids in oleoresin samples was done on the same equipment and under the same conditions of capsaicinoid characterization mentioned in point 2.6 except for the sample preparation. For sample preparation, nanoemulsions had to be disrupted. This was accomplished by mixing them with methanol (1:10 v/v) and then centrifuged at 15,000 x g for 60 min at 4ºC.
Supernatants were collected and concentrated (Genevac EZ-2.3, SP Scientific, United States) for 3 hours. Concentrated samples were resuspended in 150µL of HPLC-grade methanol and analyzed by HPLC. Separation was carried in the same column and the same solvents mentioned in point 2.6. Results were expressed as mg of capsaicin equivalents/g of extract (mg CapEq./g extract).
Entrapment efficiency was calculated as the following formula:
!"#$%&'("# !**+,+(",- (%) =2(+3ℎ#!"!#!$% '()*− 2(+3ℎ#+(,, '()*
2(+3ℎ#!"!#!$% '()* ∗ 100
2.8. In vitro studies 2.8.1. Cell culture
Primary Dermal Fibroblasts, human, adult (HDFa) (passage number 10) and macrophages from mus musculus (RAW 264.7) (passage number 19) cell lines were obtained from ATCC (ATCC, Manassas, VA). Both cell lines were cultured with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% bovine fetal serum (FBS) and 1% antibiotic (Penicilin-Streptomycin-Amphotericin) (Gibco Laboratories, Grand Island, NY, USA). These were incubated at 37ºC and CO2 at 5%.
2.8.2. Nitrite assay to determine inflammation using RAW 264.7 as model Nitrites were determined as an indicator of NO production induced by LPS.
RAW 264.7 macrophages were plated in 96-well plate plaque (5x105 cell/mL) and left to adhere for 24h. A volume of 50µl was added from different nanoemulsions formulations (0.025%, 0.050%, 0.075% and 0.10%) and oleoresins (0.10%) to the cells by triplicate and was left for incubation for 4h. Half of the plate was used as
28
control for each sample. The other half was stimulated with LPS (10µg/mL) for 24 hours. After 24h, production of nitrites was determined using Griess Reagent System (Promega, Madison, WI) and measured at 550 nm. A calibration standard curve was made to have a reference on quantification. NO inhibition percentage was calculated by taking into account cells without LPS and cell viability. Results were expressed as inhibition of NO production.
2.8.3. Cellular uptake assay
Human dermal fibroblasts (HDFa) were plated in 96-well plate plaque (5x105 cell/mL) and left to adhere for 24h. The different nanoemulsions formulations (0.025%, 0.050%, 0.075 and 0.10% v/v) and oleoresins (0.10% v/v) were added to the cells by triplicate and left for incubation for 24h. The amount of capsaicinoids in each supernatant was quantified in Nanodrop (ThermoFisher Scientific, Waltman, MA) . Results were calculated by dividing the results of the sample over the control. This number represented the non-permeated cells. Therefore, cellular uptake was calculated by resting 100 minus the number of non-permeated cells.
2.8.4. COX-2 inhibition as inflammation marker
COX-2 activity was determined by evaluating the supernatants with COX-2 Fluorescent Activity Assay Kit (Cayman Chemical Ann Arbor, MI). Results were expressed as inhibition % of COX-2 production.
2.9. Statistical analysis
Analysis of data was done by evaluating each sample by triplicate. Results were analyzed on Minitab LLC software (Pennsylvania State University, University Park, PA, USA) using one-way analysis of variance (ANOVA) and compared by Tukey’s HSD tests. Differences of p<0.05 were considered statically different.
Mean and ± standard deviation is expressed in each sample.
