Recent research has substantially focused on residual subproducts containing chemical compounds with bioactive properties. Even though there are some culinary or medicinal uses of Capsicum seeds, there is still a seed mass waste from pepper processing. Many pepper leading producer countries generally lack the facilities and infrastructure required for such processing technologies and so, pepper seeds are usually either destroyed or employed as landfilling or as animal feed. This involves an inadvertent economic loss for producers as well as a detrimental environmental impact. However, there is a hidden potential within the pepper processing industry related to valorization of pepper seeds to obtain added value by-products and thus reduce generated waste. Pepper seeds are a good source of antioxidants, carotenoids, phenolic acids, flavonoids, and vitamins C, E, and A and are also rich in volatile compounds, among others. The unique alkaloids of this genus are capsaicinoids and capsainoids, which have been linked to many beneficial biochemical and pharmacological effects including anti-oxidative or anti-inflammatory activities. Other prominent bioactive compounds of peppers seeds include saponins, lectins, and polyunsaturated fatty acids. In this context, an overview of the biological properties, extraction systems, and possible industrial application of bioactive compounds of pepper whole fruit and seeds is presented.
- Capsicum spp.
1.1 Characteristics of the genre
Amid the species
Although the characteristics of each species differ from each other (Table 2), it can be said that the genus
|Species||Flower color||N° flower||Seed color||Calyx constriction||Distribution|
|White with yellow spot||1–2||Tan||Absent||South America|
Thus, its physical appearance defers from one species to another. For example,
Being a cold sensitive plant, the best conditions for production are between 7 and 29°C and an annual precipitation of 0.3–4.6 mm. It grows best in well-drained, sandy or silt-loam soil and a soil pH of 4.3–8.7. Hot and dry weather is also desirable for fruit ripening . To carry out its cultivation it is necessary to seed or transplant the peppers, harvesting 3 months after planting .
Capsicumseeds sources and production
Chili (a variety of
On the basis of extracts obtained from pre-ceramic in the Coxcatlan caves, it is believed that the domestication of
Of the five domesticated species of
Pepper quality depends on their composition, which is determined by factors such as environmental cultivation conditions, variety, ripeness, and pre-harvest and post-harvest handling and preservation . The degree of ripening required may be one of the most important factors in quality, but it will also depend on the destined market, since not the same degree of maturity is desired for all the possible uses. Notably, the moment of harvesting is also important for the maintenance of the quality as metabolic activity persists after harvesting .
1.3 Chemical composition
Regardless of the enormous consumption and production of this kind of vegetables, there is little data about the chemical composition of the different varieties (Table 3). However, the demand and cultivation of peppers, especially “hot” cultivars, has increased due to its flavoring and medicinal properties. Some of the latter have been described as anticancer, antioxidant and antimicrobial. Its edible and nutritional value is acknowledge as well, since it is rich in vitamins (A, C, B6, E), carotenoids (β-carotene), flavonoids, oils, oleoresins and alkaloids . Therefore, the compounds that can be found in this genre are carbohydrates (accounting for approximately 85% of dry weight), polyphenols (0.5% of dry weight) and important molecules such as capsaicinoids, carotenoids and vitamins . Given these facts, peppers are considered a good source of most essential nutrients .
|M. Tres venas||D||61.05||13.28||9.61||14.40||Nd||7.02|
|Arnoia green B||F||3.51||0.12||0.16||Nd||1.31||0.33|
|New Mexico 6||D||Nd||14.79||21.95||Nd||60.61||3.48|
The capsaicinoids content, which depends on the variety and maturation stage, will determinate the pungency.
Capsaicinoids are the characteristic pungent compounds of the
The characteristic red color of many peppers is determined by the presence of different carotenoids. Actually, more than 50 different carotenoids can be found in this kind of material. Some of them are capsanthin, capsorubin, and cryptocapsin which give brilliant red color (ripe fruits) or β-carotene, violaxanthin, zeaxanthin and β-cryptoxanthin which give yellow–orange color . Nevertheless, the color will depend on the state of maturity. For example, jalapeño (
Using jalapeño as an example to study composition, its main component is water, then carbohydrates (5.3%), fiber (2.3%), protein (1.2%), fat (0.1%) and minerals, being the most important potassium (340 mg per 100 g of fresh product). It also has calcium (25 mg per 100 g of fresh product), magnesium (25 mg per 100 g of fresh product), sodium (7 mg per 100 g of fresh product), iron (2 mg per 100 g of fresh product) and zinc (0.3 mg per 100 g of fresh product). As for vitamins, the most important are ascorbic acid, retinol and folic acid (72, 20 and 23 mg per 100 g of fresh product, respectively). Other vitamins that can be found in peppers are thiamine, riboflavin, niacin and pyridoxine. Moreover, jalapeños also contain important amino acids such as lysine, methionine and valine (252, 40 and 23 mg per 100 g of protein, respectively) .
Capsicumseeds as a source of bioactive compounds
Like many fruits and plants, peppers are an excellent nutritional source. As aforementioned, it has an abundance of minerals, vitamins, aminoacids, carotenoids as also phytochemicals like phenolic compounds or polyunsaturated fatty acids (PUFAs). Likewise, capsaicins are unique to the genus
Different bioactive compounds have been isolated and extracted from
|Species and varieties||Bioactive compounds|
|Major phenolic compounds||Vitamins||Pigments||Volatiles||PUFAs||Peptides||Ref.|
|4-Hydroxybenzoic acid, vanillic acid, caffeic acid, ||Nd||Nd||Nd||Nd||Nd|||
|Elagic acid, gallic acid, chlorogenic acid, caffeic acid, salicylic acid, rutin||Nd||Nd||Nd||Nd||Nd|||
|Caffeic acid, gallic acid, ferulic acid, rutin, capsaicin, dihydrocapsaicin||Tocopherols||Capsanthin, zeaxanthin, capsorubin, lutein, β-carotene, cryptocapsin||Nd||Linoleic, palmitic, oleic acids||Nd|||
|Nd||Ascorbic acid||Nd||Betulin, β-sitosterol||Oleic, palmitic, linoleic, elaidic acids||Nd|||
|Gallic acid, caffeic acid, ferulic acid, chlorogenic acid, cathechin, epicathechin capsaicin, dihydrocapsaicin||Ascorbic acid||Nd||Nd||Nd||Nd|||
|Caffeic acid, cinnamic acid, coumaric acid, capsaicin, vanillin||Nd||Nd||Nd||Nd||Phenyl-alanine|||
|Tannins, flavonoids, coumarins, polyphenols||Nd||Nd||Saponins||Nd||Amino acids|||
2.1 Phenolic compounds
Phenolic compounds, also referred as phenolics, are secondary metabolites that may be found in a wide spectrum of plant species. They are synthesized as a result of adaptation to biotic and abiotic stress through the phenylpropanoid pathway playing an important role in plant development, because they act as a defensive mechanism that eases plant growth against harsh conditions [33, 34]. Based on recorded knowledge, phenolic compounds exhibit numerous potential health benefits that are already well described in scientific literature and are currently a major current focus of nutritional and pharmacological research [35, 36]. Phenolic acids and flavonoids are the main phenolic phytochemicals found in peppers. Likewise, capsaicinoids are synthetized in the same biochemical pathway and exhibit some similar properties such as antioxidative activity .
The yield of phenolic compounds recovered from an extraction can be very different, depending largely on the extraction method, the conservation of vegetal material and maturity state . Generally, fresh raw material preserves the highest quantities of phenolic compounds . Several factors contribute to yielding disparity, such as heterogeneous genotypes, growing and harvesting conditions of the samples . Some of the most prominent non-capsaicinoid phenolic compounds because of their valuable health benefits are phenolic acids like gallic acid, caffeic acid, chlorogenic acid or ellagic acid (Figure 2). Flavones are another important group of phenolic compounds, being some of the most prominent kaempferol, quercetin, luteolin or rutin (Figure 3) . These phenolics have demonstrated great health benefits and many of them are commercially available in purified products extracted from other plant species [39, 40]. Even so, their concentration differs among species but not much among varieties . Other phenolic compounds are reviewed in the next paragraphs as in the case of some carotenoids or vitamins.
Regarding capsaicinois, they are synthesized naturally in the placenta of pepper fruits by enzymatic transformation of vanillylamine, the phenolic portion of the molecule, which confers this alkaloid its antioxidant capacity . The seeds are not the primary source of capsaicinoids but they may absorb them because they are in close proximity to the placenta, which is the richest capsaicin fraction . Their presence in the seed and the high concentrations they achieve, has been observed to rise the riper the pepper is . It is also confirmed that fresh seeds yield more capsaicin than dry seeds, which suggests that surface capsaicinoids are sensitive to heat and/or oxidation . “Hot” pepper cultivars attribute their pungency to high levels of capsaicinoids whereas non-pungent or “sweet” peppers (e.g., bell pepper) have very low capsaicinoids quantities .
Capsinoids are other non-pungent capsaicin analogues noticeably found in
Besides, it is worth mentioning that the well-known flavone chrysoeriol (Figure 4), present in a multitude of vegetables and at least in
2.2 Fatty acids
Peppers are fruits rich in polyunsaturated fatty acids (PUFAs), and their seeds show even greater concentration per gram . The main fatty acids are indeed PUFAs . These PUFAs, which are in the whole fruit and seeds, are mainly linoleic acid, palmitic acid α-linolenic acid and stearic acid [16, 48]. Moreover, peppers appear to have very low levels of saturated fatty acids. Linoleic acid has the highest concentration (≈70%) and the other fatty acids show much lower levels [48, 49]. PUFAs and specifically linoleic acid and α-linolenic acid, are recognized as essential fatty acids and are precursors of other important fatty acids in metabolism like arachidonic acid and eicosapentanoic acid or prostaglandins that in whole contribute to normal physiological performance .
Chlorophylls and carotenoids constitute another group of valuable pepper nutrients that affect its color, but they also have important antioxidative, anti-inflammatory effects and promote immune response [22, 51, 52]. Zeaxanthin, β-carotene, violaxanthin, lutein and β-cryptoxanthin are the pigments with highest concentration among
As it has been mentioned, peppers contain several vitamins like ascorbic acid and tocopherols like α-tocopherol, γ-tocopherol and δ-tocopherol, are all isomers of vitamin E [55, 56]. These essential vitamins contribute to the normal metabolism, promote immune response and also have antioxidative bioactivity [52, 57]. Because of this, ascorbic acid is an essential vitamin that is also used as a natural food preserver . Indeed, peppers contain levels of vitamin C corresponding with those found in many citrus fruits and other vegetables considered good sources of this vitamin . Tocopherols are well-known lipophilic antioxidants and appear to exert a vital part in the normal T lymphocyte maturation, lower age-related increase in tissue inflammation and lower interleukin production [55, 58]. This makes vitamin E an important modulator to an orderly and better inflammatory reaction, among other health benefits .
2.5 Volatile compounds
Volatile compounds in
2.6 Other minor compounds
There are a few other minor and less studied compounds amid the different pepper species, because research has focused almost entirely on finding and studying phytochemicals of metabolic importance. In regard to these bioactive compounds,
Another of these minor chemicals are lectins, a group of proteins with glycoside agglutination properties. Some lectins have been described in
3. Bioactivities of the raw material
In recent years, nutraceutical and therapeutic research has focused its view towards both exotic and domestic fruits and vegetables as a source of phytochemicals with the ability to induce beneficial bioactivities . These natural chemicals have been and are already used as a main source of therapeutics in traditional and modern medicine, reaching one third of the total production of therapeutics . Furthermore, processing waste by-products of fruits and vegetables are being researched as a viable source of phytochemicals that would be affordable and reduce economic and ecological impacts of wasted by-products or taking produce out of the food market [67, 68]. Thus, fractions that are not employed in nourishment such as the placenta, seeds or leaves of many species can prove to be a valuable resource instead of end as waste or fertilizer, which is the most common use for vegetable non-edible parts .
Taking into account the aforementioned compounds present in the many different species of the genus
These bioactivities have been described in scientific reports through tissue culture and both animal and human test research as antioxidant, antimicrobial, anti-inflammatory, anticancer, analgesic or even antidiabetic [21, 22, 34]. Considering this with the fact that
Some of the most recognized bioactivities found by chemicals in the whole pepper and its seeds will be reviewed and a brief compendium of the bioactivity research can be found in Table 5.
|Activity||Bioactives||Species||Type of study||Test results||Ref.|
|Antioxidant||Capsaicin, dihydrocapsaicin||In vitro||DPPH, ABTS|||
|Total phenolic compounds||In vitro||DPPH, ABTS, ORAC|||
|Phenolic extracts||In vitro||DPPH, ferrus chelating|||
|Seed oil||In vitro||DPPH, ABTS|||
|Antimicrobial||Capsicosides A, G, D||In vitro||Gia. various yeasts|||
|Capsaicin||In vitro||Gia. |||
|Phenolic extracts||In vitro||Gia. |||
|CAY-1||In vitro||Gia. |||
|Lectins||In vitro||Gia. |||
|Total polyphenol content, capsaicins||In vitro||Gia. |||
|Jalapeño extracts||In vitro||Gia. |||
|Capsaicinoids, chrysoeriol||In vitro||Gia |||
|Residual aqueous extract||In vitro||Gia |||
|Anti-inflammatory||Capsaicin||Nd||In vitro||Inhibition of inflammatory transcription factor NF-κB and AP-1|||
|Capsaicin||Nd||In vitro||Inhibition of Ikα and Ikβ via NF-κB|||
|Capsaicin||Nd||In vitro||Inhibition of adipose tissue inflammation (interleukin 8, c-Jun)|||
Oxidative stress is caused as a result of the presence of reactive oxygen species (ROS) which may be produced in oxidative metabolism and exposure to the environment . The term ROS englobes the molecules superoxide radical (O2 2−), the hydroxyl radical (OH−) and hydrogen peroxide (H2O2). They are produced by the sequential reduction of molecular oxygen in various metabolic reactions . O2 2− is the most unstable form but it may dismutate to H2O2 by the action of endogenous superoxide dismutase (SOD) enzyme reaction or non-enzymatically . The effect of oxidative stress mainly translates into changes in the rate of metabolic reduction reactions and an increased rate of DNA mutations and cell mitosis. They are also main signals indicating cell death, which in turn triggers inflammation via the pro-inflammatory factor nitric oxide (NO), which is released by macrophages [33, 76]. Since these are both cause and result of cancer development, it is considered that high levels of ROS are detrimental to health [40, 65, 76]. All living aerobic organisms have developed defense mechanisms against oxidative stress through the synthesis of reductive biochemicals or enzymes such as SOD. The most important antioxidative biochemicals are phenolic compounds, which are prominently found in plants [52, 66]. These antioxidants present in peppers, mainly identified as the mentioned phenolic compounds, vitamins and pigments, have the potential to reduce biological oxidative stress and thus preventing the incidence of many related diseases, but also to further food preservation by inhibiting the oxidative metabolism of decomposing fungi and bacteria [33, 35, 38]. Phenolic compounds are the principal antioxidants in nature and generally show the greatest antioxidative capacities . However, high concentrations of few phenolic compounds showed pro-oxidant effects due to a concentration imbalance between ROS and the phenols, which is why it is important to maintain intake of different phenolic compounds in order to benefit from their antioxidant properties . Thus, antioxidants have a wide extent of applications, and many natural and synthetic antioxidants have been used by the food industry in order to better preserve raw or precooked products that would otherwise have a much shorter shelf-life .
The antioxidative effect of the aforementioned phenolic compounds essentially works by scavenging free superoxide and hydroxyl radicals and thus preventing high levels of ROS, NO and oxidation of sensitive biomolecules like proteins or lipids (Figure 6) [36, 40]. This results in a better physiological performance, immunomodulation and DNA mutation protection. Furthermore, the decrease of oxidative stress results in better cardiovascular health. This is due to the fact that low-density lipoprotein (LDL), as the main cholesterol carrier in the circulatory system, is susceptible to oxidation by ROS . Oxidized LDL presence has been found to be cause of atherosclerosis, a vascular ailment caused by the formation of plaques inside the arteries that may weaken blood flow and lead to cardiovascular diseases . On top of that, pepper and its seeds are described to be one of the richest vegetables in phenolic compounds . Then, it should be taken into account the antioxidant properties of the many phenolic acids, capsaicinoids, vitamins and pigments present among the
Pepper seed extracts and selected pigments, phenols, capsaicins and capsiates have been tested against the most common microorganisms present in foods and/or potential pathogens. The major antimicrobial effect of capsaicinoids has been found to be against common opportunistic and pathogenic fungi like
Capsaicinoids appear to inhibit, fungi, and both Gram positive and negative bacterial growth in several studies. In all of them, capsaicin and/or dihydrocapsaicin showed to stop or slow colony development in a significant degree [24, 35]. Some interesting results of antimicrobial tests with capsaicinoids reported that it inhibits the growth of
Even though capsaicinoids possess these interesting antimicrobial properties, the positive correlation between antimicrobial activities, concentration and its pungency and potential irritation effects make them less suitable when it comes to applying this compound as a food preserver . Thus, it is interesting to study the effects on this area of alternative chemicals found in pepper fruit and seeds. CAY-1, chrysoeriol and lectins have been previously mentioned as such.
CAY-1 is found in
Chrysoeriol is another potent antimicrobial compound present in a variety of plants as well as in peppers . Several studies have presented that chrysoeriol is capable of greatly inhibit the growth of
Certain lectins isolated from peppers have showed antifungal properties, inhibiting growth of some common opportunistic fungi like
Furthermore, a recent study analyzed the antiadhesive capacity of natural peptides from
Although an anti-inflammatory action is carried indirectly by the antioxidant capacity of other compounds to scavenge ROS and inhibit NO production, it is known that capsaicins and capsiates directly induce an anti-inflammatory effect through the activation of the transient receptor potential vanilloid 1 receptor (TRPV1) [24, 86]. TRPV1 is an ion channel receptor located in several glia that gives sensations of heat, but is also relevant in pain perception and induces this sensation, as well as inflammation . By activating this receptor, capsaicins cause the well-known pungency sensation, as well as irritation in high enough concentrations. However, after activation by capsaicinoids, the excited neurons become resistant to further stimuli . Hence, after a brief burning sensation, capsaicin may act as a local analgesic in neuropathic pain .
Research on capsaicinoids has showed that these molecules can also decrease inflammation of adipose tissue linked to obesity [73, 75]. It seems that capsaicin is able to inhibit or at least partially decrease the production of pro-inflammatory signals like interleukin 8 (IL-8), nuclear factor kappa-light-chain-enhancer of activated B cells (NF\kappaB) or active protein 1 (AP-1) [74, 80]. This anti-inflammatory effect has the potential to make inflamed tissue less prone to tumor development as well as reduce the infection caused inflammation .
Still, the pungency of capsaicins poses a setback for using them without potentially hazardous secondary effects. That is why capsinoids may be a good alternative to induce similar effects to capsaicin, even though they are not found in so many pepper species [41, 45].
Although the importance of researching capsaicinoids bioactivities in pepper is due to its unique presence in this genre, the also present phenolic compounds, vitamins and pigments also bear significant anti-inflammatory properties [25, 88, 89]. Indeed, the antioxidative properties of phenolic compounds could be a key factor in reducing age-related extended tissue inflammation [25, 58]. Nonetheless, further research assessing the suitability of using as anti-inflammatory drug these phytochemicals, and specifically capsaicin, is needed.
4. Extraction systems for bioactive compounds from
The bioactive compounds obtained from nature are secondary metabolites produced by the organisms. The concentrations of each type of compound are very variable, so in order to have enough quantities, the development of new and advanced technologies is needed.
Although there are several extraction methodologies, there is a demand for more appropriate and standardized extraction strategies. For choosing one method over another, it must be taken into account the quantitative and qualitative characteristics of the compound of interest. In addition, to improve the efficiency of a method it is important that the nature of the source, the different parameters of the method and the possible interaction are taken into account. Experiments carried out with peppers can be seen in Table 6.
4.1 Conventional extraction systems
Maceration (MA) is a type of solid–liquid extraction, in which the solid would be peppers. This raw material is characterized by having several compounds soluble in the liquid phase. The type of molecules extracted will depend on two factors: the type of starting material and the type of solvent used.
In order to carry out this process, the sample is suspended in the solvent at the desired temperature during the chosen time while stirring. Once the process has been carried out, the sample is centrifuged and the supernatant, which is where the compounds of interest are found, is filtered. Therefore, the process of extraction consists of two stages. On the first (washing) there is a rapid transfer of the compounds from the surface of the solid to the solvent. On the second one (transfer), the matter passes from the interior to the exterior of the solid by diffusion being this step the limiting stage. This method is used, for example, for the isolation of phenolic compounds of vegetable origin for their subsequent use in the food, pharmaceutical and cosmetic industries .
Water can be used as an extraction solvent, but it comes with the drawback is that it principally extracts the hydrophilic compounds present in vegetable materials. Several studies showed that water is not a very suitable solvent because its high polarity does not allow the extraction of capsaicinoids that are non-polar. However, it has the advantages or being safe, cheap and the simplest form to obtain essential oils. This technique can also be done with other solvents, being the ratio of solvent another parameter to take into account. Polarity is the reason why the most common solvents used are methanol, ethanol, water or a mixture of them .
Another parameter to optimize will be temperature. This makes it possible to differentiate two types of MA: cold maceration and heat maceration. Maceration with or without stirring, mild heating or heating under reflux are also possible variations to the method.
It is a quite old simple method with the inconvenience of long extraction times and large amounts of sample and solvents. Additionally, high temperatures can destroy thermolabile compounds such as phenolic compounds. In a study in which the effect of MA and ultrasound was related, it was detected that they have similar extraction yield of oleoresin; nevertheless, MA needs longer extraction times with the consequent loss of quality due to their higher times of exposure to high temperatures. It also was observed that n-hexane was a better solvent than ethanol .
4.1.2 Heat assisted extraction
This procedure is also a type of solid-liquid extraction in which the extraction is usually carried out in thermostatic and sealed water baths so that the solvent does not evaporate. The determining parameters are extraction time, temperature and the proportion of solvent that is used as well as the solid-liquid ratio. Due to the application of high temperatures (as far as 100°C), it is not the best extraction process for this type of matrix because certain compounds present in peppers (vitamins, phenolic compounds, etc.) are degraded by heat.
4.1.3 Cold pressure extraction
Cold pressure extraction is one of the oldest techniques of extraction for obtaining oils. It consists of mechanical pressing with the absence of heating. By using this method, little to no heat is generated, however it gives low yields. This technique was applied to Capia pepper seed, resulting in a lower extraction yield compared to traditional Soxhlet extraction with hexane. Moreover, the final oil was unpopular among consumers .
4.1.4 Organic solvent extraction
Organic solvent extraction (OSE) allows the extraction of many compounds (oils, fats and proteins). Normally, after the extraction process another step is done. It consists of concentrating the extract by removing the solvent under atmospheric or reduced pressure. OSE is the most extensively used technique to obtain oleoresins from peppers. These oleoresins are. Usually used as color additives . In this method, the most determining limitation will be the solvent properties (polarity). When choosing the solvent, it must also be taken into account if the legislation allows its use. In the EU is regulated in Commission Regulation N° 231/2012.
In order to improve these conventional extraction techniques, other methods have been developed (ultrasound assisted extraction, pressurized hot water extraction, negative pressure cavitation-assisted extraction and pulsed electric) to partner with them and solve some of the inopportuneness of traditional techniques. In this way the extraction efficiency is substantially improved.
4.2 Green extraction techniques
This group includes several techniques with the aim of not only preventing pollution but also reducing sample preparation costs by, for example, lowering solvents consumption.
4.2.1 Microwave-assisted extraction
Microwave-assisted extraction (MAE) is based on the heating of solvents that are in contact with solid samples with the use of microwaves. This allows the partition of compounds of interest from the sample into the solvent. Microwave energy accelerates a great variety of chemical reactions as well as the extraction of organic compounds from different matrices . During the process, electromagnetic energy is transformed into calorific energy by two mechanisms: ionic conduction and dipole rotation . It is a method that could be an alternative for avoiding thermal degradation and oxidation, with no influence on cell integrity and shape. However, due to thermal stress and localized high pressures, cell rupture is more rapid than with another techniques, which is an inconvenience, for example, in the extraction of volatile oils .
Some of the advantages of this technique are simplicity, effectiveness, low processing time, low solvent consumption and energy, no generation of secondary waste and can be used for larger volumes . Another advantage of this method is that it produces a uniform heating, so extraction is simultaneous regardless of the area where the compound is . By using this method, lipids, pigments, carbohydrates, vitamins and proteins can be extracted . Moreover, it is characterized by having a superior extraction rate of volatile compounds in
The selectivity and efficiency of MAE largely depends on the dielectric constant of the extraction solvent mixture, which defines its chemical polarity and thus what compounds will be extracted . It can be expected that by using water as a single solvent, the quantity of capsaicinoids extracted decrease due to its negligible polarity . Therefore, the parameters to take into account in this type of extraction are time, power, temperature and type of solvent and its ratios.
Several experiments can be seen in a summarized form in Table 7, which shows the variation between the values of the parameters among different experiments with solvents of different polarity. It is worth highlighting the values obtained for capsaicinoids using ethanol  as solvents since it is the one with the highest yield.
|Capsaicinoids||Green||MAE||Acetone (30%)||0.48 mg/g|||
|Green||MAE||Acetone (pure)||5.3 mg/g|||
|Acetone (100%)||3.92 mg/g|||
|Green||UAE||Acetone (100%)||0.31 mg/g|||
|Methanol (100%)||2.88 mg/g|||
|Other phytochemicals||Green||UAE||Methanol (80%)||—|||
|Volatiles||55||10||250||n-hexane (pure)||42 compounds|||
|Capsaicinoids||21||15||300||Acetone (30%)||0.48 mg/g|||
|50||5||800||Methanol (100%)||0.32 mg/g|||
|—||20||320||Ethanol (99.5%)||5.3 mg/g|||
|76||1||500||Methanol (60%)||230 ppm|||
4.2.2 Ultrasound assisted extraction
In every extraction process there is a critical step which is the degradation of cell walls and membranes. Ultrasound assisted extraction (UAE) makes this possible by applying pressure waves that are transmitted across a medium as compression and expansion (rarefaction) cycles, finding an area with maximum pressure in the compression phase and one with minimum pressure in the rarefaction phase. This pressure difference makes the cavitation phenomenon possible, which breaks the structure to make the compounds accessible and therefore available to extract them .
This technique has been applied to extract different bioactive compounds from herbs or algae due to its ease to disrupt their cell walls despite its resistance which differentiates this method from the previous ones, since strong disruption of the cell wall is achieved and as a result the extraction of intracellular materials is increased with the increment of energy input [106, 120, 124].
The ultrasonic technique, as well as the other green techniques, has been proven to have several advantages such as reduction of solvents consumption, temperature and time, very important parameters in the extraction of thermolabile and unstable compounds as it has no effect on the chemical structure and biological properties. Furthermore, UAE has low equipment investment and easy implementation, so it can be basically industrially employed in local companies . Moreover, there are studies that showed that this method also achieves a greater supercritical extraction of pungent compounds from ginger owing to physical effects on the surface of particles .
There are several parameters to take into account to optimize the method which include ultrasound power intensities, frequency, wavelength and time. There are several studies about it (Table 8). One of them studied the influence of several of these parameters on the extraction of capsaicinoids, observing that their release was very fast in the first 5 min of the process and then decreased. Temperature influence was also observed since an increase of 15°C (up to 45°C) improved the extraction using 95% ethanol (v/v) as a solvent. Higher temperatures did not lead to significant improvements . The effect of the solvent was also studied, being the most common solvents used for extracting capsaicinoids methanol, ethanol, acetone, acetonitrile and water. It was proved that the yield of extraction is worse with the addition of water. Methanol and ethanol have similar recoveries but ethanol was better than acetone, the best concentration of ethanol being 95% [100, 106].
|Capsaicinoids||20||—||10||40||Acetone (100)||0.31 mg/g|||
|40||—||10||24||Methanol (100)||2.88 mg/g|||
|31.5||—||20||76||Methanol (60)||200 ppm|||
|—||—||40||25||Acetone (100)||3.92 mg/g|||
4.2.3 Enzyme-assisted extraction
Enzyme-assisted extraction (EAE) is an extraction system that allows the avoidance of processing conditions like temperature or drastic pH changes and so, maintain the quality and yield of multiple biomolecules .
This type of extraction supports isolation for recovering bioingredients from different plant materials. An enzymatic pre-treatment before applying traditional methods will help to isolate high yields of bioingredients due to enzyme assisted extraction facilities. This is due to the fact that degradation of cell walls and membranes is the critical step of extraction which . Among its advantages over traditional methods are high selectivity, overall efficacy, eco-friendly procedures, low-energy consumption, minimal usage of harsh chemicals, maximum yield, low to no wasteful protection or deprotection steps, easy recovery, and process recyclability . However, it also presents some drawbacks such as the cost of enzymes, requirement of holding tanks that may require long term incubation, lack of knowledge about optimal or compatible enzyme formulations for cell disruption and inability to completely hydrolyse the bonds in plant cell wall .
The factors to take into account in this technique are fundamentally temperature, pH, and type of enzyme. A range of enzymes (lipases, carbohydrase, celluloses, proteases, pectinases) have been widely used as specific catalysts. Each enzyme has different substrates. For example, cellulase and hemi-cellulase have their greater hydrolysing activity on the cellulose found in plant cell walls, hence their name. This enzymatic processing increases the permeability of the cell wall, resulting in a better recovery of some compounds like volatile oil and resin, which are prone to degradation when extracted with more disruptive methods .
Several studies (Table 9) show that viscozyme L is an enzyme with a superior recovery rate of bioactive compounds fractions like total carotenoid content, total phenolic content, total flavonoids and total antioxidant activity with high total suspended solids (TSS). It also has at this moment the better extract yield [95, 96]. Among its applications is the extraction of pigments or capsaicinoids. In almost all cases the pH of the medium is 4.5 as it is the pH of the sample and it is in the range of optimal activity of the different enzymes .
4.3 Emerging technologies for extraction
Due to the disadvantages of traditional techniques, there is an interest in the development of new extraction techniques. Some of the most sought-after features include shortened extraction time, automation or reduced organic solvent consumption.
4.3.1 Hydrostatic high-pressure extraction
Hydrostatic high pressure (HHP) is considered an emerging technology that have been applied in the preservation of food since the end of eighties . The first food products treated with this method began commercialization in Japan in 1990 . This method is established on the application of high pressures (100–900 MPa) to the product of interest.
Among the advantages of this type of extraction there is the possibility to conduct it at room temperature, meaning no thermal degradation and derived bioactivity loss from extracted components. It also does not modify chemical structures of the different compounds of interest’s independent form of molecular weight . Moreover, in comparison with conventional techniques it is faster, gives higher extraction yields and fewer impurities  giving, unlike other preservation technologies as thermal treatment, uniform and nearly instantaneous effects throughout the foodstuff and thus independent of foodstuff geometry and equipment size which makes an easy scale-up from laboratory findings to full-scale production possible .
This method can be applied to multiple matrices for the extraction of natural compounds  like fruit and vegetables for different targets as for example carotenoids , antioxidants  and pigments .
The main parameters to be taken into account are pressure, time, temperature and type and quantity of solvent, studying each parameter for each variety in particular.
HHP can change physiological and biochemical properties of pepper based on a study carried out with
4.3.2 Supercritical-fluid extraction
In supercritical-fluid extraction (SFE), the fluid must reach temperature and pressure above the critical point, so as the fluid behaves like liquid and gas simultaneously which makes extraction easier. This technology has been used in a wide diversity of fields (food, pharmaceutical, chemical and fuel industries) due to its advantages, there is an absence of toxic residue in the final product which allows not only the extraction valuable active compounds (fatty acids, pigments, polyphenols and vitamins) free of solvents, but also to remove undesirable compounds (pollutants, toxins and pesticides) . Additional advantages are great extraction selectivity, short processing times, requirement of minimal solvents, low degradability of the extracted product and the fact that the remaining biomass can be treated with other techniques in order to continue extraction.
The most important conditions are temperature, pressure and co-solvent. The selection of each parameter will depend on the specific compound searched . The most used solvent is CO2 due to its thermodynamics and heat transfer properties. Moreover, it has a low critical point (31°C, 73 bar). Furthermore, the polarity of CO2 can be modified by the use of co-solvents such as ethanol, and in this way also extract polar components.
Studies demonstrate that the extracts obtained with this technique are better than natural spice for flavoring purposes as SFE could reduce aflatoxin in the final products . In addition, numerous bibliographic references show that carotenoid extraction is better at higher pressures .
Furthermore, when analyzing the extraction of β-carotene and capsaicin at the same time, capsaicin shows lower solubility, yet the solubility of β-carotene did not change in the presence of capsaicin, a factor to bear in mind when designing the separation process of coloring and hot components from paprika . Several pepper species have been used to obtain natural compounds of interest using supercritical fluid extraction including capsaicinoids, oleoresins, pigments, tocopherols and even aflatoxins (Table 10) observing large variations between the pressures applied in each of the methods referred to in the bibliography. The greatest amount of studies is related to the specie
|7.84 × 10−3||55||1||—||70||7.1 × 10−4|||
|7.84 × 10−3||55||1||—||250||7.1 × 10−4|||
|15||60||2.15 × 10−3||90||2.5||0.5|||
4.3.3 Pulse electric field extraction
Pulsed electric field (PEF) consists of a non-thermal method that is extensively used in food processing applications due to, among other things, its ability to kill microorganisms in liquid foods. This method involves the application of short duration electric pulses, making several pores on the cell membrane in what is called electropermeabilization or electroporation. This makes it possible for the selective recovery of intracellular components with low energy consumption as the dielectric breakdown theory explains. According to this theory, the membrane of the cell has a low-dielectric constant that when exposed to a strong electric field provokes ion migration, forming free charges of the opposite sign which accumulate at both membrane sides, generating a potential difference across the membrane which depends on the size and shape of the cells and the concentration of cells in suspension. This difference in charges makes cell walls undergo a compression, reducing the membrane thickness that results in the formation of micropores and increasing permeability (electroporation) [119, 141]. It can be used directly or as a pre-treatment prior to solvent extraction . The parameters to take into account with this method are number of pulses, length of the pulses, energy input and biomass concentration.
Among the advantages of PEF is the short extraction time (usually under 1 s), efficiency at low temperatures, decreased energy losses and a successful cell wall breakdown . The disadvantages are that membrane changes can be reversible, air bubbles make the process less effective and the efficiency of the method depends on electric field strength and electrode gap .
Further research is necessary since there is no data on the extraction of compounds with this technique. However, there are studies of the application of PEF for the production of dehydrated products or juices. As for drying, a study  done with red bell pepper at 320 J/kg, 2.0 kV/cm, 1 Hz and 30 pulses of 400 μs reported good results. Another study  with the same aim used different extraction conditions, which were 2.5 kV/cm, 100 Hz and 30 μs as the pulse time. As for juice production,
5. Seed valorisation and industrial application
As it is being already done by some factories and companies in pepper growing countries, seeds, stalks or peels are extracted through whole fruit processing with or without semi-automated equipment. This is mainly done by the food industry in order to take away the non-edible parts of the vegetable to elaborate commodities as pepper powders (e.g., paprika), sauces, jams or pickled peppers. This way, stalks, seeds and placenta are usually removed and discarded.
But in many cases, placenta and seeds are subjected to extraction techniques in order to obtain essential oils to treat chronic ailments via topical administration in traditional remedies . Even so, it is clear that pepper seeds are a multivalent source of many important compounds that could be used in different applications.
The elevated phenolic concentrations found in seeds make them an excellent target for extraction and use of these phenolic compounds, whether it is as antioxidant supplements, as food coating in order to increase shelf-life or as a potential source of functional animal feed. Moreover, the implementation of an extraction process in the food industry is dependent on the type of compounds of interest, as the yields of phytochemicals obtained may differ greatly. It should be noted that highly efficient and sustainable extraction methods continue to be a focus of research both in industry and academia .
Also, antimicrobial potential of phytochemicals present in pepper should be considered. Even though this would involve highly specialized equipment and maintain strict aseptic conditions, there is promise in their use as food preservers from common food-borne pathogenic microorganisms besides their antioxidative capability. Furthermore, their use as alternative antibiotics has hardly been researched and the safety of their administration or clinical use has not yet been assessed.
Regarding their nutrition attributes, the high linoleic acid and palmitic acid present in the seeds could lead to use them as a primary source of cooking oil, or as a main component in foods such as margarine, or preserver in certain pickled products . Also, as they are a rich natural source of vitamins E and C, extracts of these vitamins could also be incorporated as constituents of many juices and other foods. Pepper-processing industry could find very rewarding to investigate on the yet unknown beneficial properties of its by-products and obtain additional profit by integrating existing extraction and purification technologies into their process chain.
It is clear that the wide spectrum of historical applications of peppers is supported by the latest findings on the properties of peppers. The diversity among
Many thanks to MICINN for the financial support for the Ramón & Cajal researcher of M.A. Prieto, to the Regional Government of Galicia to the Regional Government of Galicia for “Programa de axudas á etapa predoutoral da Xunta de Galicia” for the pre-doctoral researchers of A.G. Pereira and the authors are also grateful to Interreg España-Portugal for financial support through the 0377_Iberphenol_ project.