Patent Application
20230414692
The present invention relates to the field of biology, botany, nutrition and biomedicine. Specifically, it relates to obtaining an ethanolic extract of vegetable origin to be used as an antitumor agent, from mature seed meal of Solanum melongena, optionally defatted.
Cancer is defined as a set of related diseases in which certain cellular alterations cause a loss of control of the proliferation process that leads to excessive cell division and the ability of these malignant cells to spread to other tissues. Cancer, in general, has a high incidence in the world population and requires the development of new, more effective treatments, especially in advanced stages of the disease in which patients have a very poor prognosis. Within the field of oncology, colorectal cancer (CRC) is considered a real public health problem due to its current prevalence and incidence projections in the coming years.
According to data from 2018 from the Spanish Society of Medical Oncology (Sociedad Española de Oncologia Médica or SEOM), CRC ranks third among cancers with the highest incidence in both sexes and worldwide. In Spain, CRC is the most frequently diagnosed cancer (year 2019) in both sexes, being the second most frequent in both men and women. Regarding its mortality rate, CRC was the second leading cause of cancer-related deaths in both sexes as of December 2019, according to the National Institute of Statistics. This mortality has a clear growth trend that has been shown to be related to lifestyle and diet.
Among the risk factors for the development of CRC are: i) age, since it is diagnosed most frequently in patients over 50 years of age (90% of cases) without other pathologies or predisposing diseases; ii) dietary factors, which play an essential role and are under continuous investigation; in fact, excessive alcohol consumption, overweight and obesity, and certain types of food (processed meat) have been linked to this pathology; iii) predisposing diseases, especially the presence of intestinal polyps or inflammatory bowel disease, which means that these patients should be considered at high risk for the development of CRC; iv) a history of CRC; v) so-called genetic or family factors, given that in 25% of cases there is a family history and in 10% a hereditary component, and vi) lastly, lifestyle, with physical inactivity being an important factor.
Despite the fact that the treatment and prevention of CRC have undergone great advances in recent years, the results, in terms of cure or reduction in incidence, are far from satisfactory. We must emphasize that, in the most advanced phase of the disease, in which patients develop distant metastases (especially in the liver), the results of the different applied therapies tested are especially poor. These results are dramatically reflected in the prognosis of these patients, who have a very low survival rate. The currently approved treatments for CRC range from surgery, when the tumor is resectable, to the use of chemotherapy based on different cytotoxics such as oxaliplatin, irinotecan, 5-fluorouracil, capecitabine, Utefos, TAS-102, Raltitrexed (either alone or in combination) or radiotherapy. In recent years, new biological drugs active against CRC have been developed, including the monoclonal antibodies cetuximab, panitumumab, bevacizumab and a recombinant fusion protein (aflibercept) that has very specific therapeutic indications (Loree et al., 2017; Nappi et al., 2018; Bregni et al., 2020). Nonetheless, and as the mean survival of these patients clearly indicates (15 to 20.5 months), the results are very limited. Therefore, improving the prognosis of these patients and improving their survival requires the development of new strategies that add therapeutic activity to preventive action (Idrees et al., 2019; Reglero et al., 2019).
The development of new therapeutic and prevention strategies covers a large number of research fields that range from nanotechnology, gene therapy, immunotherapy using strategies that activate the immune system or the use of new compounds or extracts of plant or animal origin, which may be active in the treatment of CRC or adjuvant to current therapies to improve the response to treatment. In this context, the study of the effect of plant extracts or derivatives on the viability, proliferation and survival of tumor cells has gained great interest in recent years (Goyal et al., 2017). In fact, this line of research has been endorsed and supported by the National Cancer Institute (USA) since 1960, the year in which the antitumor and preventive activity of different plant extracts against certain carcinomas began to be evaluated (Huang et al., 2013). Plants in general, and their extracts in particular, have numerous applications in medicine since they are i) a direct source of therapeutic agents, ii) a raw material for the manufacture of more complex semi-synthetic drugs, iii) for providing the chemical structures of their active principles, which can serve as a model for the preparation of synthetic drugs and iv) for the use of said principles as taxonomic markers in the search for new drugs. These possible applications are due to the phytochemicals present in the plants and their extracts, of which more than 5,000 have been identified in seeds, fruits, roots, tubers, leaves, etc., among which are phenolic compounds, carotenoids, vitamins, alkaloids, terpenes and terpenoids, nitrogenous compounds and organosulfur compounds (Thapliyal et al., 2018).
Although they have been extracted from plants or parts thereof, the possible activity of many phytochemical compounds has been studied individually, after being isolated or, at most, by analyzing the activity of fractions obtained in chromatographic processes in which their presence has been demonstrated. Thus, for example, Akihisa T. et al. (2011) isolated salannin from the seeds of the Azadirachhta indica tree and demonstrated that salannin and certain other limonoids exhibit a potent inhibitory effect on the melanogenesis process. To isolate these compounds, they carried out an extraction by reflux for 3 hours with n-hexane in order to subsequently isolate the limonoids present by chromatography. Salannin also possesses antitumor activity in neuroblastoma and osteosarcoma cell lines (Cohen et al., 1996) with an IC50 of 133±6 μM in the NIE.115 neuroblastoma cell line and 89±12 μM in the 143B.TK osteosarcoma cell line.
Moreover, Kaempferol 3-sophorotrioside is related to antioxidant and hepatoprotective activity (Toshiyuki et al., 2001).
Ting et al. (2017) developed extracts containing myricomplanoside. For this purpose, they produced an ethanolic extract (95%) from the seeds of Astragalus chinensis in order to subsequently make fractions and obtain some compounds isolated by chromatography. These fractions, one of which contained myricomplanoside, exhibit an antioxidant, anti-inflammatory and antiproliferative activity.
In turn, the group of capsianosides is characterized by interfering with the function of the cytoskeleton by modulating the reorganization of actin filaments (Hashimoto et al., 1997).
Phenolic compounds, in general, have attracted the interest of the scientific community due to their great structural diversity as well as their wide bioactivity. These phenolic compounds hold essential functions in the reproduction and growth of plants, act as defense mechanisms against pathogens, parasites and predators, and are also responsible for giving plants their color. They are not only beneficial for plants, but also play an important role in human health, as antioxidant, anticancer, antibacterial, and anti-inflammatory agents (Huang et al., 2013; Bonta et al., 2019). Due to their bioactivity, there are numerous drugs and patents whose main components are polyphenols, that is, compounds that have more than one phenolic group per molecule. This is the case of Neumentix™, (https://www.kemin.com/na/en-us/products/neumentix), a patented supplement (U.S. Pat. No. 9,839,661), obtained by Kemin, (represented in Spain by Univar) and obtained from harvested green mint (KI110 and KI42 ranges) after a drying process. This supplement, rich in polyphenols of the rosmarinic, salvianolic and caftaric acid types, has been shown, through clinical trials, to provide great benefits in cognitive performance. In addition, the polyphenols that characterize this supplement act as antioxidant agents, reducing oxidative stress, promoting neuronal growth and protecting nerve cells in the brain. Similarly, polyphenols found in grapes, blueberries, and other fruits and vegetables have been studied for their ability to lower the risk of developing neurodegenerative diseases. In addition, they have been used as a supplement to minimize the effects of age, especially memory loss, as is the case with Optimized Curcumin with Neurophenol™ (mixture of blueberry and grape extracts), from Douglas Laboratories® (Valencia, Spain: https://www.douglaslabs.es/). There are also commercialized polyphenolic extracts patented from the grape seed (Vitaflavan®, product of DRT—Les Derivés Résiniques et Terpéniques, Dax, France: https://www.vitaflavan.com/es/), from the pomace of the red grape (Eminol®) and red wine (Provinols®, from Sucren/Vitimed, distributed by Seppic, La Garenne Colombes, France: https://www.seppic.com/provinolstm-0) that have antioxidant properties.
More than 25% of the drugs used during the last 20 years are directly derived from plants, while another 25% are derived from modified natural products (Amin et al., 2009). It is worth mentioning that only between 5% and 15% of the plants used for medicinal purposes have been subject to research aimed at obtaining their bioactive compounds. This highlights the importance of the search for new drugs from plant species (Rivas-Morales et al., 2016; Wong et al., 2018).
The genus Solanum (Solanaceae) has the most species (more than 1500 registered species) of any genus in the Solanaceae family, of which the potato (Solanum tuberosum), the tomato (Solanum lycopersicum) and the eggplant (Solanum melongena) deserve to be highlighted due to their economic and cultural importance. Because the species of the genus Solanum contain various phytochemicals such as alkaloids, this genus has been used since ancient times for medicinal purposes. The alkaloids that these species present have been of great interest due to their broad biological activity, such as antimicrobial, antirheumatic, antioxidant, as well as anticancer (Jayakumar et al., 2016).
The plants of the species Solanum melongena (eggplant) seem to originate in Southeast Asia, with their main center of origin possibly being in India, a country where they have been cultivated since ancient times (at least since 2000 BC). Their ancestral cultivation in Africa and China is also known; this last country may have been a center of secondary origin in which varieties with small fruits were developed. Eggplant cultivation spread throughout North Africa, from where its cultivation seems to have spread to Muslim Spain and other warm Mediterranean countries, such as France, Italy and Greece. The numerous current varieties that have been developed coexist with ancestral varieties of Asian origin and varieties of other species, the fruit of which is also called eggplant, and with which they are closely related, such as Solanum aethiopicum, a species including varieties of ancestral domestication in sub-Saharan Africa.
Solanum melongena is one of the plant species for which therapeutic applications are known due to the research into and identification of its different bioactive compounds starting from different parts of the plant.
Thus, for example, document JP2002226387A relates to a pharmaceutical composition that contains dried eggplant powder used to topically treat wounds, burns, acne, athlete's foot, stomatitis, inflammation, tumors, etc.
Some other studies, such as those by Zhao Dong-Yin et al. (2020), describe the isolation and identification of various sesquiterpenoids and other compounds from the sepals of Solanum melongena by ethanolic extraction (70%) by reflux, followed by the isolation of the bioactive compounds by petroleum ether. These compounds were tested in vitro against cervical-uterine cancer, adenocarcinoma and gastric cancer cell lines.
Studies have also been carried out using the roots in which different compounds have been isolated that were tested in vitro in breast cancer lines. In this case, the method carried out for the development of the extract is through a reflux process with 70% ethanol and fractionation. In the studies by Yin Xin et al. in 2019, several sesquiterpenoids were isolated and none of those tested showed cytotoxicity against breast cancer (MCF-7), liver cancer (HepG2) or cervical-uterine cancer (HeLa) cell lines. Yang Bing-You's 2020 studies describe the isolation of terpenes, lignans and other compounds also from roots, some of which showed moderate cytotoxicity against the same cell lines.
As in the sepals and roots, bioactive compounds (glycoalkaloids, and, in particular, solamargine) have also been found in the skin of the fruit that have been tested, and have shown effectiveness against liver carcinoma (Fekry et al., 2019) and lung cancer (Shen et al., 2017), and which were obtained by methanolic and ethanolic extraction (70%) and using HPLC (70%). Finally, anthocyanins and delphinidin derivatives from eggplant skin, which were isolated by extraction carried out by immersion in methanol, have been shown to have antioxidant activity in colon cancer cell lines (Jing et al., 2015).
The anticancer activity of glycoalkaloids has already been observed by Lee et al. in 2004, who carried out a study on different glycoalkaloids from potatoes, eggplants and tomatoes and their activity on HT-29 colon cancer cells and HepG2 liver cancer cells. The major glycoalkaloids identified in Solanum melongena were solamargine and solanine. In general, the glycoalkaloids were shown to have some anticancer activity, although their effectiveness was greater in trials with the liver cancer cell line than against colon cancer cells.
Thus, although some studies analyze the presence of bioactive compounds in different parts of the plant of the Solanum melongena species and analyze, individually for each compound, its activity in different types of tumors, there is little research in relation to the molecular mechanisms by which these compounds act, studies that would be essential to elucidate the pathways by which these functional extracts, or isolated compounds, could act against tumor cells. Among the few works that address aspects of the molecular activity of the extracts, there is the study carried out by Nishina et al. (2015), wherein a bioactive compound, dioscin, was isolated from a methanolic extract from Solanum melongena L., which acts as an inhibitor of melanogenesis by deregulating the phospho-CREB protein, which binds and activates MITF (Microphthalmia-associated transcription factor involved in the development of melanocytes and osteoclasts).
Furthermore, although there are studies focused on other parts of the plant, the seeds seem to have received less attention: there are no recently published articles that study the antitumor activity of functional extracts from Solanum melongena seeds or isolate bioactive compounds therefrom. Only one study published in 1985 describes the isolation of 3 saponins from Solanum melongena seeds by refluxing with methanol for 4 hours at 65° C. and extract concentration under reduced pressure. However, this study did not report antiproliferative activity of these compounds (Kintia and Shvets, 1985).
Given the need to develop alternative therapeutic strategies against CRC, it would be interesting if new anticancer agents could be found that act thereon, preferably with selective antitumor activity. For simplicity purposes, it would be interesting if said activity could be found in a plant extract, preferably from a plant with edible parts, such as the eggplant, whose extract exhibits such an activity, without the need to isolate the bioactive compounds present therein (even though said isolation is possible), and if the extract could be obtained by means of a methodology that would facilitate its use as an anticancer agent at concentrations in which said extract would show reduced toxicity against non-cancer cells. It would also be a preferable additional feature if said extract contained more than one component that exhibited anticancer activity (or therapeutic activity in general) per se, which would allow the preparation of pharmaceutical compositions combined with two or more of said bioactive compounds. In addition, it would be helpful to know, preferably in some depth, the molecular and cellular mechanisms by which said extracts cause tumor cell death.
The present invention provides a solution to this problem, which also include several of the above-mentioned desired additional advantages.
In one aspect, the invention relates to a method for obtaining an ethanolic plant extract from mature seeds of Solanum melongena, which comprises the steps of:
Preferably, the method of the invention is carried out under the following conditions:
It is very specifically preferred that the defatting be carried out prior to obtaining the meal, at a temperature between 40 and 50° C. and with an extraction speed of 2 to 3 kg of seeds/hour.
In another preferred embodiment, a new extraction is carried out on the residue resulting from the first extraction, specifically on the precipitate resulting from obtaining an initial extract after centrifugation, applying the following sub-steps:
The method of the invention may include an additional final step in which the ethanol from the ethanolic extract obtained is partially or totally evaporated.
In another aspect, the invention relates to an ethanolic extract from mature Solanum melongena seeds, particularly with a high polyphenol content. Preferably, said extract will be an extract obtainable by the method of the present invention.
The ethanolic extract, according to the tests of Example 1, has a total polyphenol content ranging between 11.16 and 27.59 μg gallic acid equivalents/mg extract. Preferably, the total polyphenol content ranges from 12.06 to 27.59 μg gallic acid equivalents/mg extract (which are the extreme values determined from extracts from defatted meal). The case in which the content of total polyphenols ranges between 11.16 and 18.41 and/or the reducing capacity ranges between 5.33 and 6.31 μg gallic acid equivalents/mg extract (values obtained in extracts of variety S0506) is also of interest.
In another possible embodiment, compatible with the previous ones, the ethanolic extract comprises at least salannin and/or capsianoside II. Preferably, in addition to the two previous compounds, the ethanolic extract comprises at least one more bioactive compound selected from the group of kaempferol 3-sophorotrioside, myricomplanoside and aryllatose B, or combinations of these three compounds, with particular preference that the ethanolic extract comprise, in addition to salannin and capsianoside, myricomplanoside and/or aryllatose B, or aryllatose B and kaempferol 3-sophorotrioside. More preferably, the ethanolic extract comprises at least the five previous compounds: salannin, capsianoside II, myricomplanoside, aryllatose B and kaempferol 3-sophorotrioside.
As in the case of the method of the invention, and in the other aspects of the invention that are described below, the embodiments that correspond to extracts obtained from meal of mature seeds of Solanum melongena varieties, preferably defatted, are preferred. Thus, in the case of the ethanolic extract of the invention, it is preferred that it meet the definition based on the biochemical parameters obtainable from mature seeds of Solanum melongena varieties, that is, the extract where:
The extracts of the invention obtained from mature seeds of sample S0506 are also preferred, both from defatted and non-defatted meal, so those where:
In any of the definitions, it is preferred that the extract have been obtained by the method of the present invention, in its most general definition, or in any of its possible embodiments; included within these embodiments is the possibility of carrying out the optional final additional step in which the final, partial or total, evaporation of the ethanol is carried out. It is preferred, again, that the step of defatting the mature seeds by cold mechanical pressing has been carried out before carrying out step a) of grinding the seed and step b) of extracting the meal obtained and, especially, that each of the possible steps (defatting, grinding and extracting per se) are carried out with the defining characteristics expressed when describing the possible embodiments of the method of the invention, including carrying out a second extraction on the precipitate resulting from centrifugation that gives rise to the initial extract.
A pharmaceutical composition is also an aspect of the present invention, which will be considered a pharmaceutical composition of the present invention which comprises an extract of the present invention in its formulation, in any of the possible embodiments that have been described above, such an example being that of the extracts obtained by the method of the present invention in which the final, partial or total, evaporation of ethanol has been carried out. The composition may be a combined pharmaceutical composition that comprises at least one anticancer agent in addition to those present in the extract. In another possible embodiment, also compatible with any other embodiment, the composition may also comprise one or more pharmaceutically acceptable excipients and/or carriers.
It can also be considered that the above aspect of the invention implies that the use of an extract of the present invention for the preparation of a pharmaceutical composition is also included within the present invention, particularly if it is intended for the treatment of cancer, especially if it is selected from the group of colorectal cancer, pancreatic cancer and glioblastoma.
An extract of the present invention, or a pharmaceutical composition of the present invention, for use in the treatment of a type of cancer which is selected from the group of colorectal cancer, pancreatic cancer and glioblastoma are also aspects of the invention. More specifically, the cancer may be selected from the group of colon adenocarcinoma, pancreatic adenocarcinoma, and glioblastoma multiforme. From aspects of the invention, the preferred ones are both those referring to the extract and to the pharmaceutical composition, the extracts obtained from mature seeds, especially defatted, but also without defatting, and the pharmaceutical compositions prepared therefrom.
The aspects related to the therapeutic use of an extract of the invention or of a pharmaceutical composition of the invention can also be defined, or are related to, a method of treatment of a subject suffering from a cancer selected from colorectal cancer, pancreatic cancer and glioblastoma, which comprises administration of a pharmaceutical composition of the invention, or of a therapeutically effective amount of an extract of the invention. The subject, as in the definition of the extract of the invention or the pharmaceutical composition of the invention for therapeutic use, can be any mammal, preferably a human being.
Another aspect of the invention refers to the use of the extract of the present invention as an active ingredient to be incorporated into food supplements. “Food supplement” is defined as a product that is added to a diet and that contains substances of natural origin with a nutritional or physiological function that are called functional ingredients. It is generally taken orally and may come in different forms: pills, tablets, capsules, powder sachets, ampoules of liquid, and dropper bottles.
Also considered an aspect of the invention is a food complement or supplement that comprises the extract of the invention as an active or bioactive ingredient. This supplement may also be called a nutraceutical, which is understood as a dietary supplement or complement, presented in a non-food matrix (pills, capsules, powder, etc.), of a concentrated bioactive natural substance, preferably of plant origin, which has a favorable effect on health greater than that which the food itself could have. In other words, nutraceuticals are the components of the food, or parts of it, that provide a proven and added benefit for health, capable of providing medical improvements, in the prevention and treatment of diseases such as colorectal cancer, pancreatic cancer and glioblastoma.
The present invention is based on the methodology for obtaining and the in-depth analysis of ethanolic extracts from a plant species of the genus Solanum (Solanaceae), more specifically, obtained from the meal of mature seeds of Solanum melongena (eggplant).
At present, extracts obtained from different parts (sepals, roots, fruits, fruit skin, etc.) of Solanum melongena can be found in the existing literature. Such extracts are generally obtained with highly toxic organic solvents and/or extraction processes of great complexity and duration that can increase the toxicity of the wide variety of components (flavonoids, coumarins and terpenoids) produced. This is the case, for example, in the study by Kintia and Shvets in 1985, commented on in the previous “Background of the invention” section, in which an extract from the seeds of said plant was obtained, but in which methanol was used under reflux, a compound known to have a toxicity greater than that of ethanol at lower concentrations, and to potentially cause dizziness, nausea, vomiting, damage to the nervous system and even death. In this study, several new saponins were isolated and characterized, but they were not reported to have any activity against colon, pancreatic, or glioblastoma cancer. In fact, there are currently no studies of bioactive compounds with antitumor activity against colon cancer obtained from Solanum melongena seeds.
Moreover, studies of extracts from other parts of the plant (sepals, roots, etc.) obtained with refluxing ethanol have focused on the isolation of certain compounds present in the extracts and, in the case of having carried out antiproliferative tests, on testing the possible cytotoxicity of said compounds, individually, against certain cancer lines, among which, in general, colon, pancreatic or glioblastoma cancer lines were not included.
In response to the possible drawbacks presented by the extracts from different parts of the plant described in the prior art, the present inventors have developed an extract that exhibits favorable bioactivity and can be obtained from mature seeds of the plant, using simple extraction processes of short duration with non-toxic solvents, while allowing adequate extraction yields. This new extract also presents a more specific composition of active and low-toxic substances that are the Solanum melongena polyphenols.
The present invention is based on:
The results obtained from the research disclosed in this application show that:
Thus, the ethanolic extraction methodology provided by the present invention allows obtaining a non-toxic extract for use in biomedicine, particularly as an antitumor agent. Said methodology represents an enormous advantage for the purpose of its application in patients, since it is based on the use of ethanol, a solvent that is used regularly and at low concentrations for the administration of active principles.
Given the scientific interest that underlies the Solanum melongena species, ethanolic extracts from mature seeds of different varieties of this species, donated by the plant breeding company Agrointec Solutions SL, belonging to the Cellbitec Business Group (www.cellbitec.com) were studied as described in the trials presented below.
Seeds of four varieties of different types of the Solanum melongena species were used, covering the widest spectrum of characterization of this species, following the recommendations of the International Union for the Protection of New Varieties of Plants (UPOV, www.upov.int) defined in document TG/117/4 (https://www.upov.int/edocs/tqdocs/es/t117.pdf), the samples being those listed below:
The method for obtaining the ethanolic extract that was developed from the meal of the mature seeds, both non-defatted and defatted, of Solanum melongena, is schematized in
Following the extraction protocol described, ethanolic extracts were obtained from the defatted and non-defatted mature seed meal of Solanum melongena. The objective of this double process was to compare the activity of the extracts, removing the high percentage of lipid compounds from the mature seeds without defatting (see
To determine the yield, the ethanolic extract was divided into 1 mL aliquots for ethanol evaporation and subsequent lyophilization of the remaining water in the extract. In order to remove the ethanol from the ethanolic extracts, it was evaporated under vacuum using a Savant DNA 120 evaporation system (Thermo Scientific) for 60 minutes. After evaporation of the ethanol, the aliquots were frozen in liquid nitrogen with the remaining extract and lyophilized using a TELSTAR Cryodos-50 lyophilizer where they were kept for 24 hours. After lyophilization, the dry weight of the extract was calculated based on the difference with the container that contained each aliquot and said dry weight was referred to a volume of 1 mL of initial extract, subsequently to the total volume of extract obtained, and finally to the grams of starting seed meal for the preparation of the extract.
Total polyphenols were determined by means of the Dewanto et al. technique (2002), as described by Kapravelou et al. (2015), which uses a standard line of gallic acid with concentrations between 0 and 500 μg/mL. Furthermore, the reducing capacity of Fe3+ to Fe2+ by the different extracts was determined spectrophotometrically by means of Duh et al. (1999) technique, as described by Kapravelou et al. (2015), which uses a standard line of gallic acid with concentrations also between 0 and 500 μg/mL.
Table 1 shows the yield and total polyphenols of the extract from the non-defatted and defatted mature seed meal of the Solanum melongena S0503, S0504, S0505 and S0506 varieties. As can be seen in the aforementioned table, the yields obtained in the analyzed cases, for all the varieties of Solanum melongena, showed a great variability, with those of the extracts from defatted mature seeds being significantly higher than those of the extracts from the non-defatted mature seeds.
melongena
Furthermore, the antioxidant capacity was analyzed including biochemical studies of total polyphenols and reducing capacity. Regarding total polyphenols, the ethanolic extracts from defatted and non-defatted meal, for all the varieties tested, present very homogeneous values, being, in general, and with the exception of sample S0505, higher in the ethanolic extracts from defatted mature seeds (with values ranging between 12.06 and 27.59 μg gallic acid equivalent/mg extract for the ethanolic extract from common eggplant from defatted mature seeds) than in the ethanolic extracts from non-defatted meal (11.16 μg gallic acid equivalent/mg extract in S0506). These results made it possible to corroborate that the process of elimination of lipidic compounds, in addition to increasing the performance of the functional extracts, does not alter or eliminate the bioactive compounds of interest; additionally, they confirmed that the above mentioned method made it possible to purify the functional extract.
The results obtained from sample S0506 deserve to be highlighted (which is done throughout the present specification), as said sample has one of the highest yields (42.6 mg/g meal in the ethanolic extract from non-defatted mature seeds and 71.12 mg/g meal in the ethanolic extract from defatted seeds). The value of total polyphenols determined in the ethanolic extract from defatted mature seed meal (18.41 μg gallic acid equivalent/mg extract) is one of the highest among the extracts tested. In addition, as will be seen later in Example 2, it is one of those with the greatest antitumor activity (see Table 10). For all these reasons, as will be mentioned later, it was selected to be the subject of specific studies carried out to elucidate the molecular mechanisms of cellular action.
The reducing capacity tests for the biochemical determination of the antioxidant capacity were carried out with the extracts selected from sample S0506, obtaining values of 5.3 μg gallic acid equivalents/mg extract for the extract from non-defatted mature seed meal, and 6.31 μg gallic acid equivalents/mg extract for the ethanolic extract from defatted meal (Table 2). Therefore, the antioxidant capacity of the extracts prepared with defatted mature seed meal was higher based on the fact that the polyphenols are obtained in greater quantities.
The chromatographic studies carried out made it possible to determine the compounds present in the ethanolic extract from the mature seed meals of Solanum melongena. These studies used Waters ACQUITY H-Class Ultra Performance Liquid Chromatography (UPLC) together with a Waters SYNAP G2 Q-TOF Mass Spectrometer.
Tables 3, 4, 5, 6 and 7 below indicate the main compounds identified in each of the extracts of the Solanum melongena varieties and the chromatographic data of each of them (for compounds with more than one chromatographic peak, only the data of one of the peaks is indicated). Said compounds, and their number in the CAS (Chemical Abstract Service) registry are: triglycidyl trimellitate (CAS No: 7237-83-4), myricomplanoside (CAS No.: 123442-26-2), Kaempferol 3-sophorotrioside (CAS No.: 80714-53-0), Arylatose B (CAS No.: 137941-45-8), Quercetin 3-rhamninoside (CAS No.: 522-12-3), Swertiajaponin 3′-O-gentiobioside (CAS No.: 76166-51-3), Quercetin 3,7-diglucoside (CAS No.: 6892-74-6), Quercitrin (CAS No.: 522-12-3), Ramontoside (CAS No.: 133882-75-4), Macrostemonoside J (CAS No.: 159935-09-8), Salannin (CAS No.: 992-20-1), Mudanpioside J (CAS No.: 262350-52-7), Cudranian 1, Capsianoside III (CAS No.: 121961-81-7), Protodioscin (CAS No.: 55056-80-9), Capsianoside II (CAS No.: 121924-04-7), Macrantoside I (CAS No.: 90850-94-5), Dioscin (CAS No.: 19057-60-4), Asparanin D (CAS No.: 83931-89-9), Kalambroside A (CAS No.: 160472-99-1), Citrusin B (CAS No.: 105279-10-5), Chlorogenic acid (CAS No.: 327-97-9), Quercetin-3,4′-O-di-β-glucopyranoside (CAS No.: 29125-80-2), Myricitrin (CAS No.: 17912-87-7), Macrostemonoside J (CAS No: 159935-09-8), Blumeoside C (CAS No: 94657-28-8), Pikuroside (CAS No: 231280-24-3), Kaempferol 3-(2G-xylosylrutinoside)-7-glucoside (CAS No: 131559-51-8), Gypenoside LVI (CAS No: 109145-67-7), Vismione D (CAS No: 87605-72-9), Volubiloside B (Cas No: 485807-79-2), Nonadecanoic Acid (CAS No: 646-30-0).
Among the compounds present in the vast majority of the extracts, from both non-defatted and defatted mature seed meals, Salannin (C34H44O9), Kaempferol 3-sophorotrioside (C33H40O21), Myricomplanoside (C22H22O13), Arillatose B (C22H30O14) and Capsianoside II (C50H84O25) stand out (
As it was observed, the ethanolic extracts of the varieties, from both non-defatted and defatted mature seed meals of Solanum melongena, share, for the most part, several of the bioactive compounds described above. This is logical, since the different samples used genetically belong to the same plant species and the active compounds are part of important metabolic pathways, although it should be noted that, morphologically, the fruits, as indicated in the UPOV TG/117/4 protocol, can be quite varied while all the phenotypic varieties belong to the same species, that is, Solanum melongena.
Due to the lack of studies on the antitumor activity of the bioactive compounds present in the mature seeds of the Solanum melongena species in colon cancer, and the limited knowledge about the independent activity of the main compounds that integrate it, the research disclosed in the present application was conducted, not only on the antitumor properties of the ethanolic extracts of the invention in colon cancer, but also focused in depth on the performance of these compounds and their combined action.
In order to determine the antitumor capacity of the extracts, the cell lines T84 (human colon adenocarcinoma cell line), HCT15 (human colon adenocarcinoma cell line resistant to chemotherapy) and MC38 (murine colon adenocarcinoma cell line) were cultured. As a control, the HepG2 line was selected (a human hepatocyte cell line whose great differentiation makes it suitable as a model for the study of human hepatocytes in various types of assays, including assays on drug or active compound activity, in which they are often used as controls).
The ethanolic extracts were previously evaporated to avoid the toxicity caused by ethanol on cell lines. In addition, once evaporated, a portion was lyophilized to learn the amount of extract obtained and quantify its concentration (mg/ml), based on which the different concentrations to be tested will be calculated. The cell cultures were exposed to increasing concentrations of the evaporated ethanolic extracts from both the non-defatted and defatted mature seed meals of all types of Solanum melongena, which allowed determining the inhibitory concentration 50 (IC50) (concentration of the extract at which the extract inhibits 50% of the cell population) using the Sulforhodamine B technique. The results obtained are shown in Table 8.
Solanum melongena (S0503, S0504, S0505 and S0506)
T84: human colon adenocarcinoma cell line; HCT15: human colon adenocarcinoma cell line resistant to chemotherapy; MC38: murine colon adenocarcinoma cell line. IC50: concentration that inhibits 50% of the cells.
As can be seen in the previous table, for the ethanolic extract from non-defatted mature seeds of the S0506 variety, the IC50s were the following: 23.88 μg/ml in T84, 37.07 μg/ml in HCT15 and 48.58 μg/ml in MC38. For the extracts from defatted mature seed meal of S0506, the IC50s were: 35.07 μg/ml in T84, 29.63 μg/ml in HCT15 and 39.3 μg/ml in MC38. It should be noted that the IC50 of the ethanolic extract from defatted meal of S0506 in the HepG2 human hepatocyte line was 99.87 μg/ml, which leaves an acceptable therapeutic range for conducting further in vivo research. Furthermore, very similar IC50s were observed in the rest of Solanum melongena varieties.
In view of the results, it can be observed that the ethanolic extracts from non-defatted and defatted mature seed meals of Solanum melongena have a high antiproliferative activity, with the IC50 being very low and homogeneous among the different samples tested. This fact is noteworthy, since it indicates that the defatting process does not excessively alter the antitumor capacity of the extract obtained and, in some cases (such as the activity on HCT15 cells of the extract obtained from mature seed meal of sample S0506), even increases it. In addition, the difference between the IC50 of the tumor lines (T84, HCT15 and MC38) and the non-tumor line (HepG2) is relevant, being significantly higher in the latter.
Due to the fact that the extracts from the mature seed meals, both non-defatted and defatted, have a similar antitumor activity and that the extracts of the defatted mature seed meal have a higher yield and a higher concentration of polyphenolic compounds, it was decided to use the ethanolic extract from defatted mature seed meal to carry out the rest of the molecular tests that are detailed below. In addition, the ethanolic extract from defatted meal from sample S0506 was selected for these tests, as a representative of the other extracts, due to its lower IC50 value for the HCT15 line and to its high yield.
Lastly, and based on previous results in colon cancer cell lines, the ethanolic extracts, from both the defatted and non-defatted meals of all varieties, were tested on glioblastoma multiforme and pancreatic adenocarcinoma cell lines. For that purpose, cell lines A-172 and LN-229 (human glioblastoma cell line), SF-268 and SK-N-SH (human chemotherapy-resistant glioblastoma cell lines) in addition to Panc-1 (human pancreatic adenocarcinoma cell line) were cultivated. Using the same method described above, the IC50 of the cell lines under study were determined. The results are presented in Table 9.
For the ethanolic extract from non-defatted meal of S0506, the IC50 was 23.89 μg/ml in SF-268, 25.70 μg/ml in SK-N-SH, 24.15 μg/ml in A-172 and 21.22 in LN-229, while in the line derived from pancreatic adenocarcinoma Panc-1, the IC50 was 45.29 μg/ml. For the ethanolic extract from defatted meal of the S0506 variety, the IC50s were higher: 35.36 μg/ml in SF-268, 37.75 μg/ml in SK, 36.11 in A-172, 37.96 in LN-229 and 49.02 in Panc-1. In the remaining samples, in contrast, the extracts obtained from defatted meal generally gave lower IC50 values than those from non-defatted meal.
These results allowed us to conclude that the ethanolic extracts of all Solanum melongena varieties, both from defatted and non-defatted meal, in addition to presenting a great antitumor capacity against colon cancer, also have high antitumor activity against cell lines of glioblastoma and pancreatic adenocarcinoma.
To elucidate the mechanisms of action of the extracts of the present invention, the cell death pathway (apoptosis), mediated by caspases, mainly caspase 8 (extrinsic pathway), caspase 9 (intrinsic pathway) and caspase 3, was studied by means of the Western Blot technique, using endogenous β-actin as control.
For this purpose, cells from the colon tumor line (T84) were cultured with an IC50 of the ethanolic extract obtained from the defatted mature seed meal of S0506 and, after 72 hours, the cells were collected to proceed with the protein extraction.
To carry out the Western Blot assay, 40 μg of protein from the cells treated with the ethanolic extract, as well as from the control cells (T84 without treatment) were loaded on an SDS-PAGE electrophoresis polyacrylamide gel in a Mini-Protean II cell (Bio-Rad, Hercules, CA). Once the proteins were separated by electrophoresis, they were transferred to a nitrocellulose membrane to which 20 V were supplied at room temperature for 1 hour. These membranes were treated with blocking solution (PBS-Tween+5% powdered milk) for 1 hour, then, after 2 washes with PBS-Tween, they were incubated with the primary antibody [rabbit anti-caspase-3 polyclonal IgGs (dilution 1:500), anti-caspase-8 (1:1000 dilution) and anti-caspase-9 (1:1000 dilution); Santa Cruz Biotechnology, Santa Cruz, CA]. They were incubated overnight at 4° C. Once the incubation time had elapsed, two washings were carried out and incubation took place for 1 hour at room temperature with the peroxidase-conjugated secondary antibody. Finally, the proteins were detected by means of ECL (Enhanced Chemiluminescence) (Bonnus, Amersham, Little Chalfont, UK) (Ortiz et al. 2009).
Once the Western Blot was performed (see
To determine the activation of other possible mechanisms of cell death, studies of possible alterations in the polymerization/depolymerization of cell microtubules were carried out using immunofluorescence. To that end, T84 cell cultures were exposed to the ethanolic extract (IC50) evaporated from the defatted meal of S0506. After 24 hours, the cells were fixed and incubated with an anti-tubulin antibody. As can be seen in
Lastly, despite the fact that most of the cell death induced by the ethanolic extract from the defatted meal of S0506 was produced by apoptotic routes, a certain percentage of it occurred by autophagic routes. Cultures of T-84 cells exposed to the ethanolic extract (24 hours) were stained using a red fluorescent probe (LysoTracker™ Red DND-99, ThermoFisher Scientific) and observed under a fluorescence microscope. The appearance of the classic autophagic vesicles in the T84 line indicated the activation of this mechanism (
Angiogenesis or the formation of new blood vessels from endothelial cells is essential for the growth of tumors and for their expansion and the production of metastases. In the present study, the effect of the ethanolic extract on tumor cells in relation to their communication with endothelial cells and, therefore, with the formation of new blood vessels (angiogenesis) was analyzed. T84 cells were incubated with the ethanolic extract from the defatted meal of S0506 at doses of IC25 (20 ug/ml) and IC50 (30 ug/ml) for 24 hours. After washing the wells (PBS) and adding new DMEM (+10% FBS and 1% Ab) free of ethanolic extract, the culture was maintained for 48 hours in order to obtain a conditioned medium. Next, HUVEC cells (umbilical cord endothelial cell line) were seeded in 96-well plates with Matrigel. These cells were contacted with the conditioned media obtained in order to observe the formation of blood vessels.
As can be seen in
Tumors are characterized by having subpopulations of tumor cells called cancer stem cells (CSCs), whose fundamental properties are being undifferentiated and pluripotent, but which are especially characterized by having a high tumorigenic capacity and presenting resistance to treatments (chemoresistance). These CSCs are responsible, to a large extent, for clinical recurrences and for the low effectiveness of treatments with therapeutic regimens that are currently used. A large part of the most advanced lines of research in the field of basic and clinical oncology are based on the development of antitumor agents that, preferentially or selectively, affect this cell type, which would guarantee a better patient response to treatment and a greater benefit in patient prognosis. CSCs are a very heterogeneous population that are identified by certain cell markers, such as CD133, CD44, CD24, NANOG, SOX-2 and OCT-4, among others (Munro et al., 2018).
To determine the antitumor capacity of the ethanolic extract of the invention in certain subpopulations of cancer stem cells, T84 cell cultures were exposed to the ethanolic extract (IC50) of the defatted meal of S0506 for 72 hours. From the total RNA extracted from the cultures, a Real Time PCR was carried out to determine the expression of the CSC markers CD24, CD44, CD133, SOX2, OCT4 and NANOG.
To that end, cellular RNA was first extracted with Trizol (Invitrogen) reagent and quantified with NanoDrop 2000. 1 μg of RNA was used to carry out the retrotranscription process and obtain cDNA using SuperScript II Reverse Transcriptase (Invitrogen) with a universal reverse transcription primer. Real Time PCR was carried out using SYBR Green Supermix (iTaq Universal SYBR Green Supermix from Bio-Rad Laboratories, Hercules, CA). Gene expression data were normalized with GADPH. All quantitative RT-polymerase chain reaction (RT-PCR) assays were performed on an ABI 7900 (ABI) system, and the 2ΔΔCt method to calculate relative expression levels was applied.
As observed in
| Number | Date | Country | Kind |
|---|---|---|---|
| P202031034 | Oct 2020 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2021/070738 | 10/11/2021 | WO |
