PI3K Signaling Pathways as a Molecular Target for Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common type of cancer that affects the central nervous system (CNS). It currently accounts for about 2% of diagnosed malignant tumors worldwide, with 296,000 new cases reported per year. The first-choice treatment consists of surgical resection, radiotherapy, and adjuvant chemotherapy, which increases patients' survival by 15 months. New clinical and pre-clinical research aims to improve this prognosis by proposing the search for new drugs that effectively eliminate cancer cells, circumventing problems such as resistance to treatment. One of the promising therapeutic strategies in the treatment of GBM is the inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway, which is closely related to the process of tumor carcinogenesis. This review sought to address the main scientific studies of synthetic or natural drug prototypes that target specific therapy co-directed via the PI3K pathway, against human glioblastoma.

New immunodiagnostic methods for human tegumentary leishmaniasis in the last 10 years: Technological prospecting.

Leishmaniasis is a neglected disease and more than 1 billion people live in endemic areas with the risk of infection worldwide. Although it is an important epidemiological issue, the gold standard method of diagnosis requires invasive sample collection and is accompanied by a high level of sensitivity variation in results. The present study aims to conduct a patent prospection of immunodiagnostic methods for human tegumentary leishmaniasis in the last 10 years, focused on those with high sensitivity and specificity, and simple usability. We searched seven patent databases: The LENS, WIPO, EPO, USPTO, Patent Inspiration, Google patents, and INPI. Eleven patents were found that satisfy our search criteria, with six of them being registered in 2017. Most patents were registered in Brazil. The information obtained here covers the main characteristics of the immunodiagnostic methods evaluated. Moreover, our prospective study reveals the latest biotechnological advancements achieved in the immunodiagnosis of tegumentary leishmaniasis, especially in Brazil, which holds the majority of patents in this subject. However, no patent for immunodiagnostic methods was found in the last three years, which raises concerns about the present and future trends of leishmaniasis diagnosis.

LQM10, a guanylhydrazone derivative, reduces nociceptive and inflammatory responses in mice.

In the present study, we examined the antinociceptive and anti-inflammatory activities of a guanylhydrazone derivative, (E)-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2-guanylhydrazone hydrochloride (LQM10), in mice. The antinociceptive effect was determined by assessing behavioural responses in different pain models, while anti-inflammatory activity was examined in carrageenan-induced pleurisy. Intraperitoneal LQM10 administration reduced the acetic acid-induced nociceptive behaviour, a phenomenon that was unaltered by pretreatment with yohimbine, atropine, naloxone or glibenclamide. In the formalin assay, LQM10 reduced nociceptive behaviour only in the second phase, indicating an inhibitory effect on inflammatory pain. LQM10 did not alter the pain latency in the hot plate assay and did not impact the locomotor activity of mice in the rotarod assay. In the carrageenan-induced pleurisy assay, LQM10 treatment inhibited critical events involved in inflammatory responses, namely, leucocyte recruitment, plasma leakage and increased inflammatory mediators (tumour necrosis factor Like Properties of Chalchones and Flavonoid Derivatives [TNF]-α and interleukin [IL]-1ß) in the pleural exudate. Overall, these results indicate that LQM10 exhibits antinociceptive effects associated with peripheral mechanisms and anti-inflammatory activity mediated via a reduction in leucocyte migration and proinflammatory mediators, rendering this compound a promising candidate for treating pain and inflammatory process.

Obstacles to Glioblastoma Treatment Two Decades after Temozolomide.

Glioblastomas are considered the most common and aggressive primary brain tumor in adults, with an average of 15 months' survival rate. The treatment is surgery resection, followed by chemotherapy with temozolomide, and/or radiotherapy. Glioblastoma must have wild-type IDH gene and some characteristics, such as TERT promoter mutation, EGFR gene amplification, microvascular proliferation, among others. Glioblastomas have great heterogeneity at cellular and molecular levels, presenting distinct phenotypes and diversified molecular signatures in each tumor mass, making it difficult to define a specific therapeutic target. It is believed that the main responsibility for the emerge of these distinct patterns lies in subcellular populations of tumor stem cells, capable of tumor initiation and asymmetric division. Studies are now focused on understanding molecular mechanisms of chemoresistance, the tumor microenvironment, due to hypoxic and necrotic areas, cytoskeleton and extracellular matrix remodeling, and in controlling blood brain barrier permeabilization to improve drug delivery. Another promising therapeutic approach is the use of oncolytic viruses that are able to destroy specifically glioblastoma cells, preserving the neural tissue around the tumor. In this review, we summarize the main biological characteristics of glioblastoma and the cutting-edge therapeutic targets that are currently under study for promising new clinical trials.

Pre-clinical evaluation of LASSBio-1491: From in vitro pharmacokinetic study to in vivo leishmanicidal activity.

Leishmaniasis is a public health issue. It is among the top five parasitic illnesses worldwide and is one of the most neglected diseases. The current treatment disease includes limitations of toxicity, variable efficacy, high costs and inconvenient doses and treatment schedules. LASSBio-1736 was described as antileishmanial drug-candidate to cutaneous leishmaniasis, displaying plasma stability and with no preliminary signals of hepatic or renal toxicity. In this paper, we described the in vitro pharmacokinetic study of LASSBio-1491 (a less lipophilic isostere of LASSBio-1736) and it is in vitro and in vivo leishmanicidal activities. Our results demonstrated that LASSBio-1491 has high permeability, satisfactory aqueous solubility, long plasma and microsomal half-lives and low in vitro systemic clearance, suggesting a pharmacokinetic profile suitable for its use in a single daily dose. The antileishmanial effect of LASSBio-1491 was confirmed in vitro and in vivo. It exhibited no cytotoxic effect to mammalian cells and displayed good in -vivo effect against BALB/c mice infected with Leishmania major LV39 substrain, being 3 times more efficient than glucantime.

Can inflammasomes promote the pathophysiology of glioblastoma multiforme? A view about the potential of the anti-inflammasome therapy as pharmacological target.

Glioblastoma multiforme (GBM) is a malignant brain tumor with one of the worst general survivorship cases among the existing neoplasia. This aggressiveness is due to its complex molecular heterogeneity, immunohistochemistry and genetics. The current therapeutic approach brings little contribution to the improvement of the survival of the patients. Due to that, new forms of treatment have been explored, one of them being immunotherapy. In this aspect, the inflammasome pathway, which induces inflammation and immunosuppressive tumor response, contributing to the progression of the tumor, seems to be a new alternative to improve the treatment efficacy and the survival of the patients.

Leishmanicidal activity of Morita-Baylis-Hillman adducts.

Leishmaniasis is a neglected disease that affects millions of people, mostly in developing countries. Although this disease has a high impact on public health, there are few drug options to treat the different leishmaniasis forms. Additionally, these current therapies have various adverse effects, including gastrointestinal disturbances, headache, pancreatitis, and hepatotoxicity. Thus, it is essential to develop new drug prototypes to treat leishmaniasis. Accordingly, the present study aimed to evaluate the leishmanicidal activity of Morita-Baylis-Hillman adducts and their O-acetylates, carboxylic acid derivatives, and acid and ester derivatives of 2-methyl-phenylpropanoids against Leishmania chagasi. Initially, we evaluated the cytotoxicity of 16 derivatives (1-16G) against J774A.1 macrophages. Eight derivatives (2G, 4G, 5G, 7G, 9G, 10G, 13G, and 15G) showed no cytotoxicity at up to the maximum concentration tested (100 µM). When evaluated for antileishmanial effect against promastigote forms, 1G, 6G, 8G, 10G, 11G, 13G, 14G, 15G, and 16G displayed significant toxicity compared to the control (0.1% DMSO). Additionally, the compounds 1G, 5G, 7G, 9G, 11G, 13G, 14G, and 16G reduced macrophage infection by amastigotes. Thus, we conclude that these derivatives have antileishmanial effects, particularly 1G, which showed activity against promastigotes and amastigotes, and low toxicity against macrophages.

LASSBio-596: a New Pre-clinical Candidate for Rheumatoid Arthritis?

Pain and inflammatory disorders are significant health problems because of prevalence and associated disabilities. In this context, LASSBio-596 is a hybrid compound able to modulate TNF-α and phosphodiesterases 4 and 5, exhibiting an anti-inflammatory effect in the pulmonary inflammatory model. Aiming at a better description of the activities of LASSBio-596, we initially conducted nociception tests (acetic acid-induced abdominal writhing, glutamate, and formalin-induced nociception and hot plate test) and later inflammatory tests (acute, peritonitis; and chronic, arthritis) that directed us to this last one. In the abdominal writhing test, there was a dose-dependent inhibition, whose response occurred at the maximum dose (50 mg/kg, p.o.), used in the subsequent tests. LASSBio-596 also inhibited nociception induced by chemical (glutamate by 31.9%; and formalin, in both phases, 1st phase: 25.7%; 2nd phase: 23.9%) and thermal agents (hotplate, by increased latency for pain at two different times). These effects were independent of the motor function, legitimated in rotarod. As there was a response in the inflammatory component of nociception, we performed the peritonitis test, in which migration was inhibited by LASSBio-596 by 39.9%. As the inflammatory process is present in autoimmune diseases, we also performed the arthritis test. LASSBio-596 reduced paw edema from the 15th day to the 21st day of treatment (no liver changes and with fewer paw injuries). In addition, LASSBio-596 decreased serum levels of TNF-α by 67.1%. These data demonstrated the antinociceptive effect of LASSBio-596 and reinforces its anti-inflammatory property (i.e., RA), amplifying the therapeutic potential of this molecule.

Fibrinolytic Enzymes From Extremophilic Microorganisms in the Development of New Thrombolytic Therapies: Technological Prospecting.

Extremophilic microorganisms from a wide variety of extreme natural environments have been researched, and many biotechnological applications have been carried out, due to their capacity to produce biomolecules resistant to extreme conditions, such as fibrinolytic proteases. The search for new fibrinolytic enzymes is important in the development of new therapies against cardiovascular diseases. This article aimed to evaluate the patents filed about protease with fibrinolytic activity produced by extremophilic microorganisms whose use is aimed at the development of new drugs for the treatment of cardiovascular diseases. The prospecting was carried out using data on deposits and patent concessions made available on the technological bases: European Patent Office (EPO), United States Patent and Trademark Office (USPTO), World Intellectual Property Organization (WIPO), Instituto Nacional de Propriedade Industrial - Brazil (INPI), The LENS and Patent Inspiration. The International Patent Classification and subclasses and groups for each document were also evaluated. Although 382 patents were selected using terms related to extreme environments, such as "thermophile" and "acidophiles", few were related to clinical use and were mainly performed using Bacillus subtilis and Streptomyces megasporus strains. A highlight of nattokinase was produced by Bacillus subtilis GDN and actinokinase by Streptomyces megasporus SD5. The low number of patents on enzymes with this profile (extreme environments) revealed a little-explored field, promising in the development of new microbial thrombolytic drugs, such as fibrinolytic enzymes with less adverse effects.

Design and Synthesis In Silico Drug-like Prediction and Pharmacological Evaluation of Cyclopolymethylenic Homologous of LASSBio-1514.

Acylhydrazones are still an important framework to the design of new bioactive compounds. As treatment of chronic pain represents a clinical challenge, we decided to modify the structure of LASSBio-1514 (1), previously described as anti-inflammatory and analgesic prototype. Applying the homologation as a strategy for molecular modification, we designed a series of cyclopentyl- (2a-e), cyclobutyl- (3a-e), and cyclopropylacylhydrazones (4a-e) that were synthetized and evaluated in murine models of inflammation and pain. A comparison of their in silico physicochemical and drug-like profile was conducted, as well as their anti-inflammatory and analgesic effect. Compounds 4a (LASSBio-1755) and 4e (LASSBio-1757) displayed excellent in silico drug-like profiles and were identified as new analgesic lead-candidates in acute and chronic model of pain, through oral administration.

Leismanicidal Activity of Propolis Collected in the Semiarid Region of Brazil.

Objective: The aim of the current study is to investigate the chemical composition, cytotoxic effect, and leishmanicidal activity of propolis collected in the semi-arid region of Bahia, Brazil. Methods: EtOH extract, hexane, EtOAc and MeOH fractions from propolis were analyzed by ultra-performance liquid chromatography coupled with diode array detector and quadrupole time-of-flight mass spectrometry. The identification was based on the exact mass, general fragmentation behaviors and UV absorption of the flavonoids. The in vitro cytotoxic effect and leishmanicidal activity of ethanolic extract, hexane, ethyl acetate, and methanolic fractions of propolis were evaluated. Results: Five triterpenes and twenty-four flavonoids were identified. The propolis did not present toxicity to the host cell up to the maximum concentration tested. In addition, all tested samples showed statistically significant activity against promastigotes of Leishmania chagasi and Leishmania amazonensis. Regarding the activity against amastigote forms of L. amazonensis, the hexane fraction, presented statistically significant activity with IC50 of 1.3 ± 0.1 µg/ml. Conclusion: The results support the idea that propolis can be used for future antileishmania studies.

Pigments from Antarctic bacteria and their biotechnological applications.

Pigments from microorganisms have triggered great interest in the market, mostly by their "natural" appeal, their favorable production conditions, in addition to the potential new chemical structures or naturally overproducing strains. They have been used in: food, feed, dairy, textile, pharmaceutical, and cosmetic industries. The high rate of pigment production in microorganisms recovered from Antarctica in response to selective pressures such as: high UV radiation, low temperatures, and freezing and thawing cycles makes this a unique biome which means that much of its biological heritage cannot be found elsewhere on the planet. This vast arsenal of pigmented molecules has different functions in bacteria and may exhibit different biotechnological activities, such as: extracellular sunscreens, photoprotective function, antimicrobial activity, biodegradability, etc. However, many challenges for the commercial use of these compounds have yet to be overcome, such as: the low stability of natural pigments in cosmetic formulations, the change in color when subjected to pH variations, the low yield and the high costs in their production. This review surveys the different types of natural pigments found in Antarctic bacteria, classifying them according to their chemical structure. Finally, we give an overview of the main pigments that are used commercially today.

Costus spiralis (Jacq.) Roscoe leaves fractions have potential to reduce effects of inflammatory diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Since drugs currently used to manage pain and inflammatory conditions present several side effects, the investigation of new anti-inflammatory and antinociceptive agents from folk-medicine plants is an important approach. Costus spiralis (Costaceae) has been used in Brazilian medicinal teas to treat urinary infection, cough, inflammation, arthritis, among others. AIM OF THE STUDY: The current study focused on investigating anti-inflammatory and antinociceptive effects of fractions from C. spiralis leaves using animal models. MATERIALS AND METHODS: Adults Swiss mice were used in the following experimental models: acetic acid-induced abdominal writhing, formalin-induced nociception, hot plate, zymosan-induced peritonitis, and arthritis induced by complete Freund's adjuvant. RESULTS: The presence of steroids was confirmed in all fractions. Flavonoids, condensed tannins and saponins were observed in EFL. In methanolic fraction leaves (MFL), the presence of flavonoids and pentacyclic triterpenoids was confirmed. Orally administered leaf fractions significantly reduced abdominal writhing. Fractions were ineffective in the neurogenic stage of the formalin test, but in the inflammatory stage, ethyl acetate fraction levaes (AcFL), ethanolic fraction leaves (EFL), and MFL significantly reduced paw licking time by 69.6 ± 11.9%, 58.2 ± 9.4%, and 79.6 ± 8.3%, respectively. In the hot plate test, the reaction latency was similar for treated animals and controls. However, in the peritonitis test, cell migration was significantly reduced in animals treated with chloroform fractions leaves ClFL (61.8 ± 11.4%), AcFL (58.7 ± 8.3%), EFL (39.2 ± 5.0%), and MFL (64.8 ± 4.4%). This was similar to the result observed in the chronic inflammation model, this time only the chloroform fraction was able to reduce paw edema. CONCLUSION: Our results show that leaf fractions of Costus spiralis are capable of modulating peripheral nociceptive and inflammatory responses without effects on central nervous system being potential substrates for phytochemical purification, structural and mechanistic studies.

Semicarbazone derivatives as promising therapeutic alternatives in leishmaniasis.

In the present study, we investigated the in vitro and in vivo leishmanicidal activity of synthetic compounds, containing a semicarbazone scaffold as a peptide mimetic framework. The leishmanicidal effect against amastigotes of Leishmania amazonensis was also evaluated at concentration of 100 µM-0.01 nM. The derivatives 2e, 2f, 2g and 1g, beyond the standards miltefosine and pentamidine, significantly diminished the number of L. amazonensis amastigotes in macrophages. These derivatives were also active against amastigotes of L. braziliensis. As 2g presented potent leishmanicidal activity against the amastigotes of L. amazonensis in macrophages, we also investigated the in vivo leishmanicidal activity of this compound against L. amazonensis. Approximately 105L. amazonensis promastigotes were subcutaneously inoculated into the dermis of the right ear of BALB/c mice, which were subsequently treated with 2g (p.o. or i.p.), miltefosine (p.o.) or glucantime (i.p.) at 30 µmol/kg/day x 28 days. Thus, a similar reduction in the lesion size was observed after the administration of 2g through oral (63.7 ±â€¯10.1%) and intraperitoneal (61.8 ±â€¯3.7%) routes. A larger effect was observed after treatment with miltefosine (97.7 ±â€¯0.4%), and glucantime did not exhibit activity at the dose administered. With respect to the ear parasite load, 2g diminished the number of parasites by p.o. (30.5 ±â€¯5.1%) and i.p. (33.3 ±â€¯4.3%) administration. In addition, 2g induced in vitro apoptosis, autophagy and cell cycle alterations on L. amazonensis promastigotes. In summary, the derivative 2g might represent a lead candidate for antileishmanial drugs, as this compound displayed pronounced leishmanicidal activity.

Synthesis and evaluation of the antibiotic and adjuvant antibiotic potential of organotin(IV) derivatives.

A series of organotin(IV) derivatives was investigated in vitro for their antibiotic and adjuvant antibiotic properties (efflux pump inhibitors) against Staphylococcus aureus strains that overexpress efflux pump proteins for norfloxacin (SA-1199B), erythromycin (RN-4220) and tetracycline (IS-58). Most organotin(IV) compounds showed significant antibacterial activity with small Minimum Inhibitory Concentration (MIC) values, some of which were close to 1.0µg/mL (3.1µM), but this feature was also associated with substantial cytotoxicity. Nevertheless, the cytotoxicity of these organotin(IV) compounds can be overcome when they are used as antibiotic adjuvants. Their remarkable adjuvant antibiotic properties allow potentiation of the action of tetracycline (against IS-58 strain) by up to 128-fold. This likely indicates that they can act as putative inhibitors of bacterial efflux pumps. These results reinforce organotin(IV) complexes as promising antibacterial agents, and many of these complexes, if associated with antibiotics, can act as potential adjuvant antibiotic candidates.

Usual normalization strategies for gene expression studies impair the detection and analysis of circadian patterns.

Recent studies have shown that transcriptomes from different tissues present circadian oscillations. Therefore, the endogenous variation of total RNA should be considered as a potential bias in circadian studies of gene expression. However, normalization strategies generally include the equalization of total RNA concentration between samples prior to cDNA synthesis. Moreover, endogenous housekeeping genes (HKGs) frequently used for data normalization may exhibit circadian variation and distort experimental results if not detected or considered. In this study, we controlled experimental conditions from the amount of initial brain tissue samples through extraction steps, cDNA synthesis, and quantitative real time PCR (qPCR) to demonstrate a circadian oscillation of total RNA concentration. We also identified that the normalization of the RNA's yield affected the rhythmic profiles of different genes, including Per1-2 and Bmal1. Five widely used HKGs (Actb, Eif2a, Gapdh, Hprt1, and B2m) also presented rhythmic variations not detected by geNorm algorithm. In addition, the analysis of exogenous microRNAs (Cel-miR-54 and Cel-miR-39) spiked during RNA extraction suggests that the yield was affected by total RNA concentration, which may impact circadian studies of small RNAs. The results indicate that the approach of tissue normalization without total RNA equalization prior to cDNA synthesis can avoid bias from endogenous broad variations in transcript levels. Also, the circadian analysis of 2-Cycle threshold (Ct) data, without HKGs, may be an alternative for chronobiological studies under controlled experimental conditions.

Antinociceptive activity of Tibouchina pereirae, an endemic plant from the Brazilian semiarid region.

The anti-nociceptive activity of an extract of Tibouchina pereirae Aubl (AETP) was investigated using two models of chemically induced pain, viz. the acetic acid-induced writhing and the formalin test, respectively, with dipyrone and indomethacin as reference drugs, respectively. In the acetic acid-induced writhing test, AETP application (100 mg/kg) caused a significant reduction of writhing produced by acetic acid. In the formalin test, AETP reduced the formalin effects significantly only in the late phase. These findings thus indicate the involvement of AETP only in peripheral antinociceptive mechanisms. In addition, AETP exhibited good antioxidant activity (EC50 approx. 15 µg/mL) in the DPPH free radical scavenging assay.

Involvement of ß adrenergic receptors in spasmolytic effect of caulerpine on guinea pig ileum.

Previously, we demonstrated that caulerpine has spasmolytic effect on guinea pig ileum. The aim of this study was to investigate pathways of its spasmolytic action. We test caulerpine against phasic contractions induced by carbachol in the circular layer of guinea pig ileum and this alkaloid did not inhibit these contractions, indicating that caulerpine did not interfering with the mobilisation of Ca2+ from intracellular stores. Additionally, the spasmolytic effect of caulerpine did not involve K+ channels. Furthermore, we observed that α2-adrenergic receptors were not involved in the spasmolytic effect of caulerpine, since the relaxation curve induced by caulerpine was not shifted in the presence of yohimbine (α2-adrenergic antagonist). However, in the presence of propranolol (ß-adrenergic antagonist), the relaxation curve induced by caulerpine was right-shifted, resulting in a fivefold increase in EC50. Thus, a possible mechanism for the spasmolytic action of caulerpine is the activation of ß-adrenergic receptors.

Chemical constituents from Sidastrum paniculatum and evaluation of their leishmanicidal activity

AbstractSidastrum paniculatum (L.) Fryxell, Malvaceae, is popularly known in Brazil as “malva-roxa” or “malvavisco”. The species is found mainly in Northeast region where it is used by locals to treat spider bites and bee stings. Aiming to identify the chemical compounds from S. paniculatum secondary metabolism and to contribute to the chemotaxonomic knowledge of Malvaceae family, a phytochemical study of S. paniculatum was carried out. Besides that, the isolated compounds were evaluated for antileishmanial activity against promastigotes of Leishmania braziliensis. By using chromatographic techniques the study resulted the isolation of eight compounds: 3-oxo-21β-H-hop-22(29)-ene; sebiferic acid; sitosterol 3-O-β-d-glucopyranoside/stigmasterol 3-O-β-d-glucopyranoside; phaeophytin a; 132(S)-hydroxyphaeophytin a; 132(S)-hydroxy-(173)-ethoxyphaeophorbide a and 7,4′-di-O-methylisoescutellarein. The structure of all isolated compounds was elucidated by spectroscopic analysis, including two-dimensional NMR techniques. In addition, the isolated compounds phaeophytin a; 132(S)-hydroxyphaeophytin a; 132(S)-hydroxy-(173)-ethoxyphaeophorbide a and 7,4′-di-O-methylisoescutellarein exhibited antileishmanial activity against promastigotes of L. braziliensis.

Antimicrobial Analysis of an Antiseptic Made from Ethanol Crude Extracts of P. granatum and E. uniflora in Wistar Rats against Staphylococcus aureus and Staphylococcus epidermidis.

INTRODUCTION: Surgical site infection remains a challenge for hospital infection control, especially when it relates to skin antisepsis in the surgical site. OBJECTIVE: To analyze the antimicrobial activity in vivo of an antiseptic from ethanol crude extracts of P. granatum and E. uniflora against Gram-positive and Gram-negative bacteria. METHODS: Agar drilling and minimal inhibitory tests were conducted for in vitro evaluation. In the in vivo bioassay were used Wistar rats and Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 14990). Statistical analysis was performed through variance analysis and Scott-Knott cluster test at 5% probability and significance level. RESULTS: In the in vitro, ethanolic extracts of Punica granatum and Eugenia uniflora and their combination showed the best antimicrobial potential against S. epidermidis and S. aureus. In the in vivo bioassay against S. epidermidis, there was no statistically significant difference between the tested product and the patterns used after five minutes of applying the product. CONCLUSION: The results indicate that the originated product is an antiseptic alternative source against S. epidermidis compared to chlorhexidine gluconate. It is suggested that further researches are to be conducted in different concentrations of the test product, evaluating its effectiveness and operational costs.