Palo Negro (L. rivularis) is an endemic and native shrub of Chile, belonging to the Asterácea family. This family has important active components such as acetylenes, sesquiterpene, and sesquiterpene lactones. L. Rivularis possesses Leptocarpin (LTC); a sesquiterpene lactone that has been researched for many years, possessing medicinal properties such as antitumor, antineoplastic, and apoptotic activity. Quercetin can also be found as another of its relevant compounds, as it has antiviral, hypoglycaemic, anti-inflammatory, antioxidant, preventive properties for cardiovascular and anticancer diseases. The studies below highlight the use of Palo Negro infusion in its entirety, due to the synergy of the compounds it possesses.

Leptocarpin and infusion 

Alvarez (2005), who studied the hypoglycaemic effect of L. Rivularis infusion, points to a procedure called TTOG (Oral test in response to glucose tolerance). This procedure was developed to classify carbohydrate tolerance in general, showing the ability of B cells in the pancreas to secrete insulin and tissue sensitivity.

From this study, it can be seen that this infusion works by modifying the insulin response, after administering a specific load of glucose (2 grams per pound) in diabetic rats. Therefore, an increase in tolerance response, resulting from a peripheral restoration to insulin, or inhibition of intestinal glucose consumption could result in an increased insulin production by B cells in the pancreas.

 

In conclusion, infusion of Leptocarpha Rivularis was shown to be a viable alternative for the control of hyperglycemia and hypercholesterolemia (characteristics of NIDDM (non-insulin-dependent diabetes mellitus)), without side effects and without altering parameters in healthy patients. In addition, analyses related to AIM (immunomarked area) show an increase in the amount of insulin related to Langerhans Islets in diabetic rats treated with the infusion. However, it is not possible to determine whether this amount is sufficient for the hyperglycemia control state found in diabetic rats.

Martínez et al (2006) studied the acid reaction of LTC, a component with anti-tumor activity that formed two new isometric products. The structures were established by a spectroscope and confirmed through ROESY experiments and theoretical studies by molecular mechanisms. The in vitro cytotoxicity of these isometric components was less active than LTC, showing the importance of chemical structure with the presence of the oxyrane ring, influencing biological activity. Cytotoxic activity was measured in six lines of cell cancer.

Following the study, DNA and RNA synthesis is not affected by LTC and synthetic products. Therefore, its biological activity is not at this level. LTC produces a noticeable effect on protein biosynthesis to a different extent in cancerous cell lines.  Also, the effectiveness of the results could determine that LTC is a naturally sourced antineoplastic agent that can be used in different types of neoplasm.

 

Regarding the cytotoxic and apoptotic effects of LTC, Bosio et al (2015) researched sesquiterpene lactones in pharmacological studies, because they had a number of biological activities such as anticancer, anti-inflammatory and antimicrobial. LTC has been widely used in folk medicine by Mapuche people. Previous research has shown that LTC decreases cell viability in cancer cell lines, for example: HT 29, PC 3, DU-145. MCF7 and MDA MB-231. This inhibitory effect is dose-dependent with IC50 in the range of 2.0-6.4 um. However, the LTC effect on human dermal fibroblasts decreases. Thus, the effect has been related to apoptosis showing that LTC produces morphological changes, significant chromatin condensation and fragmentation in cancerous lines compared to control cell lines. As additional support of apoptosis, it has been demonstrated by flow cytometry that LTC induces depletion in the mitochondrial membrane’s potential and, consequently, a release of cytochrome C, and an increase in caspase-3 activity has also been observed. Finally, the LTC effect on Nf-kB activation has been studied, and results indicate that cells treated with LTC exhibit a significant reduction in Nf-kB activity at concentrations as low as 7um.

Zheng et al (2017) stated that in the clinical treatment of neoplasm, chemotherapeutic drugs (which could either be natural products or chemical derivatives) are widely and gradually used. Up-to-the-date, the search for new products with profitable biological activity to our health has been focused in new medicinal plants. The Asteracea family possesses many bioactive compounds such as polyacetylenes, diterpenes, sesquiterpenes and lactones that express several clinical benefits: antitumor, anti-ulcer, anti-inflammatory, neurocytotoxic and cardiotonic activity. Moreover, it has the ability to intervene in critical biological processes such as cell- signaling, cell proliferation, apoptosis, and mitochondrial metabolism.

Due to these reasons, they concluded that LTC suppresses osteosarcoma proliferation, migration, and invasion. IGF-1R is one of the LTC objectives in the osteosarcoma suppression, which provides clinical treatments for the osteosarcoma as a new molecular target and drug. Other approaches are necessary to show IGF-1R’s role as an LTC host in osteosarcoma suppression as well as animal experiments.

 

Jiménez Gonzáles et al (2018), pointed out the identification of organic compounds in UHPLC/ESI/MS is the first step in the majority of spotting techniques for the characterization of biologically active metabolites in natural sources. From this view, the L. Rivularis, which is used in Chilean folk medicine regarding gastrointestinal ailments and cancer avoidance, showed antioxidant and inhibitor properties in regards to cholinesterase. Phenolic compounds and many others were spotted in Palo Negro infusion for the first time. Its active compounds could either emerge as natural antioxidants, or as raw material for the inhibitor-insulation of AChE, apart from being a complementary source against Alzheimer and others related. This study highlights the Quercetine presence, a phenolic compound belonging to the flavonoids.

Olea et al (2019) suggests that phytochemical studies have shown that L. Rivularis contains a high SQL percentage, highlighting LTC as the main component. The LTC has a similar structure to other SQL, being highly cytotoxic; It also induces apoptosis and acts as an efficient NF-kB inhibitor. Moreover, micellar polymers application as conductive vehicles for anticancer drugs has been extensively studied over the past two decades.

As a conclusion, the cytotoxicity of both ethnic extracts and ethyl acetate obtained from L. Rivularis were evaluated against several cancer cell lines: PC-3 (prostate cancer), MCF-7 (breast cancer), HT-29 (colon cancer) and other non-tumor cell lines such as human colon epithelial cells (CoN). The results indicated both extracts exhibited activity in HT-29, MCF-7 and PC-3 cell lines, with IC50 values in the range of 11-16 ug/mL. A comparison of these IC50 values with those previously reported by leptocarpine suggests the existence of a synergistic effect on the activity of EA extract (ethyl acetate). This effect can be attributed to the presence of other lactones that are minor components inside this extract.

Finally, it is worth to say that none of the L. rivularis extracts show activity in non-tumor cell lines, suggesting that these selectively act in tumor cells.

Uquiche et al (2019) states that nowadays it is being sold in pharmacies and alternative medical stores as a product to make infusions. The bioactive compounds in the plant have shown an inhibitory and cytotoxic effect in cancer cells.

This study also emphasizes the concept of Lipoxygenase; an enzyme involved in regulating inflammatory responses by leukotriene biosynthesis, which have been linked to inflammatory diseases including cancer. Phenolic compounds exert an anti-inflammatory effect by inhibiting lipoxygenase, through their activity at the active site of the enzyme. As  a consequence, a high-modifying concentration does not necessarily end up in an improvement in the extract efficiency and phenolics’ total extraction

Mena (2008) assessed the LTC the antitumor, anti-proliferative, and cytotoxic potential, a sesquiterpene lactone; its likely action mechanism and its influence on the ability and expression of multi-drug resistance proteins on two different cell lines. One of them, HL60 (Human Promyelocytic Leukemia) and U937 (Human Monocytic Leukemia) along with normal and ex vivo leukocytes obtained from primary leukemia cells cultured to determine the medicinal effect of Leptocarpine on cell viability in different cell lines.    

The cytotoxic and anti-proliferative effect of Leptocarpine was studied by exclusion of blue triptan and TTT reduction for 24 hours in concentrations between ranges from 0 to 100 uM. LTC successfully produced a drop in cell viability of about 70% compared to controls in leukesmic cells, inducing apoptosis at a p- 21WAF-1 and dependent forms associated with intrinsic routes (Bcl-2). LTC also induced negative variations in the expression of some MDR transporters such as MRP1 and MDR1.

Bosio (2014) concludes the following: LTC significantly decreases cell viability in cell lines studied depending on dose and time. In addition, LTC has a high selection level (2.95-9.23), inducing chromatin condensation and fragmentation in the cell lines studied. Moreover, it increases mitochondrial membrane’s permeability, inducing the specific activation of caspase 3 in the studied cell lines (except cell lines DU-145, MCF-7 and DHF).

LTC also induces the cell cycle arrest in the phase (G0/G1) in the cell lines studied. It inhibits nuclear localization and DNA binding with transcription factor NF-kB, and apparently inhibits the HIF-1a transcription factor’s nuclear localization.

Summarizing, it can be stated that the LTC behaviour in an acid environment allows us to think that this molecule is sensitive to acid conditions, and therefore as possible medicine for the future, but it should not be administered orally; unless it is protected from gastric acids. This is why it is transformed into heliangolidas structures with less biological activity. It can also be claimed that LTC inhibits cell viability in various cell lines regarding rat studies. Another LTC characteristic is that it is dose-and-time dependent, as well as its high selectivity in terms of tumour cells where early apoptosis occurs.

 

Jiménez- González et al (2018) performed analysis of the L. rivularis, where the presence of quercetin, a flavonoid that has been extensively studied, was detected.

This compound has medicinal properties described. 

Batiha et al (2020), conducted bibliographic review on pharmacological activity, biochemical properties of quercetin. This compound belongs to flavonoids, which are an essential component of cosmetic, pharmaceutical, food and medicinal formulations. They also highlight that this polyphenol is found in a wide variety of vegetables and properties have been shown to be anticancer, antiviral, for the treatment of allergies, metabolic and inflammatory disorders, cardiovascular diseases and arthritis. Recent bibliographic compilation of Zhang et al (2020) on preclinical for the quercetin behaviour in Alzheimer, point out that the studies conducted are incomplete and information is lacking to accurately determine the compound’s applicability for the treatment of Alzheimer's, but at first, it gives hope of being effective for this treatment.

Due to current events, quercetin is being intensively researched for COVID-19 treatment and prevention, Derosa et al (2020) point out that this compound has previously been used in Chinese folk medicine for the treatment of several diseases and as an antiviral, due to its ability to inhibit virus replication. Quercetin is a viable alternative for COVID-19 treatment, as it can be incorporated within food to ease and massify its consumption.

Although there are studies that have investigated Palo Negro , there is still a lot of information that needs to be put under evaluation, as well as methods and procedures that can positively impact LTC effectiveness  and its possible insertion into pharmaceutical medicine and not just in folk/alternative medicine due to its anticancer potential. In addition to gathering information referred to other active compounds that this plant possesses, Kimun is developing extraction and purification processes regarding the compounds previously mentioned in Palo Negro. All the research, development and innovation carried out in Kimun is possible due to the 10% reinvestment in terms of profits that are obtained.