Reactive Oxygen Species (ROS)
In Five Ayurvedic Ingredients
a preliminary review
by Saagar Dhanjani
This pilot review draws on journal articles published in five journals to survey recent studies on plants and minerals considered part of Ayurvedic practice. A total of 68 articles were tagged, while ten articles were read and considered for inclusion in the review. This study was originally designed to evaluate how effective ayurvedic treatments were in the treatment of cancer. Reactive Oxygen Species (ROS) is a common factor in apoptotic behavior and, therefore, a standard chemotherapeutics target. As a result, the effect ayurvedic treatments have on ROS was explicitly examined in this study. A total of five ayurvedic treatments (three herbs, one naturally occuring substance, and one herbo-metallic/mineral preparation) were studied: Piper Nigrum, Mineral Pitch, Flacourtia Indica, Azadarichta Indica, and Manikya Bhasma. There are several different naming systems used when identifying these Ayurvedic ingredients, such as Latin, Sanskrit, English romanization, and common name. Five different cancer cell lines were studied: MCF-7, HT-29, Huh-7, E6-1, HT-29, among different Ayurvedic treatments.
Reactive Oxygen Species (ROS) have been shown to be elevated in almost all types of cancers. (Liou & Storz, 2010) ROS are radicals, ions, or molecules that contain oxygen. ROS radicals contain an unpaired electron in their outermost electron shell. (Liou & Storz, 2010) Examples of these radicals include superoxide (O2-), hydroxyl radical (OH-), peroxyl (LOO-), and alkoxyl radicals (LO-). (Li et al., 2016) However, some ROS do not contain any unpaired electrons in their outermost shell. Examples of these include hydrogen peroxide, peroxynitrite (ONOO-), hypochlorous acid (HOCl), and ozone (O3). (Li et al., 2016) ROS can also be referred to as reactive oxygen intermediates (ROIs), reactive oxygen metabolites (ROMs), oxygen radicals, and reactive nitrogen species (RNS). (Li et al., 2016) RNS, for example, is a collective term to include oxides of nitrogen or nitrogen-containing reactive species like nitrogen dioxide radical and peroxynitrite. Because these compounds almost always contain oxygen, they can be considered under ROS.
High levels of ROS in cancer cells can be a result of increased metabolic activity, mitochondrial dysfunction, peroxisome activity, increased activity of oxidases, cyclooxygenases, lipoxygenases, thymidine phosphorylase, or by crosstalk with infiltrating immune cells. (Babior, 1999; Storz, 2005; Szatrowski & Nathan, 1991) Normally, intracellular levels of ROS are maintained by either non-enzymatic molecules such as glutathione, flavonoids, and vitamins A, C, or E or antioxidant enzymes. (Liou & Storz, 2010) However, in cancer cells, macrophages and neutrophils aim to increase the generation of ROS to induce apoptosis. NAPDH oxidase produces a large amount of superoxide, which then leads to hydrogen peroxide formation. (Babior, 1995, 1999) Because inflammation has shown to be critical to tumor growth, macrophages, in an effort to induce apoptosis, generate nitric oxide during inflammation processes. (Balkwill, 2009; Berasain et al., 2009; Xiao & Yang, 2008) This nitric oxide reacts with superoxide to produce peroxynitrite radicals that contribute to tumor cell apoptosis. (Cui et al., 1994) Similar to how our body fights cancer cells, chemotherapeutics also focuses on increasing cellular ROS to irreparable levels to induce tumor apoptosis. (Trachootham et al., 2009) Examples of well-known chemotherapeutics that accomplish this are gemcitabine with trichostatin A, epigallocate-3-gallate, capsaicin, sulindac, and benzyl isothiocyanate. (Donadelli et al., 2007; Gahr et al., 2007; Lee et al., 2009; Qanungo et al., 2005; Sahu et al., 2009; Shankar et al., 2007, 2008; Srivastava & Singh, 2004; Zhang et al., 2008) This study aims to provide a review of ayurvedic herbs that are also ROS-producing reagents.
Herbs with the Potential to be ROS Producing Reagents
Keywords: Piper Nigrum, MCF-7 cells, HT-29 cells, cytotoxicity, DNA fragmentation
Ayurvedic practices have been using Piper Nigrum for the treatment of many medical disorders. (Chaveerach et al., 2006, 4) Grinevicius et al. (de Souza Grinevicius et al., 2016) showed Piper Nigrum’s (Black Pepper) ability to be a ROS-producing reagent. Piper nigrum fruits were first harvested at a farm in São Mateus, Espírito Santo, Brazil. The fruits (145 g) were dried and ground, and extracted with 93% ethanol. Then cytotoxicity was tested with two cell lines: human breast carcinoma (MCF-7) and colon cancer (HT-29). The cytotoxicity was tested by tetrazolium salt (MTT) assay, and the contents of ROS were measured by dichlorofluorescein diacetate. After 24 hours of treatment with Piper Nigrum, the proliferation of MCF-7 cells was reduced by 50% when compared to the negative control (Dulbecco’s modified Eagle medium plus dimethyl sulfoxide). The Piper Nigrum ethanolic extract also demonstrated the ability to cause DNA fragmentation. The break in DNA strands compromises the genetic information it contains, and therefore often leads to apoptosis. In addition, intracellular ROS overproduction in MCF-7 cells was observed with Piper Nigrum treatments, along with increased activity in superoxide dismutase (SOD), glutathione reductase, and catalase which are all antioxidant enzymes. These increases in ROS are the reason for DNA fragmentation and subsequently apoptotic activity in tumor cells. Images of Piper nigrum as a flower and as a powder are displayed in figure one.
Figure 1. “Black Pepper - Piper Nigrum L.,” Living Proof L.L.C., accessed August 16, 2021, https://www.alivingproof.com/products/black-pepper.
Keywords: Bitumen, fluorescent dye, dose dependence, liver cancer cell line
Mineral Pitch (MP), also known as Bitumen, is an ayurvedic medicinal mineral used in the Himalayan regions (see figure 2). Pant et al. (Pant et al., 2016) demonstrated its effects on the production of ROS. The study collected the herb from Matela, a village in Bajhang District in north-western Nepal. Huh-7 cells were incubated for 24 hours with different concentrations of MP (0, 50, and 100 µg/mL). Apoptosis was determined by TUNEL assays, while MTT assays determined cell proliferation. Formation of ROS and nitric oxide were also observed by 2’,7’-dihydrochloroflurorescein acetate (DCFH-DA) and the Griess reagent. Incubation with MP led to significant decreases in cell proliferation with a 55.8, 65.3, 70.3, and 73.3% decrease for 100, 200, 500, and 1000 µg/mL of MP, respectively. The percentage of apoptotic cells were 21.7, 63, 77.3, 80.1% for 100, 200, 500, and 1000 µg/mL of MP respectively. These were all significant increases when compared to the untreated cells. ROS production increased 30, 38, 49.8% in cells incubated with 100, 200, and 500 µg/mL of MP, respectively. Nitric oxide production had percentages of 24.7, 60.27, 61.2, and 65.33 for 20, 200, 500, and 1000 µg/mL, respectively. SOD activity was significantly decreased by 20 and 34% with concentrations of 500 and 1000 µg/mL of MP, respectively. The decreases in antioxidant levels may be an explanation for the augmentation of ROS.
ayurvedic herbs that are also ROS-producing reagents.
Figure 2. Dyet, Dave. “Category: Asphaltum.” Wikimedia Commons, https://commons.wikimedia.org/wiki/Category:Asphaltum
Keywords: Flacourtia Indica, apoptosis, HCT116, survivin, caspase-3
Another study described Flacourtia Indica (FIM), also known as Baichi, effects on ROS production. (Park et al., 2014) Flacourtia Indica was collected from Sreepur, a city in central Bangladesh. Parts of the plant have long been used in Ayurvedic medicine. After being sun-dried for fifteen days, the plant was extracted with methanol. Human cancer cells (HCT116) were then incubated with FIM for 24, 48, or 72 hours. FIM reduced cell viability and increased apoptotic activity in proportion to increasing concentration. Treatment with FIM also showed strong evidence in its ability to induce apoptosis. FIM decreased survivin levels, a protein that inhibited cellular apoptosis and increased the expression of caspase-3, cytochrome c, and Parp (a caspase substrate). Caspases cleave many cellular substrates, which allows them to be a moderator for cellular apoptosis. Using a DCF-DA fluorescence staining method, the accumulation of ROS levels was also examined. ROS levels increased in a concentration-dependent manner and were significantly greater in comparison to the control. Figure three displays FIM as a flowering plant.
Figure 3. Bera, Bubai. “Flacourtia Indica.” Wikimedia Commons, 21 Oct. 2021, https://commons.wikimedia.org/wiki/File:Flacourtia_indica_bubai03.jpg.
Keywords: Neem, Azadarichta indica leaves, HT-29, E6-1, cell viability, ROS levels, mitochondrial instability
Azadirachta indica leaf extracts have also demonstrated the potential to selectively increase intracellular ROS levels in cancer cells. Also known as “Neem,” Azadirachta indica is a medicinal plant (see figure 4) that has long been used in Ayurvedic practices. Roma et al. (Roma et al., 2015) demonstrated Neem’s selective induction of apoptosis. The dried neem leaf powder was purchased from Premier Herbal Inc in Toronto. Human T-cell leukemia cells (E6-1) and colon cancer cells (HT-29) were supplemented with either aqueous or ethanolic Neem leaf extracts (NLE). Cell viability was reduced upon treatment with aqueous and ethanolic NLE in both leukemia and colon cancer cells. The effective concentration (EC-50) value for ethanolic NLE, or the concentration required to induce a response that is halfway between the baseline and maximum, is 0.04 mg/mL in leukemia cells and 0.4 mg/mL in colon cancer cells. The EC-50 value for aqueous NLE in leukemia and colon cancer cells, on the other hand, was 0.5 and 3 mg/mL, respectively. Interestingly, when normal cells were treated with NLEs, there was no significant reduction in cell viability. After treatment for 48 hours, the cancer cells also showed an increase in Hoechst staining, which is an indicator of chromatin condensation and apoptosis. Oxidative stress marker, H2DCFDA, was used to determine ROS levels after incubation with NLEs for 48 hours. A greater increase in whole ROS levels was observed in cells treated with NLEs compared to the cells treated with the positive control (Paraquat). In addition to whole-cell ROS levels, increases in mitochondrial ROS levels were observed. Mitochondrial instability is a major component of apoptosis, which shows that the increase in ROS levels causes induction of apoptosis.
Figure 4. Jah, Dhee. “Neem Plant.” Wikimedia Commons, 21 Oct. 2021, https://commons.wikimedia.org/wiki/File:Neem_plant.jpg.
Keywords: Manikya Bhasma, comparative study, statistical measurement, apoptosis
Finally, a study on Manikya Bhasma (MB) demonstrated its ability to be a ROS-producing reagent. (Jha & Trivedi, 2021) Mankiya Bhasma is a herbo-metallic/mineral preparation that contains ruby, orpiment, and arsenic sulfide. The MB was collected from a Baidyanath store in Guwahati city . Osteosarcoma cells (MG-63) were treated with MB for 48 hours. An MTT assay showed reduced cell viability proportional to MB concentration. Interestingly, the IC50 value or the concentration required for 50% inhibition in white blood cells (WBC) was almost five times the IC50 value for cancer cells. For example, the IC50 value for WBC was 873.81± 14.12, while the IC50 value for MG-63 was 105.73 ± 6.15. This demonstrates MB as a promising chemotherapeutic that can selectively target cancer cells. Like every herb studied previously, MB also led to a large increase in ROS and subsequently induced apoptosis. After treatment with MB, 21.25% of MG-63 cells were in the early apoptotic stage, 26.01% were in the late apoptotic stage, while 36.01% were healthy cells. On the other hand, 92% of MG-63 cells remained healthy when not treated with anything.
 The Manikya Bhasma in this study was purchased at a market in Guwahati. Because this is a preparation and not just a herb, a more reliable sample would have been collected at an academic institute or under controlled conditions.
All these Ayurvedic ingredients are dynamic and are used for a variety of ailments, including fever, cough, digestive issues, memory, inflammation, and impotence. However, these treatments are still not considered chemotherapeutic due to the lack of clinical studies. The studies on Piper Nigrum, MP, and FIM above used ethanolic extracts in their experiments. More research needs to be done in order to test the safety of ethanolic extracts of these ayurvedic ingredients in living organisms. It is important to note that the Ayurvedic ingredients examined in these studies were presented as individual herbs. However, Ayurvedic literature such as Sarangdhar Samhita highlighted the concept that individual plants are not adequate to achieve a desired therapeutic effect. Combining multiple herbs are necessary to not only attain the desired therapeutic effect, but also reduce toxicity (Parasuraman, 2014). Therefore, polyherbal treatments of the Ayurvedic ingredients discussed in this study should also be used in clinical studies. While many consider Ayurvedic medicine to be an “alternative” practice, research studies such as the ones above show its effectiveness. More clinical research needs to be done so that Ayurvedic medicine can be looked at as a “primary” practice instead of something that is complementary or alternative.
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