Roshni Shivvedi1, 2, Satish shilpi2, Shweta Kumar1, Rajesh Singh Pawar1
1Pharmacognosy and Phytochemistry Laboratory, VNS Group of Institutions, Bhopal, (M.P.)-462044, India
2Pharmaceutics Laboratory, Ravishankar College of Pharmacy, Bhanpur Sqaure, Bhopal (M.P.)-462037, India
*Address for Corresponding Author
Ravishankar College of Pharmacy, Bhopal (M.P.), India
Objective: This study was aimed to evaluate antioxidant activity of the Cannabis sativa extract (aqueous) using eukaryotic model Sachharomyces cerevisiae (both wild BY 4743 and knockout Δtrx2 Δctt Δcta) against H2O2 induced oxidative stress. Material and Methods: The antioxidant activity was investigated using viability assay, growth curve, spot assay and level of mitochondrial ROS determined by MitoSOX staining method. Results: The study showed the significant (**P<0.01) antioxidant activity of ACS. The treated yeast cells were able to maintain a higher level of growth as compared to the control. In viability assay the H2O2 stressed group showed 35% (wild) and 33% (Δtrx2) % viability of yeast cell while treated ACS 400µg/ml+H2O2 group showed improved % viability i.e. 50% (wild) and 57% (Δtrx2) after incubating for 30 minutes. In spot assay also ACS (400 µg/ml) showed a protective effect against challenge which is indicated by the growth in extract treated group. Compared to the negative control group the extract-treated group showed less fluorescence indicating the ability of the extract to scavenge mitochondrial superoxide and thereby protecting the cells. Conclusion: In this study, we found that aqueous extract of C. sativa exhibited marked free radical scavenging and antioxidant activity against hydrogen peroxide radicals in vivo. This extract can be used for the management of diseases like cancer, diabetes, neurodegenerative disease etc.
Keywords: Antioxidant, Hemp, Saccharomyces cerevisiae, ROS, oxidative stress
Oxidative stress is an imbalance between antioxidant and pro-oxidant resulting in irreversible cellular damage. ROS (reactive oxygen species) produced by a living organism as a result of normal cellular metabolism, they function in physiological cell process at low to moderate concentrations, but they produce adverse reaction at high concentration to cell components such as lipids, proteins, and DNA. RNS (reactive nitrogen species) are a family of antimicrobial molecules derived from nitric oxide and superoxide produced by the enzymatic activity of inducible nitric oxide synthase and NADPH oxidase respectively (Valko et al., 2006). Overproduction of free radicals can cause oxidative harm to biomolecules, (lipids, proteins, DNA), ultimately leading to several chronic diseases like atherosclerosis, cancer, diabetics, rheumatoid arthritis joint pain, post-ischemic perfusion damage, myocardial infarction, cardiovascular diseases, chronic inflammation, stroke and septic shock, aging and degenerative diseases in humans (Uttara et al., 2009). Herbs are traditionally defined as any part of a plant that is used in the diet for their aromatic properties with low nutritional value. But, more recently herbs are used as a source of various phytochemicals which possess powerful antioxidant activity. The demand for herbal medicines is superior to allopathic medicine due to their safety, fewer side effects, efficacy and good belief of the public in herbal medicines and their products. The secondary metabolites of plants are used as drugs and show many biological activities like antifungal, anticancer, antibacterial and antioxidants (Golla et al., 2014). Cannabis sativa Linn.is an annual herbaceous plant, (Synonym: Indian hemp, Ganja, Marihuana), the biological source is dried flowering tops of the cultivated female part of the plant C. sativa Family Cannabaceae (Kokate and Gokhl 2006). Plants growing in the tropical parts of India, Africa, and America produce narcotic substance whereas plant growing in temperate climates don't produce opiate substances and it is helpful in the generation of fiber and oil (Mohamed et al., 2009). More than 525 constituents have been identified from C. sativa L. The most particular class of C. sativa constituents are the C-21terpenophenolic cannabinoids. Other phenolic constituents include flavonoids, spiro indans, dihydro stilbenes, phenanthrenes, dihydrophenanthrenes, Tetrahydro cannabinoids, sitosterol-3-O-D-glucopyranosyl-6′-acetate (Zuradi et al., 2006). It has been found that the cannabinoids in C. sativa particularly tetrahydrocannabinol (THC) and cannabidiol (CBD) showed potent antioxidant properties in various pre-clinical applications (Russo et al., 2006). The selected plant C. sativa possess various activities like antioxidant activity (Chen et al., 2012). The various parts of the plant are used in the treatment of several diseases, pain, whooping cough, asthma, sedative-hypnotic (Frankhauser et al., 2002) neurodegenerative diseases (Gerra et al., 2010) chemotherapy-induced nausea and vomiting, multiple sclerosis (Vara et al., 2011) anticoagulant (Iuvone et al., 2004) and glaucoma (McAllister et al., 2007) anticancer agents (Levndal et al., 2006; Costa 2004). This study was aimed to evaluate the antioxidant property and H2O2-induced oxidative stress protection activity of the aqueous extract of C. sativa and explore the underlying mechanism of action.
Material and methods
The dried plant materials were obtained from authorized outlet (Rajkumar Sahu Contractor) Bhopal. The plant was authenticated by Dr. Zia-ul-Hasan. A voucher specimen of the plant has been deposited (418/BOT/ Safia/16). The plant material was ground to a coarse powder before extraction.
Preparation of the extract
Coarsely powdered plant parts (aerial parts) of C. sativa (35 gm) was defatted with petroleum ether (40-60ºC) and successively extracted with methanol and distilled water for 36 hrs using Soxhlet apparatus. The extracts were evaporated to dryness under reduced pressure by rotary evaporator and the resulted dried residue was stored in airtight containers for further use (Uddin et al., 2014).
Chemicals and reagents
The chemicals used in the study were YPD (Difco Laboratories Detroit, Michigan), hydrogen peroxide (30%) (Merck, Mumbai), ascorbic acid (Sigma-Aldrich Mumbai), MitoSOXTM (Molecular probes, Invitrogen, USA), mounting solution (Vector Laboratories, Burlingame, CA), propidium iodide (Sigma-Aldrich, Mumbai). All other chemicals and reagents used in this work were of analytical grade.
Yeast strains, media and growth conditions
The Saccharomyces cerevisiae wild-type strain BY 4743 and deletion strains (Δtrx2, Δctt, Δcta) strains were purchased from Open Biosystems (Thermo Scientific) were grown up to OD600 0.6 to 1 in liquid YPD media (1% yeast extract, 2% glucose and 2% peptone) using incubator shaker 30ºC and 180 rpm.
Preliminary phytochemical screening
The various phytoconstituents of the aqueous extract of C. sativa (ACS) (aerial parts) were analyzed to detect the carbohydrates, alkaloids, flavonoids, saponins, tannins, phenolic compounds, terpenoids, steroids, proteins and mucilage using standard tests (Tiwari et al., 2011).
The aqueous extract (ACS) at different concentrations (200 µg to 1600 µg) were evaluated for toxicity in S. cerevisiae-BY 4743 (Garay et al., 2016).
Growth curve of yeast (BY 4743, Δtrx2) under stress in presence of various extracts/ ascorbic acid
The cultures of BY 4743 and Δtrx2 (OD600=0.1), were transferred to different wells of a microplate. Further different concentrations of 10µl ACS (400, 800, 1600 µg/ml) and 10 µl ascorbic acid (AA) (10 mM) were added in duplicate. Finally, 10µl H2O2 (40 mM) was added to all the wells except the control and incubated at 30ºC for 24 hrs in a microplate reader (BioTEK, USA) with continuous shaking at medium intensity. OD600 was measured at every hour. Normal YPD was used as a blank (Wu et al., 2011).
The cultures of BY 4743 and Δtrx2 (OD600=0.1), was taken (1 ml) in a microtube in quadruplet and added ACS (400 µg/ml)/ AA (10 mM) all the tubes were incubated for 2 hrs, 23°C, at 180 rpm. Further, 40 mM H2O2 (100 µl) was added to all the tubes to get a final concentration of 4 mM, and incubated for 1 hr, 23ºC at 180 rpm. The cells were harvested (12,000 rpm, 30 sec) and washed with sterile water. Finally, the cells were suspended in 1ml of sterile water. Further serial dilutions (10-1, 10-2 and 10-3) were made of a 96 well microtiter plate, and spotted (2.5 µl) onto YPD agar plate. Plates were incubated 23oC/48 hrs, then imaged by UVP Multidoc It (Bench Top Transilluminator) (Spencer et al., 2014).
Viability assay by plate method
A single colony of both wild strains BY 4743 and deletion strain Δtrx2 was inoculated with YPD and were grown at 30ºC at 180 rpm for overnight. Adjusted OD600~0.6, both the cultures were divided into four groups each containing 1 ml. Except the control group, all the groups were treated with 4 mM H2O2. The test groups were treated with ACS (400µg/ml) and the standard group was treated with AA (10 mM). All the tubes were incubated for 3 hrs at 30ºC in dark. After incubation, OD600 was checked and an equal number of cells were taken from each tube. The cells were harvested, washed and finally suspended in 1000 µl of water. 10µl was taken from the cell suspension and diluted to 1000µl, from which 20 µl was spread on a YPD agar plate in triplicate. Incubated at 30ºC for 48 hrs and the viable cell was expressed as the colony-forming unit. Colonies were counted after growth and then imaged after 48 hrs by UVP multi-doc It (Bench top Transilluminator) (Iracema et al., 2007).
Viability assay by propidium iodide staining
The cultures were prepared by inoculating single colony of both wild strains BY 4743 and deletion strain Δtrx2 with YPD at 30ºC at 180 rpm for overnight. Then, the culture of both the yeast strains was adjusted to an OD600~0.6. The cultures were divided into four groups each having 1 ml. All the tubes were incubated for 3 hrs at 30oC, in dark. After incubation, the cells were pelleted and washed thrice with Milli-Q water. The cells were suspended in 200µl of PBS buffer and mixed with 0.2 µl of propidium iodide, to get a final concentration of 1 µg/ml from the stock (1mg/ml). After 5 min incubation in dark, the cells were harvested and washed with PBS. Finally, the stained cells were suspended in an appropriate amount of PBS. About 20µl cell suspension was mounted on a slide. The cells were visualized under a fluorescence microscope (Zeiss Apotome) at 100 X (Liesche et al., 2015).
Mitochondrial ROS Estimation by MitoSOX staining
The yeast BY 4743 strain and Δtrx2 were stained by MitoSOX Red to determine the mitochondrial superoxide. The culture was prepared by inoculating single colony of both the strains in YPD at 30ºC, 180 rpm for overnight. The overnight culture (OD600~0.6) was divided into four groups, normal control, standard (AA 10mM), negative control (4 mM H2O2) and test (ACS 400µg/ml+H2O2). All the cultures were incubated for 3 hrs at 30ºC, 180 rpm. The cells were harvested (12000 rpm, 30 sec), washed and suspended in 200 µl Milli-Q water. The cell suspension was mixed with 0.2 µl of MitoSOX Red from the stock (5 mM), to get a final concentration of 5µM. After 20 min incubation in dark, the cells were harvested and washed with PBS (3 times) to remove media and were finally suspended the stained cells in an appropriate amount of PBS. About 20 µl cell suspension was mounted on a slide. The cells were visualized under a fluorescence microscope (Zeiss Apotome) at 100 X (Liesche et al., 2015).
Preparation of the extract
The calculated % yield of plant extract was found to be 4.58 % (1.60 g) of petroleum ether, 9.2 % (2.7 g) of methanolic extract and 1.28 % (0.35 g) of water extract.
Preliminary phytochemical screening
According to preliminary screening alkaloids, flavonoids, glycosides, tannins, saponins, terpenes were present in the aqueous extract of C. sativa (Table 1).
Table 1. Phytochemical screening of C. sativa
Name of phytoconstituents
Aqueous extract of CS (Cannabis sativa)
(+) Present, ( -) Absent
In the toxicity study, the growth of S. cerevisiae- BY 4743 cells (untreated and treated) was monitored with ACS in different concentrations (200, 400, 800 and 1600µg/ml) as shown in the (Figure 1). All the treated groups exhibited normal growth pattern and there was no toxicity of extract observed at tested concentrations. So, these concentrations of plant extract were used for further studies.
We performed liquid growth assay of BY 4743 and Δtrx2 (Figure 2) at concentration 400, 800 and 1600µg/ml of ACS. From the growth curve, it was observed that H2O2-induced stress was overcome by ACS (400 µg/ml) in both the wild and Δtrx2 strains of S. cerevisiae. After 12 hrs, the optical density of yeast cells treated with ACS (400µg/ml) was even greater than the control group and found to be significant (**P<0.01). AA was used as the standard antioxidant and it showed significantly (**P<0.01) improved growth of stressed cells comparable to the control.
Figure 2. (A) growth curve of wild (4743), (B) growth curve of Δtrx2. Determination of arresting concentrations of oxidants on BY 4743 yeast cells and Δtrx2 yeast cells (OD600~ 0.01) were challenged with H2O2 (4 mM) CTL- control group, H2O2 hydrogen peroxide (4 mM) containing group, AA –ascorbic acid (10mM) containing group, ACS different concentration 400 µg, 800 µg and 1600 µg/ml cell density was monitored every hour for h at 30ºC. Growth curve shows that treatment of ACS 400 µg/ml was able to overcome the H2O2 stressed cell growth (** P<0.01) (** P<0.01).
We performed spot assay on wild strain as well as H2O2 sensitive deletion strains (Δctt, Δcta, Δtrx2). All the strains of yeast treated with ACS (400µg/ml) + H2O2 were able to overcome the stressing effect and showed a similar growth pattern as that of normal group (Figure 3).
Figure 3. (A) Normal: contain yeast culture, (B) Negative control: cells were stressed with H2O2, these showed growth less than other groups (C) positive control: under H2O2 stressed and treated with AA these showed more cells than negative group (D) Test group contain H2O2 stressing agent and test drug ACS (400 µg/ml), here the deletion strains showed similar growth pattern as that of normal control group
A viability assay is an assay to determine the ability of cells or tissues to maintain or recover viability. Cell viability can be determined by various methods. We evaluated the cell viability by plate method and propidium iodide staining method.
The viability assay was performed to study the protective activity of ACS against H2O2 induced oxidative stress in two yeast strains (BY 4743 and Δtrx2). The percentage of viable cells was calculated using the number of colony-forming units (c.f.u.) where the control group is taken as the reference (100%). After 48 hours, 35% viability (wild BY 4743) and 33% cell viability (Δtrx2) of yeast cells was observed in the H2O2 stressed group, while the cells treated ACS (400 µg/ml) + H2O2 showed an improved % viability i.e. 50% cell viability (BY 4743) and 57% cell viability (Δtrx2)(**P<0.01) (Figure 4).
Figure 4 (A) CTL- control, (B) H2O2-Negative control (Hydrogen peroxide), (C) AA-Ascorbic acid, (D) ACS-test group cell viability assay by plate method in wild and Δtrx2 this graph showed % cell viability test group showed in wild 50% (**P<0.01) and Δtrx2 57% (**P<0.01) viable cell that will be comparable more than negative control. A&A1 -Control group (BY 4743) C&C1-positive control (H2O2+AA); B&B1 -Negative control (H2O2) D&D1-Test group (H2O+ACS)
Propidium iodide (PI) is an intercalating agent and a fluorescent molecule to evaluate cell viability. PI is commonly used for identifying dead cells in a population and as a counter stain fluorescent techniques. PI enters rupture cell membrane and impaired cell wall showed more fluorescence. After staining with PI in both strains BY 4743 (Figure 5) and Δtrx2 (Figure 6) H2O2 stressed cell, a decreased viability was observed for both the strains (BY 4743, 57.15±1.8 and Δtrx2, 43.75±9.72). At the same time, ACS (400µg/ml) treated group showed an increased viability (BY 4743, 92±1.9 and Δtrx2, 93±1.5), similar to AA (10 mM) treated group (BY 4743, 95.35±2.227 and Δtrx2, 96±1.4849), compared to negative control. The results indicate a significant (**P<0.01) scavenging effect against the H2O2-induced stress thereby protecting the cells from oxidative stress (Figure 7).
Figure 5. (A) Control group; (B) negative control stressed by (4 mM) H2O2; (C) positive control stressed by 4 mM H2O2 or group treated with 10 mM AA; (D) test group stressed by 4 mM H2O2 or treated with ACS 400 µg/ml. The negative control group showed more staining than the positive control or test group. ACS showed the same effect as AA. A&A1-Control group (Δtrx2) C&C1-positive control (H2O2+AA); B&B1-Negative control (H2O2+yeast culture) D&D1-Test group (H2O2+ACS)
Figure 6. (A) Control group; (B) negative control stressing by 4 mM H2O2; (C) positive control stressing by 4 mM H2O2 or group treated with 10 mM AA; (D) Test group stressing by 4 mM H2O2 or treated with ACS (400 µg/ml). The negative control group showed more staining than the positive control or test group. ACS same effect comparable to as AA
Figure 7. This graph show % viability (A) Control group; (B) negative control stressing by (4 mM) H2O2; (C) positive control stressing by 4 mM H2O2 or group treated with 10 mM AA (D) test group stressing by 4 mM H2O2 or treated with ACS 400 µg/ml. Showed significant (**P<0.01) radical-scavenging H2O2 effect. A&A1-Control group (BY 4743) C&C1-positive control (H2O+AA); B&B1-Negative control (H2O2) D&D1-Test group (H2O+ACS)
MitoSOX Red reagent is a fluorogenic dye particularly targeted to mitochondria in live cells. The oxidation of MitoSOX Red reagent by superoxides produces red fluorescence. The production of superoxides by mitochondria can be visualized in fluorescence microscopy by utilizing the MitoSOX Red reagent. MitoSOX Red reagent permeates live cells, where it selectively targets mitochondria and is quickly oxidized by superoxides (Girgih et al., 2011). In this study, the treated cells-both strains BY 4743 (Figure 8) and Δtrx2 (Figure 9) were stained with MitoSOX to determine the amount of mitochondrial ROS. The negative control (H2O2 stressed) group showed more number of fluorescent cells which were evident by the high percentage of mitochondrial superoxides. While in the positive control, (AA + H2O2) showed less number of fluorescent cells (BY 4743, 6.4 % ± 0.070 and Δtrx2, 8.7 % ± 0.353). The test (ACS 400 µg/ml+H2O2) treated group showed the lesser number of fluorescent cells than the negative control group (BY 4743, 5.1 % ± 0.7071 and Δtrx2, 3.59 % ± 0.417), the percentage was found to be even less than that of AA. The results indicate that ACS was capable of scavenging the mitochondrial superoxide in a significant manner (***P<0.001) and thereby showed the protective effect against H2O2- induced oxidative stress (Figure 10).
Figure 8. (A) control group; (B) negative control stressing by 4 mM H2O2 ; (C) positive control stressing by 4 mM H2O2 or group treated with 10 mM AA (D) test group stressing by 4 mM H2O2 or treated with ACS 400 µg/ml MitoSOX stain was used for the detection of mitochondrial ROS level and the m-ROS was stained by red fluorescence. A&A1-Control group (Δtrx 2 ) C&C1-positive control (H2O+AA); B&B1-Negative control (H2O2+yeast culture) D&D1-Test group (H2O+ACS)
Figure 9. (A) control group; (B) negative control stressed by 4 mM H2O2 (C) positive control stressed by 4 mM H2O2 or group treated with 10 mM AA; (D) test group stressing by 4 mM H2O2 or treated with ACS 400 µg/ml MitoSOX staining was used for the detection of mitochondrial ROS level and the m-ROS was stained by red fluorescence.
Figure 10. This graph shows % stained cells by MitoSOX staining; A: control group; B: negative control stressing by 4 mM H2O2 C: positive control stressing by 4 mM H2O2 or group treated with 10 mM AA D: test group stressing by 4 mM H2O2 or treated with ACS (400µg/ml) significant result (*** P<0.001; ** P<0.01).
Hemp seed protein hydrolysate showed effective in vitro antioxidant properties. The ability of the peptides to scavenge hydroxyl and DPPH radicals, reduce ferric metal ions and chelate transition metal ions is indicative of their potential use for managing metabolic disorders that arise from excessive levels of ROS (Hong et al., 2015). It was demonstrated that the ethanolic extract of Hemp seeds, effectively inhibit H2O2 mediated oxidative stress and propose its beneficial effect as a therapeutic agent in preventing oxidative stress mediated diseases (Gavamukulya et al., 2014). The extract of Hemp seed hull, is proven to be a rich source of natural antioxidants, and its consumption as dietary supplements, help to prevent oxidative stress (Chen et al., 2012). Antioxidants are the substance that reduces damage due to free radical, C. sativa has long been known for its psychotropic effect. But recently research disclosed the wild range of pharmacological activity like anti cancer, anti diabetic etc (Girgih et al., 2011). The current study aimed to evaluate antioxidant activity of the C. sativa extract (aqueous) using eukaryotic model S. cerevisiae (both wild strain, Δtrx2) against H2O2 induced oxidative stress. The ACS (400,800µg/ml) was able to overcome the stressing effect of H2O2 and the treated cells showed a growth curve similar to that of normal control. We found that, surprisingly, ACS at low concentration (400 µg/ml) showed better protection compared to that of higher concentration (800, 1600µg/ml). So, we can infer that ACS is effective even at low concentration. In the viability assay, the % viability of the extract treated cells were increased approximately twice and twice times for BY 4743 and Δtrx2 respectively. On staining with propidium iodide the measured viability of the live yeast cell treated group was improved viability levels were twice and twice the time for BY 4743 and Δtrx2 respectively. The ACS also reduced mitochondrial ROS in yeast cells both BY 4743 strain and Δtrx2. In addition, the antioxidant properties of ACS are a promising strategy for therapeutic effects by avoiding disorders in the normal redox reactions in healthy cells. These activities of C. sativa extract may be attributed to the presence of various phytochemical constituents found to be such as alkaloids, flavonoids, glycoside, tannin, and saponins, terpenes which were reported earlier and confirmed in the present study. Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids. Phenolic compounds have been related to antioxidant activity, some studies have emphasized specific classes such as flavonoids and tannins. The higher phenol content in the aqueous extract would partly contribute to its higher antioxidant activity. This study provides experimental evidence and supports the medicinal use of this plant and lends pharmacological credence to the ethnomedical use in the traditional system of medicine. Also, this study demands further studies to elaborate its use, active constituents, and safety.
In this study, we have shown that ACS exhibited significant free radical scavenging activity and antioxidant activity against hydrogen peroxide radicals in vivo. This extract showed that regulation of redox state is critical for cell viability; activation, proliferation, and organ function and protect the oxidative stress and oxidative stress generated disease. The results show that the synergistic action of the phytoconstituents present in ACS has a lot of potential for the management of diseases like cancer, diabetes, neurodegenerative disorders etc.
One of the author's want to acknowledge All India Institute for Technical Education (AICTE), New Delhi, India for providing JRF grant (GPAT; 355118413) for the completion of M. Pharm. dissertation work. Author's want to acknowledge Department of Chaperon Biology Lab, Indian Institute of Science Education & Research (IISER), Bhopal, India for providing the experimental and instrumental facilities to carry out the research work.
Conflict of Interest
Authors have no conflict of interest.
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