INTRODUCTION:

Cancer or malignant neoplasm is known otherwise, as a disease when cells are in a state of uncontrolled proliferation and growth and are known to not only intrude on and destruct adjacent tissues (invasion) but also for its capability to spread to other parts of the body which is via lymph or blood circulation¹. Cancer accounts for about 28% of deaths and is a major public health problem in many developed and developing countries. The most common cancer in men affects prostate glands (about 27%) and breasts in women (also about 30%)².

Deaths in patients suffering from cancer are reportedly due to modifiable risk factors, common ones being smoking, tobacco and alcohol consumption and diet factors lacking in nutrition³. In dеvе1opеd countries, obesity and overweight are also reasons for cancer⁴, and in low and middle-income countries transmission of human papilloma virus through unprotected sex is a leading factor for cervical cancer⁵.

Plant-derived constituents have been a great attraction at present as an alternative source of battling several diseases either by inhibition or prevention⁶. Compared to synthetic chemotherapeutic agents the plant-derived natural chemicals possess relatively low toxicity, have high target specificity, and are mostly ingestible. Several clinically useful anti-cancer agents such as (vinblastine, vincristine paclitaxel)⁷, (camptothecin, topotecan, irinotecan, etoposide)⁸ and were acquired or developed from phytochemicals, which have been proved inevitable and valuable resources to uncover novel anti-cancer chemicals.

Oxygen though is vital for the aerobic life process only 5% or more of the inhaled oxygen is converted to reactive oxygen species (ROS)⁹. Under normal circumstances, the free radicals which are defined as chemical species possessing an unpaired electron positively charged, negatively charged, or electrically neutral; are detoxified by the antioxidants present in the body and there is equilibrium between the ROS generated and detoxified by the antioxidants present¹⁰. Free radicals are acquired from four elements including oxygen, nitrogen, sulphur, and chlorine¹¹. When the generation of ROS overtakes the antioxidant defense of the cells, the free radicals start attacking the cell proteins, lipids, and carbohydrates and this leads to several physiological disorders¹². Overproduction of reactive species such as superoxide anion (O2•-), hydroxyl radical (OH•), hydrogen peroxide (H₂O₂), singlet oxygen (1O₂), and nitric oxide (NO) is reported to cause oxidative stress¹³. Free radicals have been implicated in the pathogenesis of diabetes, liver damage, nephrotoxicity, inflammation, cancer, cardiovascular disorders, and neurological disorders and in the process of aging¹⁴.

Among rheumatoid disorders, rheumatoid arthritis is known to be the major ailment¹⁵. Rheumatoid arthritis is an autoimmune disease marked with the inflammation of joints, destruction of articular synovial proliferation¹⁶. Inflammation comprises of both systemic and local responses and it produces avascular tissue response on the living tissues¹⁷ and noted as the response on injury to cells and body tissues through infections, chemicals, thermal factors, and mechanical injuries¹⁸. Auto antibodies such as Rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA), are responsible for clinical manifestation of Rheumatoid arthritis identified for many years¹⁹. Though several synthetic drugs are available as anti-inflammatory agents; the use of medicinal plants has always been an alternative. Therefore, a continuous effort to explore and identify medicinal plants for their anti-inflammatory activity is in practice for decades. The most abundant mediators in inflammatory cells are histamine, serotonin, bradykinin, prostaglandins, etc. and prostaglandins are omnipresent substances that indicate and modulate cell and tissue responses involved in inflammation²⁰.

Free radicals that damage cells lead to inflammation and therefore it is understood that Inflammation and oxidation are closely related²¹. Depletion of immune system antioxidants, gene expression genes alterations leading to the production of abnormal proteins, and disorders such as hero sclerosis, gastritis, cancer, arthritis, ischemia, tissue injury, central nervous system injury, and AIDS are caused by Free radicals²². Plentiful naturally obtained chemical compounds have been reported that suppress nuclear factor-ĸβ mediated inflammation pathways²³ along with COX-pathway in our body which are basic pathways for inflammation. The lysosomal enzymes released during inflammation produced a variety of disorders and are said to be related to acute or chronic inflammation²⁴. The Diclofenac drugs are reported to inhibit these lysosomal enzymes or stabilize the lysosomal membrane²⁵. Since the HRBC membrane is similar to components of the lysosomal membrane, the prevention of hypotonicity-induced HRBC membrane lysis is taken as a measure of the anti-inflammatory activity of drugs.

Ochna obtusata is also known as Mickey mouse plant belonging to the family Ochnaceae is a perennial herb with stout rot stock, simple leaves, alternate and obovate. The flowers are pink or slightly purple in panicles and the fruit drupes with greenish-black colour²⁶. Several different extracts of Ochna obtusata have been reported to have antidiabetic²⁷, antidiuretic²⁸, antiulcer²⁹, anticholinergic³⁰, hepatoprotective³¹, antiglucosidase, antioxidant and antihyperlipidemic³², neuroprotective³³ activities which are due to its total phenolic, flavonoid, and terpenoid contents.

MATERIALS AND METHODS:

Preparation of Plant Extracts:

The fresh leaves of Ochna obtusata were collected from the forest of Tirumala Hills for the study which was authenticated by Dr. Madhava Shetty, taxonomist, Department of Botany, Sri Venkateswara University; Andhra Pradesh. The collected leaves were shade dried at room temperature, pulverized by a mechanical grinder, and sieved to obtain coarse powder which was used for successive extraction by the Soxhlet extraction method using petroleum ether, chloroform, ethanol, and methanol and then filtered. The ethanolic extract was used for the study since it was proved to have potential activity due to its phytoconstituents as confirmed by preliminary phytochemical studies reported earlier. The extract was later allowed to cool and concentrated under reduced pressure using a Rota-vapor to obtain a semi-solid mass.

In Vitro anti-cancer activity by MTT assay:

Cell Culture:

All the ingredients like DMEM (Dulbecco's modified Eagles medium), MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide], Phosphate Buffered Saline (PBS), and Fetal Bovine Serum (FBS) were purchased from Sigma Chemicals, Hyderabad. Other additional apparatus like 25cm²and 75cm²flask and 96 well plates required for the study were also purchased from Sigma Chemicals, Hyderabad.

Cell Lines:

Various cell lines like HepG2 human hepatocarcinoma cells (liver hepatocellular carcinoma), HCT116 human colon carcinoma cells (human colorectal carcinoma), and MCF-7 human breast adenocarcinoma cells (Michigan Cancer Foundation-7) were obtained from NCCS, Pune for studying the antineoplastic activity of Ochna obtusata and the cells were maintained in Dulbecco's modified Eagles medium supplemented with 10% Fetal Bovine Serum and the antibiotic doxorubicin (0.5μg/ml) as the standard reference. The cells were cultivated in an atmosphere of 5% CO₂air at 37^OC.

MTT assay³⁴

The anti-cancer assay of plant extracts was performed using MTT assay on various cell lines. Ethanolic concentrate of 100 mg was dissolved in DMSO and stored in -20^OC till its usage³⁵. The cell lines were plated in 96 well plates of 100µl at the concentrations of 5 X 10³cells/well which was with the help of a hemocytometer and incubated overnight. After incubation, 100μl of ethanolic extracts (5, 10, 25, 50, 75, and 100μg/ml) and the reference compound doxorubicin³⁶ (0.5μg/ml) in a culture medium was added to the cells and incubated for 48 hours. After incubation, the drug solution is discarded, and fresh media with MTT solution (0.5μg/ml) was added to each well, and plates were incubated at 37^OC for three hours. After this, it is observed that precipitates are formed in the well due to the reduction of the MTT salt to chromophore formazan crystals by the cells with metabolically active mitochondria. The absorbance for all the samples was measured at 570nm and this was done in triplicate and average absorbance was used to calculate the percentage inhibition of the plant extract. The percentage growth inhibition was calculated using the following formula and IC₅₀ values generated from the dose-response curves for each cell line.

[Abs _Control– Abs _sample]

% Inhibition = ------------------------------- X100

Abs _control

In Vitro Anti-Inflammatory Activity:

HRBC Membrane Stabilization Assay³⁷:

The anti-inflammatory activity of leaves extract of Ochna obtusata was determined by the HRBC membrane stabilization method. Blood was collected from healthy human volunteers not taken any NSAIDS for 2 weeks before the experiment and mixed with an equal volume of Alsever’s Solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid and 0.42% NaCl in water). The blood was centrifuged at 3000rpm and packed cells were washed with isosaline (0.85%, pH 7.2), and 10% v/v suspension was made with isosaline. The assay mixture contained the drug; 1ml phosphate buffer (0.15M, pH 7.4), 2ml of hyposaline (0.36%), 0.5 ml of SRBC suspension. Diclofenac was used as the reference drug and Instead of hyposaline, 2ml of normal saline was used as a control. All the assay mixtures were incubated at 37^OC for 30 minutes and centrifuged. The haemoglobin content in the supernatant solution was estimated using a colorimeter at 560 nm. The percentage of haemolysis was calculated by assuming the haemolysis produced in the presence of distilled water as 100%. The percentage of HRBC membrane stabilization or protection was calculated using the following formula:

Optical density of drug treated sample

% Protection = 100 ------------------------------------- X 100

Optical ensity of control

Protease Inhibition Assay³⁸

The reaction mixture 2ml containing 0.06mg trypsin, 1ml 20Mm Tris HCl buffer (pH 7.4) and 1ml of different concentration of plant extracts (50, 100, 150, 200, 250µg). The reaction mixture was incubated at 37ºC for 5 minutes and 1ml of 0.8% (W/V) casein was added. It was incubated at 37ºC for 20 minutes followed by the addition of 70% perchloric acid to arrest the reaction. The cloudy suspension was centrifuged and the absorbance of the supernatant was recorded at 210 nm against buffer as blank. Aspirin was taken as a standard drug for comparative study. The percentage inhibition of protease inhibition was calculated as per the formula below:

[Abs _Control – Abs_sample]

Percentage Inhibition = -------------------------------- X 100

Abs _Control

In Vitro Anti Arthritic activity:

Protein Denaturation Assay³⁹:

The reaction mixture (5ml) consisting of 0.2ml of egg albumin (from fresh hen’s egg), 2.8ml phosphate-buffered saline (pH: 6.4), and 2ml of varying concentration of plant extracts. A similar volume of double distilled water served as a control. Then the mixtures were incubated at 37±2°C in an incubator for 15 minutes and then heated at 70ºC for 5 minutes. After cooling, their absorbance was measured at 660nm by using the vehicle as blank. Diclofenac at the final concentration of (1mg/ml) was used as a reference drug and treated similarly for determination of absorbance. The percentage inhibition of protein denaturation was calculated as per the formula below:

[Abs _Control – Abs_sample]

Percentage Inhibition = ---------------------------- X 100

Abs _Control

RESULTS AND DISCUSSION:

In Vitro Anti-cancer Activity:

Anti-cancer activity of EEOO was determined by MTT assay and the study revealed that the extract was effective on HepG2 (human hepatocarcinoma cell lines as compared to HCT116 human colon carcinoma cells (human colorectal carcinoma), and MCF-7 human breast adenocarcinoma cells (Michigan Cancer Foundation-7). The plant extract exhibited significant toxicity in a dose-dependent manner in HepG2 as compare to the other two cell lines. The IC₅₀values of HepG2 were found to be 57.46μg/ml as compared to HCT116 and MCF-7 of 55.10μg/ml and 54.39μg/ml as tabulated in Table 1. The effect of EEOO on HepG2, HCT116, and MCF-7 were depicted in Figure 1, Figure 2, and Figure 3.

Table 1: IC₅₀ values of Ethanolic extract of Ochna obtusata on Various Cell Lines

S. No

Sample Name

HepG2

(μg/ml)

HCT116

(μg/ml)

MCF-7

(μg/ml)

EEOO

57.46

55.10

54.39

Standard (Doxorubicin)

24.01

24.08

20.37

Figure 1: Effect of EEOO on HepG2 Cell Lines

Figure 2: Effect of EEOO on HCT116 Cell Lines

Figure 3: Effect of EEOO on MCF Cell Lines

In vitro Anti-Inflammatory activity:

HRBC membrane stabilization Assay:

The anti-inflammatory activity of EEOO was evaluated by the HRBC membrane stabilization method and the results are depicted in Figure 4. The actual fact of using erythrocyte membrane is because of its similarity to the lysosomal membrane⁴⁰. Maximum inhibition of 73.35% was observed in the case of EEOO taken at a concentration of 500µg/ml; as compared to Diclofenac taken as standard which was found to be 83.38% of plant extract. It was also observed that all various other concentrations exhibited significant anti-inflammatory activity. It was therefore concluded that the EEOO possesses anti-inflammatory activity.

Figure 4: HRBC Membrane Stabilization Assay of EEOO

Proteinase Inhibition Assay:

The EEOO leaves exhibited anti-inflammatory activity that was studied by using inhibition of Proteinase and the results are illustrated in Figure 5. Under the protein denaturation assay, it was observed that the absorbance of plant extracts solution increased concerning the standard. The percentage inhibitions of plant extracts at 250µg/ml were found to be 92.18% and 71.39% in the case of standard and plant extract respectively. Recent evidence shows that Flavonoids are proved to be effective proteinase inhibitors thereby safeguarding the tissues from injury, thus exhibiting anti-inflammatory property⁴¹.

Figure 5: Protease Inhibition Assay of EEOO

Protein Denaturation Assay:

The anti-inflammatory activity of EEOO was studied by evaluating its capability of inhibiting protein denaturation and the results are illustrated in Figure 6. The mechanism behind the denaturation of protein involves the alteration in electrostatic, hydrogen, hydrophobic, and disulfide bonding⁴². It was observed that EEOO was able to act as agents that prevent protein denaturation thereby indicating itself to possess anti-inflammatory activity which was exhibited by an increase in the absorbance compared to the control. It was observed that at a concentration of 1000µg/ml, the percentage inhibition of standard drug was 87.22% and that of the plant extract at the same concentration was 74.31% thereby exhibiting significant anti-inflammatory activity. This may be because of various Flavonoids in the EEOO since Flavonoids inhibit regulatory enzymes or transcription factors responsible for controlling mediators involved in inflammation⁴³.

Figure 6: Protein Denaturation Assay of EEOO

CONCLUSION:

From the above study, it was observed that EEOO is a strong anti-cancer, anti-inflammatory and anti-arthritic agent. The phytoconstituents present in the EEOO possess anti-cancer activity which was observed by inhibition of cell growth of cancerous cells. They are also responsible for, neutralizing enzymes thereby protecting the RBC membrane from lysis and inhibiting protease enzyme thereby confirming its anti-inflammatory activity. The anti-arthritic activity can be confirmed due to its ability to prevent protein denaturation in the study. Therefore it may be concluded to perform the in vivo studies to understand its perspective.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation

ACKNOWLEDGEMENTS:

The author is grateful to the Management of Guru Nanak Institutions, Hyderabad for providing the necessary facilities to carry out the work. The author would also like to express his deep gratitude to the Principal (School of Pharmacy) and all his colleagues, School of Pharmacy, Guru Nanak Institutions Technical Campus for supporting the study. The authors are also grateful to the Principal and distinguished Faculty, GITAM Institute of Pharmacy, GITAM Deemed to be University; Visakhapatnam for all support extended to accomplish the study.

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Received on 09.07.2021 Modified on 21.08.2021

Research J. Pharm. and Tech 2022; 15(9):3999-4004.

DOI: 10.52711/0974-360X.2022.00670