In 2020, there are projected to be 18.1 million cancer cases worldwide, based on the report of Global Cancer Statistics it showed that in case of men and women approximate 9.3 million and 8.8 million respectively cancer incidents were identified.²⁴ In 2020, 12.5 percent and 12.2 percent of all new cases of cancer were diagnosed as breast and lung cancer, respectively. With 1.9 million new cases, colorectal cancer was the third most prevalent cancer in 2020, making up 10.7% of all new cases.²⁵ In 2020, 15.4% of all new cases of malignancy in males will be lung cancer, making it the most incident cancer type in men worldwide. Resistance to chemotherapy and reoccurring illnesses are also significant hindrances. To improve the effectiveness of treatment, new therapeutic approaches or anticancer drugs may be considered. To create anticancer drugs, a variety of plants, marine life, and microbes are used as sources. It is well recognized that bacterial and fungal metabolites are important sources of bioactive substances in microorganisms. However, there are currently not many anticancer medications made from microbial metabolites.

According to research, phytochemicals have anticancer properties, and many of them are being employed to treat this well-known illness.^26-28 Recently several research findings revealed that phytochemicals isolated from plant origin have been used as potent drugs to cure many disorders but at the same time it is associated with several practical difficulties working with plants such as slow growth, abnormal habitat with less percentage of reproducibility of targeted desired phytochemcials.^29,30 Natural substances are a well-known significant source of potent anticancer medicines that help us fight cancer. Plants, microorganisms, and marine species make up the majority of the principal sources. A variety of substances that are successfully employed as chemotherapy drugs, including daunorubicin, doxorubicin, actinomycin D, bleomycin, and mitomycin C, are mostly derived from bacteria.³¹ Numerous fungal metabolites are still being developed and tested in human therapeutic trials. For instance, the anti-angiogenesis agent fumagillin made from A. fumigates.³² It was discovered that phenylahostin, isolated from A. ustus, is cytotoxic to a number of cancer cell lines, including those from the breast, colon, lung, ovary, and leukaemia.³³ It has been demonstrated that the mycotoxin anguidine, a member of the trichothecenes family and generated by a number of Fusarium species, prevents leukaemia cells from proliferating.³⁴

Due to the production of primary metabolites that have beneficial products like enzymes, such as -amylase, amyloglucosidase, hemicellulase, and glucose, as well as citric acid, Aspergillus species are important economically.^35-38 Itaconic acid, a co-polymer used in the manufacturing of paint, is produced by A. terreus. A. terreus has the ability to produce secondary metabolites that can be exploited to produce therapeutically useful products, such as statins, a medication used to decrease cholesterol.^34,37

According to the accepted practice for biochemical testing for secondary metabolites, the phytochemical analysis for A. terreus in the current study was carried out. The results of the phytochemical study indicate that, among the various solvent extracts, the Aspergillus terreus methanol extract has demonstrated the presence of phenols and terpenoids as key phytochemical constituents. The outcomes are displayed in Table 1. Phenols in general have drawn a lot of attention due to their crucial physiological functions (such as hormones, aliphatic membrane anchors, maintaining membrane structure), ecological functions (such as defence compounds, insect/animal attractants), and their numerous uses in pharmaceutical and industrial applications, ranging from flavors and fragrances to disinfectants and lubricants.^38,39 Triterpenoids, which are these substances’ primary bioactive components, have analgesic and anticancer effects.⁴⁰

Table (1):
Phytochemical analysis of different solvent extracts of Aspergillus terreus.

+ve: Present; –ve: absent

Based on phytochemical findings, the total phenolic content of the methanol extract has been quantified using standard Gallic acid calibration curve (Y=0.001X+0.113) and it was found to be 176 µg/g. Phenols have the nature of antioxidant and possess several applications such as fight against ageing, cancer, and other diseases of the heart.⁴¹ Applications of phenolic to enhance the nutritional content and quality of food are proliferating quickly in the food business.⁴²

The methanol extract of A. terreus was tested for toxicity on the non-cancerous transformed fibroblast cell line L929 using the conventional MTT cell viability assay method before being examined for anticancer activities. In this test, the viability of the L929 cell line was assessed after exposure to methanol extract at various doses (50, 100, 150, 200, and 250µg/ml). The percentage of cell viability in the methanol extract-treated dropped as extract concentrations rose. Nearly higher concentrations of the extract have demonstrated its toxicity, with more than 50% of the cells being inhibited. Table 2 and Figure 1 present the findings. Figure 2 depicts the morphological impact of A. terreus methanol extract on the L929 cell line.

Table (2):
Cytotoxicity of methanol extract of A. terreus against non-cancerous L929 cell line.

*Results are taken in triplicates and expressed as Mean±SE

Despite considerable advancements in medical technology for its diagnosis and treatment, cancer is arguably the most deadly and progressing disease, posing a mortality danger to the entire planet. All cells are subject to oxidative stress; hence oxidation and free radicals generation leads to the incident of various tumor locations’ carcinogenesis.⁴³ Both men and women can develop lung cancer, commonly known as lung carcinoma. It is regarded as the leading global cause of death. Lung cancer can be treated in a variety of ways, including surgery, radiation therapy, hormone therapy, chemotherapy, and targeted therapy, but each of these approaches has potentially severe adverse effects. Natural remedies have long been a cornerstone of medical care. Nearly 80% of the population, on average, uses natural medicine systems for primary healthcare, according to observations3. In the present study different concentrations of the methanol extract of A. terreus (50µg, 100µg, 150µg, 200µg and 250µg) were treated to the lung cancer A549 cell line. The cell viability results shown that as the concentration of test sample was increased there is gradual decrease in the percentage of cell viability. In the methanol extract treated A549 cells the percentage of viable cells in lung cancer was found to be 85.06% at 50µg concentration and at higher concentration i.e. 250µg it was found to be 35.12 and for standard drug cisplatin it was about 65.60 (Table 3 and Figure 3). It was evident from looking at the cell viability tested methanol extract shown appreciable results and demonstrated to be more efficient against lung cancer A549 cell lines. These findings even correlate with the morphological results and the MTT outcomes are correlated. Compared to untreated cells, the test samples of treated A549 have different cellular shape like elongated, ruptured and uneven shapes whereas untreated cells had a distinct spherical shape without any cellular spaces. Contrarily, in extract treated cells it was found dose dependant manner with formation of intra cellular spaces between the cells were seen (Figure 4) with several apoptotic characteristics such as cell expansion, cell shrinkage, membrane blabbing and cells turgidity. These were all characteristics of cells going through apoptosis. The test samples may be causing the lung cancer A549 cell line to undergo apoptosis, according to MTT and microscopic examination findings.

Table (3):
Percentage of cell viability in lung cancer A549 cells treated by methanol extract of Aspergillus terreus.

The results are represented as mean± standard error

Apoptosis (planned cell death) is characterized by a number of different features such as release of protein phosphatidylserine through the plasma membrane, internucleosomal DNA breaking, membrane blabbing, chromatin condensation and fragmentation, and membrane blabbing.⁴⁴ As a result, one of the best cancer treatment methods is triggering apoptosis.⁴⁵ Phosphatidylserine (P.S.) on the plasma membranes outer layer functions as a marker site in causing the early stages of apoptosis.^46,47 The membrane’s outer layer contains phosphatidyl-serine (P.S.) is specifically binds with the targeted protein like Annexin V.⁴⁸ The process of phosphatidyl-serine translocation is ongoing. In the current work, A549 lung cancer cells that have undergone treatment were used to determine early and late apoptosis using flow cytometry. The findings of current work showed that untreated cells experienced a small percentage of apoptosis that may have been caused by natural mechanisms, whereas methanol extract treated A549 cells shown around 10.84% of cells with apoptosis and whereas for standard drug cisplatin it was found to be 16.64% (Figure 5 and Figure 6).

Gas chromatography (GC), one of the most used techniques in chromatography procedures, has emerged as one of the most crucial instruments for the separation of phytocompounds. Recently, GC-MS has solidified its position as a potent method for identifying secondary metabolites in both plant and non-plant species.⁴⁹ In the current investigation, GC-MS analysis was used to determine the potential chemicals present in the methanol extract. The gas chromatogram has nine strong peaks, according to the GC-MS findings. To identify the substances, each peak will be put through a similarity search utilizing the NIST library database. 1-Flurodecane, Methyl palmitate, Ethyl palmitate, 9, 12-Octadecanopic acid, 10-Octadecanoic acid, Methyl stearate, Octadecadeoinoate, Ethyl 9-hexadecanoate, and 1-Monoarachidin have all been detected in a methanol extract of the A. terreus (Table 4 and Figure 7). These reported compounds are known to have a variety of uses, including use in the cosmetics sector, detergents, flavouring agents, and the pharmaceutical industry. According to Sermakkani and Thangapandian,⁵⁰ 9, 12-Octadecandionoic acid has anti-inflammatory, antimicrobial, hypocholesterolemic, and hepatoprotective effects.

Table (4):
GC-MS identified compounds present in Methanol extract of Aspergillus terreus.