Reasons and purpose of research

Functional and eco-friendly cosmetics have risen in demand due to the increased use of beauty products among the younger generation that values self-development, and the trend of eco-friendly products in a rapidly aging society with increased average incomes for women, due to increased gender equality in the modern society [1].

New trends, such as “YOLO” (You Only Live Once), the latest lifestyle that emphasizes one’s happiness and encourages consumption for happiness, and “wellness,” a term that combines well-being and fitness; reflect the rising popularity of natural beauty products, cosmetic ingredients, and eco-friendly personal care products [2].

For example, a large cosmetic company has terminated the import of beauty products made with parabens [3], and there have been persistent efforts to find natural ingredients that can be used to develop functional cosmetics with whitening, anti-aging, or protective properties due to the toxicity and side effects of synthetic raw materials [4]. In addition, research on the basic anatomy and physiology of the skin needs to be conducted to develop and deliver appropriate skin formulations, essential in manufacturing functional cosmetics [5]. Physiologically active substances extracted from natural ingredients have whitening and anti-aging effects as well as superior soothing effects, and studies on their efficacy are continuously being conducted. Functional cosmetics with natural extracts are recognized as products that can satisfy the needs of people of all ages interested in societal trends and cosmetic materials. As products are directly applied to the human body, natural extracts should be usable, stable, and safe, in addition to having the ability to maintain the physiological function of the skin [1]. However, there has been limited research on physiological activities in evaluating natural extracts as cosmetic ingredients. Therefore, the current study examined graviola leaf extract, known for its chemotherapeutic effects and as functional food, to confirm its industrial value and potential as an ingredient for functional cosmetic [6].

Research on the anti-inflammatory effects of graviola leaf extract

When physical or chemical stimuli such as bacterial infection occurs, our body activates immunocytes for cellular self-defense and maintenance, and the activated immunocytes secrete cytokines and nitric oxide (NO) to neutralize the exogenic pathogens. However, an excessive immune response leads to a chronic inflammatory response. Continued inflammatory response promotes mucosal damage and causes functional disorders such as pain, swelling, and fever, eventually disrupting the body’s immune system that induces atopic dermatitis, allergies, and cancer and increases the risk for chronic systemic inflammatory diseases and autoimmune diseases. Therefore, controlling the inflammatory response is an important way of maintaining the body’s immune system and controlling various physiologic phenomena [7]. Macrophages are the main type of cells in the human body that control the inflammatory response. They are activated by external stimuli, and activated macrophages secrete inflammatory mediators such as cytokines to accelerate the inflammatory response [8]. Lipopolysaccharide (LPS), a representative mitogen of macrophages, is in the outer membrane of gram-negative bacteria, serves as an endotoxin, and can induce a sufficient level of inflammatory response even in small amounts. LPS stimulation promotes the secretion of NO, prostaglandins E, and inflammatory cytokines; including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6; in macrophages during the early stages of infection. The excessive secretion of these inflammatory factors leads to an escalation of the inflammatory response from a local to a systemic level. There has been various research on natural ingredients to control various types of early inflammation-related factors and treat systemic inflammatory responses by inhibiting the secretion of inflammatory mediators. Ethanol extracts from graviola leaves have been shown to inhibit the secretion of NO and cytokines (e.g., IL-6, TNF-α, and IL-1β), which are inflammatory mediators with anti-inflammatory effects [9].

Ingredients

Ingredients and method of extraction

The dried graviola leaves used in the experiment were purchased from Well Food Korea, which imports the leaves from the Philippines. Three liters of 70% ethanol were added to 300 g of dried graviola leaves to increase the final volume by tenfold, and the leaves were extracted over three days at room temperature. To isolate the extract, the extract was centrifuged, filtered using Whitman No. 2 filter paper, and evaporated using an evaporator (EYELA) to remove the ethanol, the extraction solvent. The concentrate was freeze-dried after the evaporation process and the final product was refrigerated to be used as a sample for this experiment.

High-temperature preservation of the sample

The graviola leaf extract prepared as above was stored at a high temperature for 1 to 5 days in a 50°C drying oven (Chang-Shin Scientific Co.) and was used after being filtered at various concentrations using a syringe filter (0.2 μm).

Cell line

RAW 264.7 macrophages, obtained from the Korea Cell Line Bank, were used. RAW 264.7 cells were used to measure the intracellular production of NO.

Cell culture

RAW 264.7 cells were cultured using a high glucose Dulbecco’s modified Eagle’s medium (Hyclone) with the addition of 1% penicillin/streptomycin (100 IU/50 mg/ml; Sigma) and 10% fetal bovine serum (Sigma). Cell cultures were maintained at 37°C, 5% CO₂, and 100% humidity in the moisture chamber (Sanyo Electric Co.).

Cell viability in graviola leaf extract using neutral red assay

A neutral red (NR) assay was performed to detect cytotoxic substances. The assay uses a cationic staining reagent that passes through the cell membrane through non-proliferation and accumulates inside lysosomes. It evaluates the degree of cytotoxicity by applying the principle of absorption and accumulation of NR dye inside the lysosome of uninjured cells and excretion of the dye only upon damage to the cell membrane or lysosome via external or chemical stimuli. The graviola leaf extract was diluted with 70% ethanol to concentrations of 6.25, 12.5, 25, 50, and 100 mg/ml, cultured at each concentration in the cells for 48 hours, and the NR assay was performed to confirm the effect of graviola leaf extract on the viability of mouse-derived RAW 264.7 cells.

The cells showed a high viability of 90% when cultured with 6.25 and 12.5 mg/ml concentrations of graviola leaf extract, 6.5% in 25 mg/ml, and 50% in both 25 and 50 mg/ml.

Measurement of nitric oxide production inhibition

The amount of NO production in cell media was measured in the form of nitrite (NO₂^-) and nitrate (NO₃^-) to measure the NO production-inhibition effect of graviola leaves [10]. RAW 264.7 cells were plated at a concentration of 5×10⁴ cells per well into a 96-well plate and cultured for a single day. Then, the cell media was removed, added to media containing 1 mg/ml of LPS at concentrations of 6.25, 12.5, 25, and 50 mg/ml, and cultured for two days. One hundred milliliters of cell culture supernatant and 100 ml of Greiss reagent were added to a new 96-well plate, allowed to react in a shaded environment for 10 minutes, and quantified using a microplate reader at a wavelength of 540 nm. The formula (c) for inhibition of NO production was calculated as follows:

Inhibition of NO production (%) optical density (O.D.) of the sample with

Anti-inflammatory effects of graviola leaf extract

Measuring the inhibition of nitric oxide production

NO, an important mediator of the inflammatory response, is a substance derived from L-arginine, produced by NO synthase, and an essential neurotransmitter in the nervous, immune, and cardiovascular systems [11]. We assessed the effect of graviola leaf extract on the inhibition of NO production by adding LPS, an inflammatory inducer, to RAW 264.7 cells, treated the cells with concentrations of 5, 10, 20 50, and 100 µg/ml of graviola leaf extract, and then measured the inhibitory effect of NO production (Fig. 1, 2). Compared to the LPS-treated cells, graviola leaf extract significantly reduced the amount of NO production by 44.6% in 50 µg/ml (p<0.05) and 40.68% in 100 µg/ml (p<0.05). NO has an important physiological role in blood pressure regulation, coagulation, and immune function against cancer cells. However, when NO exceeds a certain level, it starts causing cellular damage via inflammatory reactions, which has harmful effects on the human body [1]. The results confirmed that graviola leaf extract has an excellent effect on inhibiting LPS-induced NO production and thus can be postulated to inhibit cell damage caused by inflammation.

Figure 1. Effect of Annona muricata L extract on cell viability in RAW 264.7 cells. Values represent the mean±standard deviation of three measurements.

Figure 2. Inhibitory effect of Annona muricata L extract on LPS-induced nitric oxide production by RAW 264.7 cells. The result is represented as mean ± standard deviation. LPS, lipopolysaccharide. *p<0.05.

This study was conducted to confirm the physiological effects of graviola leaf extract and to examine its effectiveness as a potential component of functional cosmetics. The results of skin cell activation by graviola leaf extract are as follows. RAW 264.7 cells treated with extracts at various concentrations showed NO production inhibition in a concentration-dependent manner compared to LPS-induced RAW 264.7 cells. This study proposes the use of graviola leaf extract as an anti-inflammatory ingredient that can be used in acne cosmetics and cosmetics used for inflammatory skin among functional cosmetics. I also think that stabilization research on extracts should be carried out. Animal studies and clinical trials should be conducted to determine the future availability of graviola leaf extracts.

The authors have nothing to disclose.