Pharmacological and toxicological activities of the extracts of papaya leaves used traditionally for the treatment of diarrhea

2.1 Instruments

The absorbance of the prepared each concentration extract for the calculation of percentage of inhibition was measured by Shimadzu UV–visible spectrophotometer, Japan

2.2 Sample collection and identification

The leave samples were collected from the papaya producing farmer in Malacca, Malaysia. The samples were collected during the month of September 2018. Soon after collection, the leave samples packed in a plastic bag for travel and brought to the University of Nizwa Research Lab. Only the health papaya leaves were used for processing. The necessary process of the collected samples was done in the research lab 15L for extraction. The morphological features or identification of the leaves was done through website as well as the local people (https://en.wikipedia.org/wiki/Papaya).

2.3 Sample preparation

At first, the papaya leave samples were properly clean with water to eliminate filthy materials and kept at room temperature on newspaper until dried to obtain constant weight. The dried samples were crushed by using blender and made into a coarse powder. The course powder samples were kept at 4 °C in a safe place to avoid microbial and unintentional contamination.

2.4 Preparation of extracts and fractionation

The dried coarse powder (100 gm) was soaked with methanol (2 L) at room temperature for 48 h. During the extraction process the plant materials were stirred up and down using a glass rod for complete extraction. After 48 h of soaking, it was filtered by Buchner funnel to remove the unwanted coarse powder materials. Then, the filtrate was evaporated to dryness by using the usual rotary evaporator which gave an amorphous solid methanol extract (8.78 gm). From the methanol extract, approximately 7 gm was dissolved in water and transferred to the separatory funnel for fractionation (Weli et al., 2018). Various polarities of solvents such as hexane, dichloromethane, ethyl acetate, and butanol were used for successive fractionation to give hexane, dichloromethane, ethyl acetate, and butanol extracts which were evaporated to give the corresponding extracts. The water fraction was also evaporated and it considered as water extract. All the extracts were kept at 4 °C in a close container to avoid contamination as well as for further process.

2.5 Free radical scavenging bioassay

The free radical activity of the above various polarities extracts of papaya leaves was measured through 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical bioassay. DPPH bioassay was performed according to the procedure described by Al Amri and Hossain (2018). The DPPH solution was prepared by dissolving 3.2 mg DPPH in 100 ml of analytical grade methanol. Approximately 2.7 ml of DPPH solution was added to each tube followed by the addition of 300 μl of a different polarity mother extract of each concentration. The final concentration was about 400 μg/ml, 200 μg/ml, 100 μg/ml, 50 μg/ml, and 25 μg/ml. The final mixtures were shaken well by sonicator and kept in dark for incubation at room temperature for one and half hours. The absorbance of the incubated each concentration sample was measured by Shimadzu UV–visible spectrophotometer at the wavelength of 517 nm. The methanol was used as a blank while mixture of 300 μl of methanol and 2.7 ml of DPPH solutions were used as a control. Each concentration of the test samples was performed in triplicates. Finally, the percentage inhibition of each extract of various concentrations was measured according to the formula given below. The IC₅₀ values of each extract were calculated by using graphical methods. $$\text{Scavenging}\text{activity}\left(\%\right)=\left[\left(\text{Ac}-\text{As}\right)/\text{Ac}\right]\times 100$$ where

• Ac = means absorbance of DPPH

• As = means absorbance of the test sample.

2.6 Cytotoxic bioassay

The cytotoxic activity of the above various polarities prepared extracts of papaya leaves was determined through brine shrimp lethality (BSL) bioassay. BSL bioassay was performed according to the procedure described by Al Alawi et al. (2018). In this present experiment, the artificial sea water was prepared by dissolving 38.2 mg of sodium chloride in 1000 ml of water for the hatching of the shrimp eggs. The artificial sea water solution (250 ml) was used in a duo plastic chamber and the shrimp eggs were added into the duo chamber and left for 24 h under the 60-watt light bulb. After 24 h incubation, the eggs were hatched and the larvae entered into another chamber. Various concentrations of each extract were prepared by using DMSO solvent. 4.9 ml of artificial sea water was added to the all working tubes followed by the addition of 100 μl each concentration of each extract. The final six concentrations were of 500 μg/ml, 250 μg/ml, 125 μg/ml, 65 μg/ml, 35 μg/ml and 15 μg/ml. The final mixtures were shaken well by sonicator and added with 10 larvae and kept in working tubes under the light at room temperature for 24 h incubation. The DMSO solvent was used as a control without extract while mixture of 4.9 ml of artificial sea water and 100 μl of DMSO solutions. Finally, the alive larvae were calculated by magnifying glass. The IC₅₀ values of each extract were calculated by using graphical methods.

3.1 Free radical scavenging activity

All the successive fraction extracts including methanol extract at various concentrations were used for the determination of free radical scavenging inhibition by using the modified DPPH method which was described by Al Amri and Hossain (2018). Most of the extracts from the papaya leaves did not showed promising activity against the free radical scavenging activity. The percentage inhibition was within the range of 35–70%. Among the extracts of papaya leaves, the most prominent activity was obtained from water extract and the lowest was in methanol extract. The free radical scavenging inhibition of each extract at various concentrations is presented in Table 2. The IC₅₀ values of each extract of papaya leaves were calculated by using graphical methods. The highest IC₅₀ values among the papaya leave extracts was obtained from water extract and the lowest was in the hexane extract (Table 2 and Fig. 2).

Close

Fig. 2

IC₅₀ values from scavenging inhibition (%) of various papaya leaves extracts.

Export to PPT

3.2 Cytotoxic activity

All the successive fractions of methanol extract as well as a mother methanol extract from the leaves of papaya at various concentrations were used for the determination of percentage of mortality by using the modified BSL method which was described in Al Alawi et al. (2018). Most of the extracts from the papaya leaves showed highest mortality against the BSL bioassay. Among them, the highest mortality was found in dichloromethane extract and the lowest mortality was obtained from water extract. The percentage of mortality of each extract at various concentrations is presented in Table 3. The LC₅₀ values of each extract of papaya leaves were calculated by using graphical method (Fig. 3). The highest LC₅₀ values were obtained from water extract and the lowest was in the methanol extract (Table 4 and Fig. 3).

Close

Fig. 3

LC₅₀ values from percentage of mortality (%) of various papaya leaves extracts.

Export to PPT

4.1 Plant extract

The weight of various extracts obtained from the papaya leaves by using the soaking method and the percentage of yield is shown in Table 1. The weight of the methanol extract was an approximate 8.78 gm ≈ 8.78%. The methanol crude extract was fractioned by using various polarities of solvent starting from hexane. Among the fractionated extracts, the highest yield was obtained from hexane extract and the lowest yield was obtained from ethyl acetate which is presented in Table 1.

4.2 Free radical scavenging activity

The plant’s medicinal value depends on the presence of chemicals that showed positive pharmacological and physiological actions on the human body which can be protected and treating human diseases. In the past studies on papaya plant showed that the selected plant contains secondary metabolites (Ayoola & Adeyeye, 2010). They are connected with the biological and pharmacological properties like antioxidant, antimicrobial, anti-inflammatory, antidiabetic etc. (Weli et al., 2018; Al Amri and Hossain, 2018). The previous studies also showed that the selected plant has several groups of chemical such as alkaloids, terpenoids, carbohydrates, tannins, flavonoids, saponins and steroids in various polarities of extracts in consideration amounts (Wall, 2006; Ayoola & Adeyeye, 2010). Nowadays, the antioxidants from natural sources are highly and well accepted over synthetic antioxidants due to their toxicity in the human body. There are several methods available to determine the antioxidant activity. Among them, the DPPH bioassay is the most popular and well accepted method for the determination of antioxidant activity of plant products. In our experiment, the scavenging activity of various papaya extracts was measured by using DPPH bioassay. In this method, DPPH is used to measure ability to donate the electron and radical scavenging ability of products (Al Amri and Hossain, 2018). The electron is transferred from antioxidant to DPPH radical molecules (Al Amri and Hossain, 2018). Therefore, the DPPH is acts as a reducing agent and thereafter DPPH lose its deep violet colour gradually. The change of colour was measured by using UV–visible spectrophotometer at the wavelength 517 nm. The antioxidant activity as a percentage of inhibition of various extracts of papaya leaves was calculated by using the well-established formula. The range of inhibition of various extracts of papaya leaves was from 35 to 70.35%. Among the six prepared extracts from the leaves, the highest inhibition was in water extract and the lowest was in ethyl acetate extract followed by butanol > hexane > dichloromethane > methanol > ethyl acetate extract. The high activity in water extract may be due to the antioxidant guided most of the compounds present in this extract. In addition, the water is highly polar solvent and the water soluble compounds are also polar. Therefore, most of the antioxidant guided compounds are polar in nature. The LC₅₀ of all prepared extracts of the papaya leaves was calculated in various concentrations of each extract against the percentage of inhibitions (Fig. 3 and Table 4). According to Table 4, the highest IC₅₀ values were obtained from water extract (782.06 μg/ml) and the lowest was in the hexane extract (156.39 μg/ml) comparing to the original methanol extract and other extract of papaya leaves followed by water > butanol > ethyl acetate > methanol > dichloromethane > hexane extract. However, the toxicity is high in hexane and lower in water extract, followed by hexane > dichloromethane > methanol > ethyl acetate > butanol > water extract. Literatures from past studies have shown several previous reports on antioxidant activity of papaya leaves (Banu & Cathrine, 2018; Gotink and Verheul, 2010; Ang, 2012). However, our experimental results do not align with the reported data (Gotink and Verheul, 2010; Ang, 2012). The main reasons are i. our sample and extraction process were not similar ii. They prepared extract directly from the leaves while in this study fractionation process was used and iii. The presence of phytochemicals was in the extracts. Thus, these differences have made the results not aligned with the reported values.

4.3 Cytotoxic activity

The modification of the procedure described in Al Alawi et al. (2018), the lethality of various extracts of papaya leaves on brine shrimp was evaluated and the results of percentage mortality at various concentrations were shown in Table 3. Among the papaya leaves extracts, hexane, dichloromethane, ethyl acetate and methanol extracts showed promising mortality. However, the butanol and water extracts have lower mortality compared to other extracts. In addition, another indication is that the mortality of the extract is decreasing by the decreasing of concentration of each extract. It was noted that all the extracts from the leaves of papaya at 500 μg/ml produce significant percentage of mortality. The results showed that the highest mortality of about 90% was obtained from the dichloromethane extract at the concentration 500 μg/ml. However, the lowest mortality of about 40% was obtained from water extract. The dichloromethane showed that the highest mortality could be due the presence of highest number or concentration of toxic chemicals. In this result, it is clear that most of the toxic compounds are present in the dichloromethane extract and lesser number or concentration in water extract. Some correlation was observed when log concentration was plotted against the percentage of mortality (Al Alawi et al., 2018) on the graph paper. The LC₅₀ values of each extract were calculated using Microsoft Excel 2010 (Fig. 3 and Table 4). According to the Table 4, methanol leaves extract showed significant cytotoxic activity having LC₅₀ value of 118.73 μg/ml comparable to the other polarity papaya extracts. The dichloromethane and butanol also showed the potent LC₅₀ activity. From the results, it indicates that most of the toxic compounds present in the methanol extract followed by dichloromethane > butanol > hexane > ethyl acetate > water extract. Several literatures on cytotoxic activity of papaya leaves have reported significant cytotoxic activity, however, this study does not align with the reported data (Nguyen et al., 2016; Shahar et al., 2011; Garcia-Solis et al., 2009; Otsuki et al., 2010). It could be due to the sample processing, extraction pattern from the plant samples, solvent–solvent fractionation as well as phytochemicals.