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Journal of Stress Physiology & Biochemistry, Vol. 17, No. 1, 2021, pp. 13-23 ISSN 1997-0838 Original Text Copyright © 2020 by Nirmal Kumar, Priya Maran and Ravi Shankar
ORIGINAL ARTICLE
Mineral composition, Antioxidant and Antiinflammatory Activities of the Crude Extract of Leaves of Carica papaya L.
G. Nirmal Kumar, Madhu Priya Maran and S. Ravi Shankar*
Department of Botany, Madras Christian College (Autonomous), Affiliated to University of Madras, Chennai - 600 054, Tamil Nadu, India.
*E-Mail: botanyravi@gmail.com
Received August 31, 2020
Carica papaya L. (family Caricaceae) commonly known as papaya is a well-known fruit crop also having under-utilized medicinal potential. Mineral analysis shows that a significant level of potassium (207.10±0.40 mg/100g) and phosphorous (65.60±0.59 mg/100g) are present. Antioxidant activity of hexane, chloroform, ethyl acetate and ethanol crude leaf extracts were evaluated. IC50 hexane fraction (321.82±0.78) demonstrated the highest ABTS radical scavenging activity and IC50 of the chloroform fraction (623.23±1.32) showed the strongest DPPH free radical scavenging activity and their activities were stronger than that of ascorbic acid. Ethanolic extract IC50 shows significant anti-inflammatory activity (173.66±1.24) when compared with other extract fractions. The present study signifies that the leaves of C. papaya are rich in minerals and known to possess significant antioxidant and anti-inflammatory activities. This shall be further explored to identify novel bioactive substances with pharmaceutical applications.
Key words: Carica papaya L., antioxidant, anti inflammatory, mineral composition
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Medicinal plants, both wild and cultivated, have been used since the beginning of civilization as food and medicine. These plants produce a wide range of secondary metabolites as defence chemicals and they have found various therapeutic uses in medicine from time immemorial (Dilipkumar Pal et al., 2011). Papaya is a well-known plant for its edible fruit and it belongs to the family Caricaceae. They are tree-like large plant with 5-10 m tall stem and the leaves (50-70 cm diameter) are confined strictly to the top. The flowers emerge on the axils of the leaves and develop into large fruit. Fruits are large and big in size, fleshy orange in colour and seeds are numerous, black in colour, round in shape and they are covered with the gelatinous aril (Mahendra and Nikhil, 2016).
Carica papaya is commonly known as paw paw which is a rich source of carotenoids, ascorbic acid, thiamin, riboflavin, niacin, pyridoxine and vitamin K (Adetuyi et al., 2008 and Bari et al., 2006). The well riped fruits are popularly used as dessert or it can be made into a puree, wine and jam, while the unripe ones are used for cooking as raw vegetable (Matsuura et al., 2004 and Ahmed et al., 2002). Papaya is a good source of vitamin as mentioned and the amount varies between the maturation stages and varieties (Hernandez et al., 2006 and Wall 2006).
The entire plant of Carica papaya is economically valued in the various field, especially, has excellent medicinal properties. Investigation reports show that it helps to overcome heart-disease and also reduce high cholesterol levels. Papaya efficiently treats all types of abdominal, digestive problems such as dyspepsia, hyperacidity, dysentery and constipation. It is a potential source of beta-carotene that prevents the cause of some forms of cancer due to the activity of free radicals. Flowers are consumed orally to treat cough, bronchitis, asthma and chest cold. Leaves and fruits of papaya contain several proteins and alkaloids with the importance of industrial and pharmaceutical applications. Carapine, an alkaloid present in papaya is used as a heart depressant and for gastrointestinal ailment (Aravind et al., 2013).
Some of the significant medicinal properties which include inhibition of cancer cell growth, helping to cure
dengue fever, facilitate digestion, acne medicine, increase appetite, ease menstrual pain, meat tenderizer and relieve nausea. (Aravind et al., 2013). Regular consumption of ripen papayas overcomes habitual constipation and prevents premature ageing (Orwa et al., 2009). The unripe papaya fruits produce a kind of proteolytic enzyme called papain that helps in the digestion of proteins so it is used as an aid for indigestion (Ezekiel and Florence, 2012).
Biomolecules that inhibit the activity of free radicals and delay the oxidative damage of protein, lipids etc., are known as antioxidants. (Lim et al., 2006). Some secondary metabolites in the papaya were related to the antioxidant activity. Previous studies on the DPPH free-radical scavenging activity using water extract of flesh seeds of papaya indicated the most potent antioxidant activity (Kothari and Seshadri, 2010). Inflammation is a problematic biological comeback of vascular tissue to harmful stimuli, pathogens, irritants characterized by redness, warmth, swelling and pain. Many medicinal plants have shown to exhibit potent anti-inflammatory effects in the treatment of inflammation (Deepa and Renuka, 2014). Bamidele et al. (2008) reported the anti-inflammatory activities of ethanolic extract of leaves of papaya in rats using carrageenan-induced paw edema and formaldehyde induced arthritis models. The present study is envisaged to estimate the mineral content, antioxidant and anti-inflammatory activities of the crude extract of papaya leaf powder.
MATERIALS AND METHODS
Source of the Material
Leaves of Carica papaya L. were collected from in and around Tambaram, Tamil Nadu. The species was authenticated in the Centre for Floristic Research and Herbarium, Department of Botany, Madras Christian College, Tambaram, Tamil Nadu (Livingstone and Henry, 1994). Mineral Analysis
One hundred gms of fresh leaves were taken and shade-dried for seven days. After that, it was grinded, sieved and the finely powdered leaf sample was kept in an air-tight container and used for further investigation. Minerals were determined by digesting the leaf powder
in 3M Hydrochloric acid. The five inorganic elements calcium, iron, magnesium, phosphorus and potassium were determined according to the methods of AOAC (2003).
Preparation of the Leaf extract by Soxhlet extraction procedure
3gms of leaf samples were placed in the filter paper cartridge (No 89) in a standard Soxhlet apparatus and extracted with 170 ml of different solvents such as hexane, chloroform, ethyl acetate and ethanol for 2 hours. Extracts were cooled to room temperature and used at different dilutions for antioxidant and antiinflammatory activity determination. Antioxidant Activity ABTS Radical Scavenging Assay
Radical scavenging activity using ABTS was carried out according to the procedure followed by Delgado-Andrade et al. (2005). ABTS was obtained by mixing 7mM ABTS solution and 2.45 mM K2S2O8 (Potassium perfulate). Then the mixture was left undisturbed in the dark for 12 - 16 h before use. The ABTS solution further diluted with 5 mM phosphate-buffered saline (pH 7.4) to an absorbance of 0.70±0.02 at 730 nm. After the addition of 1 mL of plant extract solution of varying concentrations (10-60 Mg/mL) to 1 mL of diluted ABTS+ solution, the absorbance was measured after 30 min. The free-radical scavenging activity of the samples by ABTS was calculated by,
% = [(AO - Al)/A0]X100
DPPH Free-Radical Scavenging Assay
1, 1- Diphenyl-2-picryl hydrazyl (DPPH) assay by Raaman (2015) with slight modification was used to determine free radical scavenging activity of the plant extract. Using methanol the solution of 1 mL of 0.1mM DPPH was prepared. Various concentration of 0.1 ml of the extract was reacted with 1.9 ml of DPPH solution. The reaction content was mixed carefully and left in the dark (room temperature) for 30 mins. The absorbance reading of the extract was recorded at 517 nm wavelength. Ascorbic acid (vitamin C) is a great antioxidizing agent was used as the standard reference. The capacity of the sample to scavenge DPPH radical inhibition was calculated from the following formula:
In-vitro Anti-inflammatory Activity (Anti-denaturation assay)
Anti-inflammatory assay was carried out according to Gnana Ruby Priya et al. (2011) with minor modifications. The standard drug and leaf extracts with different solvents were dissolved in a least amount of DMS (Dimethyl Formamide) and the extract is further diluted with phosphate buffer (0.2 M) with a pH of 7.4. The concluding amount of DMF in all solutions was less than 2.5%. Test Solution (4ml) containing various concentrations of the drug was mixed with 1 ml of 1mM albumin solution diluted in phosphate buffer and incubated at 37°C in the incubator for 15 min. Denaturation was induced by keeping the reaction mixture at 70°C in a water bath for 15 min. At 600nm, the turbidity was noted and measured. Inhibition percentage was measured from reference sample (control). The diclofenac sodium which is a strong anti-inflammatory drug was used as standard reference. The percentage of inhibition was calculated using the following formula.
Percentage of inhibition = 100 X (At — Ac)/At
At = O.D. of test solution
Ac = O.D. of control
RESULTS AND DISCUSSION
Carica papaya has been used as a nutritious and medicinal plant over many centuries.
Generally, leaves, fruits and seeds of this plant are used as ethnomedicine. Because of its medicinal uses, we attempted to determine its antioxidant and antiinflammatory activities. It was also screened for its mineral content. According to Otsuki et al. (2010), numerous studies were conducted related to papaya fruits and seeds mentioning about its macro and micro mineral contents. Mineral analysis
This study made an effort to investigate the mineral composition of papaya leaves which includes potassium, calcium, magnesium, phosphorus and iron (Table 1).
The results revealed that leaves of papaya contain notable amount of potassium (207.10±0.40 mg/100g), phosphorus (65.66±0.59 mg/100g), calcium (23.10±0.99 mg/100g), magnesium (13.83±0.73 mg/100g) and iron (4.67±0.69 mg/100g). The mineral profile of Peporomia pellucida whole plant by Der-Jiun et al. (2012) shows similar results to our results of mineral composition.
Potassium is an essential mineral to both cellular and electrical functions in the body. Na and K are mainly involved in maintaining the ionic balance of the human body and tissue excitability (Indrayan, 2005). Because of the solubility of both the salts, Na plays a promising role in the transport of metabolites. Potassium salt is of great significance as a diuretic. Makanjuola and Makanjuola (2018) reported that potassium level was high both in the papaya seeds of 720.83 mg/100g and 504.33 mg/100g in the skin. The third most rich element present is calcium (Indrayan, 2005). Cells use calcium to activate certain enzymes and help to transport ion across the cellular membrane, which also plays a vital role in maintaining the regular heartbeat. Therefore Papaya is recognised as an essential source of Ca which is needed for the regulation of muscle contraction, glandular secretion and for mediating vascular contraction (Straub, 2007). Magnesium is an essential mineral for energy activity like calcium and chloride plays a role in regulating the acid-alkaline balance in the body (Bouanga Kalou et al., 2011). In our present study, magnesium is present comparatively in low amount (13.88±0.73 mg/100g). Akpanabiater et al. (1998) reported that magnesium is an important cofactor in many enzymatic reactions and in many enzyme-catalysed reactions.
Among all the micronutrients, iron has the best-described history. It is a vital micro mineral which plays an important role in the metabolism of almost all living organisms. Iron is found to be in relatively low amount (4.67±0.69 mg/100g) in papaya leaf. In humans, Iron serves to be a main constituent in a number of proteins and enzymes. (Beard et al, 1997 and Fairbanks, 1999). Deficiency of iron is a frequent nutritional problem affecting many people all over the world. It is an important proportion in the diet of pregnant women, nursing mothers, infants, convulsing patients and elderly
people to prevent anemia and other related diseases (Oluyemi et al, 2006). Similar results were obtained by Makanjuola and Makanjuola (2018) for papaya seeds 4.21 mg/100g and 2.73 mg/100g for skin respectively. Antioxidant Activity
The scavenging of the stable DPPH radical and ABTS radical is used widely to estimate the antioxidant activity of fruits, vegetables, cereal grains, wine, etc., from the phenolic compounds present in them. It is depended on the quantity of the reducing ability of antioxidants toward DPPH and ABTS. The results of the ABTS antioxidant analysis is shown in Fig. 1. The results indicate that there is significant decolourization in the ethanol and hexane extracts at 500|g and 1000|g respectively. Fig. 2 indicates the maximum inhibition percentage in the 500|g and 1000|g concentrations of all the extracts used in this study. The IC50 value of the ethanolic fraction indicates the strongest antioxidant activity (43.48±0.89) followed by ethyl acetate fraction (68.13±0.80). Kaibing Zhou et al. (2011) found that ABTS radical scavenging activity of the n-butanol fraction was significantly stronger than that of the other extracts in the seeds of papaya.Yuhahara and Iswahyuni (2018) revealed that the extracts from papaya seeds especially ethyl acetate and n-butanol contained free radical scavengers, possibly acting as principal antioxidants.
The DPPH is a stable radical with maximum absorption at 517 nm that can readily undergo scavenging by antioxidant (Lu and Yeap Foo, 2001). Porto et al. (2000) and Soares et al. (1997) found that it has been widely used to test the ability of compounds as free radical scavengers by hydrogen donors and to evaluate the antioxidant capacity. The antioxidant activity performed with the DPPH free radical scavenging method result (Fig 3) indicates there is significant decolouration in ethanol extract at 500 |g and 1000 |g concentrations. Fig. 4 demonstrates maximum inhibition percentage in ethanol and ethyl acetate extract at 500 |g and 1000 |g concentrations. According to Hanani et al. (2005) a substance can be expressed to be a strong antioxidant if it has an IC50 value <200 ppm, that has antioxidant capabilities such as those associated with phytochemical screening results that
include flavonoids. Flavonoids have been studied to have various activities such as antioxidants and antibacterials (Mohan et al., 2008).
The DPPH free radical-scavenging activities of the samples were estimated by comparing the IC50 of the extract fractions and ascorbic acid. Concentration of the sample necessary to decrease the initial concentration of DPPH by 50% (IC50) under the experimental condition was calculated. Therefore, a lower IC50 value indicates a higher antioxidant activity. According to the IC50 values, the ability to scavenge DPPH free radicals of the sample could be ranked as ascorbic acid>ethanol>ethyl acetate>hexane>chloroform fraction (Table 2). The IC50 value of the ethanolic fraction indicates the strongest antioxidant activity (127.41±0.85) followed by ethyl acetate fraction (182.76±0.82) which had stronger antioxidant activity too. Yuhahara and Iswahyuni (2018) reported a similar results that ethanolic extracts of papaya leaf have a strong antioxidant activity with IC50 value of 100.0±0.07 ppm.
Mahattanatawee et al. (2006) found that the antioxidant activity is mainly due to the presence of phenolic compounds which are the important constituents of fruit because they inactivate the lipid free radicals or prevent the decomposition of hydroperoxides into free radicals. Kaibing Zhou et al. (2011) investigated the DPPH free radical-scavenging activities of all extracts from seeds of papaya which is related to the amount of antioxidant constituents extracted from seeds of papaya by various solvents. Compounds such as ascorbic acid, p-carotene, a-carotene and different xanthophylls have been detected in banana and may have contributed to the antioxidant activity of the extracts (Kondo et al, 2005). The scavenging activity on DPPH radical was related to the ripening stage, the activity increased as a result of increasing ripening stage for each stage. These results imply that antioxidative compounds other than phenolics and flavonoids were also involved in inhibiting the DPPH radicals.
Anti-inflammatory activity
Inflammation is the immune response of body tissue to injury or infection (Bamidele et al., 2008). It is an essential component for natural immunity. The inflammatory route involves a difficult biological fall of
cellular signals that modify physiological responses and eventually resulting in the familiar clinical symptoms of pain, swelling, heat and redness (Comalada et al., 2005). The role of inflammation is to destroy invading microorganisms, inactivate toxins and to achieve healing and repair. However, it may be potentially harmful, causing life threatening hypersensitivity reactions and progressive organ damage. Due to the enormous medicinal potential of the Carica papaya, the present study made an effort to evaluate the in vitro anti-denaturation activity from leaf powder of papaya.
The results of the different solvent extracts namely chloroform, ethanol, ethyl acetate and hexane towards anti-denaturation activity are indicted in Fig. 5. Maximum inhibition is shown in the hexane extract at 1000|g and 2000|g concentration (Fig 6). The results of IC50 values (Table 3) have demonstrated that the chloroform extract have good anti-denaturation activity 40.94±0.82 followed by ethyl acetate 64.65±0.85 when compared to the other extracts. Saurabh et al. (2016) reported significant anti-inflammatory activity of papaya leaf juice (at 0.72 ml/ 100 g body weight) against carrageenan-induced rat paw edema and impaired in vivo vascular permeability with a percentage of 82.0 while inducing a percentage of 10 membrane-stabilizing activity of rat RBC. Papaya seeds (methanol and aqueous extract) have also been examined and found to display anti-inflammatory activity in in vivo study (Amazu et al., 2010; Umana et al., 2014). Unfortunately, none of the studies in in vivo investigated the anti-inflammatory or immunomodulatory effects of any part of the papaya plant in the non-polar extracts.
Many promising bioactive molecules have been isolated from papaya and investigated for their medicinal applications. The proved medicinal activity of papaya includes antioxidant, anti-inflamatory, antimicrobial, anti-fertility, antimalarial and immune modulatory etc. (Krishna et al., 2008; Tatyasaheb et al., 2014; Vij and Prashar, 2015). The leaves of C. papaya is an excellent source to treat dengue fever and it was proved to increase platelet and total white blood cell count (Ahmad et al., 2011; Sarala and Paknikar, 2014). The leaves of papaya are rich in several minerals and many studies reported that these minerals content may balance the
deficiency caused by the virus and strengthen the immune system (Sharma et al., 2013). So in this study we made a great attempt to analyse the dried crude leaf
extract of papaya to investigate the mineral profile,
Table 1 Mineral analysis of the leaves of Papaya - Macronutrients
Minerals mg/100g of leaf powder
Potassium 207.10±0.40
Calcium 23.10±0.99
Magnesium 13.83±0.73
Phosphorus 65.66±0.59
Iron 4.67±0.69
Values are represented as mean ± standard deviation (n=3).
Table 2 Antioxidant activity of the leaves of papaya by ABTS and DPPH free radical scavenging assay
Leaf extract ABTS assay (ICS0 |Jg) DPPH assay (ICS0 Hg)
Hexane 321.82±0.78 274.60±0.75
Chloroform 263.06±0.82 623.23±1.32
Ethyl acetate 68.13±0.80 182.76±0.82
Ethanol 43.48±0.89 127.41±0.85
Ascorbic acid 7.42±0.69 11.11±0.81
Values are represented as mean ± standard deviation (n=3).
antioxidant and anti inflammatory activities which serve as a preliminary source and it needs much more clinical trial on this.
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Fig 2 Antioxidant assay by ABTS radical scavenging activity using papaya leaves
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a) Hexane; b) Chloroform; c) Ethyl acetate; d) Ethanol; e) Ascorbic acid (Standard)
Table 3 Anti- inflammatory activity from the leaves of papaya
Leaf extract 1C50 Mg
Hexane 68.61±0.74
Chloroform 40.94±0.82
Ethyl acetate 64.65±0.85
Ethanol 172.32±0.77
Diclofenac Na 41.07±0.81
Values are represented as mean ± standard deviation (n=3).
Fig 4 Antioxidant assay by DPPH radical scavenging activity using papaya leaves
Fig 5 Anti-inflammatory assay of papaya leaves using egg albumin method
a) Hexane; b) Chloroform; c) Ethyl acetate; d) Ethanol; e) Diclofenac Na (Standard)
Fig 6 Anti inflammatory activity of papaya leaves using Egg albumin method
CONCLUSION
Papaya is a famous and common fruit known for its medicinal properties world wide. The present study is to investigate the potential of C. papaya in the evaluation of antioxidant and anti-inflammatory studies. Papaya extract undoubtedly offers a cheap and possibly valuable treatment for dengue. A herbal medicine being investigated to control the epic problem of dengue is the extract of the leaves of the plant. These evidences are inadequate to comment on the role of papaya leaf extract in dengue. The active compound needs to be identified and the dosage should be standardized to prove its efficacy in dengue beyond doubt. Large-scale randomised studies could establish the efficacy of this
It is also
necessary to conduct pharmacokinetic studies to ensure that the active principle is absorbed from the digestive tract. The present study will provide pavement to explore the future application of Carica papaya leaves as a raw material in the pharmaceutical industries, especially in the treatment of dengue with clearly defined outcome measures.
ACKNOWLEDGEMENT
The authors are thankful to the Principal and Secretary and the Head of the Department of Botany, Madras Christian College (Autonomous) for their support and providing necessary facilities in carrying out this study.
CONFLICTS OF INTEREST
All authors have declared that they do not have any conflict of interest for publishing this research.
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