{"id":27096,"date":"2019-06-25T11:22:11","date_gmt":"2019-06-25T11:22:11","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=27096"},"modified":"2020-04-23T04:02:54","modified_gmt":"2020-04-23T04:02:54","slug":"potential-in-vitro-and-in-vivo-antioxidant-activities-from-piper-crocatum-and-persea-americana-leaf-extracts","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol12no2\/potential-in-vitro-and-in-vivo-antioxidant-activities-from-piper-crocatum-and-persea-americana-leaf-extracts\/","title":{"rendered":"Potential In Vitro and In Vivo Antioxidant Activities from Piper Crocatum and Persea Americana Leaf Extracts"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Many medicinal plants family member from Indonesia known as potential candidate for natural antioxidant, such as mengkudu (Moringa citrifolia<\/em>),1<\/sup>\u00a0rambutan (Nephelium lappaceum<\/em> sp.),2<\/sup>\u00a0betel (Piper betle<\/em>)3,4<\/sup> and avocado (Persea americana<\/em> Mill).5\u00a0<\/sup>Red betel is commonly found in tropical area and its leaves are conventionally used by the surrounding natives as medical treatment to overcome several diseases such as diabetes, inflammation6<\/sup>\u00a0and wound healing.3<\/sup>\u00a0Beside its unique odor from its essential content, red betel leaves also possess several functional active compounds including flavonoid, alkaloid, saponins, and tannins.7<\/sup>\u00a0Many recent studies have reported that active compounds of medicinal plants exhibit hypoglycemic activity and antioxidant activity8-10<\/sup> whereas comparable to which shown by Butylated hydroxytoluene (BHT) and vitamin E.11<\/sup><\/p>\n

Several studies have been carried out to evaluate antioxidant activities of avocado fruit and its part. Antioxidant activity of avocado peel extract had been evalutaed using radical scavenging assay including Ferric Reducing Antioxidant Power (FRAP) and Oxygen Radical Absorbance Capacity (ORAC).12<\/sup>\u00a0Daiuto et al. also evaluated antioxidant activities of seed and peel parts of avocado.13<\/sup>\u00a0Avocado leaves were enriched with phenolic bioactive compounds which potential as a natural antioxidants14<\/sup> and positively contains alkaloids, flavonoids, saponins, tannins and steroids under methanolic solution to extract avocado leaves.15<\/sup>\u00a0Avocado leaves have been empirically used as a diuretic, analgesic, anti-inflammatory, hypertensive, hypoglycemic, diarrhea, sore throat and hemorrhage cure.16,17<\/sup>\u00a0Meanwhile, avocado fruit is nutritious as a preservative and antioxidant.18,19<\/sup>\u00a0Avocado flesh can be used as an anti-hyperlipidemia and has the potential to reduce the risk of metabolic s\u00edndrome.20-22<\/sup><\/p>\n

The potentials use of red betel (Piper crocatum<\/em> Ruiz & Pav) and avocado (Persea americana<\/em> Mill) leaf extracts as natural source of antioxidant must be completed by the understanding of its safety and possible side effects.23<\/sup>\u00a0Moreover, we also carried out antioxidant activity assays of red betel and avocado leaf extracts to shed light on the safety of its potential application as natural source of antioxidant without any further negative side effects. We have employed 1,1-diphenyl-2-picrylhidrazyl (DPPH) in so called cytotoxicity assay. The specimens\u2019 resistance on DPPH will provide the insight of their capability to overcome free radicals without necessarily distinguish the radical\u2019s type.24<\/sup>\u00a0The aim of this research was to determine the antioxidant activities of Persea americana<\/em> Mill and Piper crocatum<\/em> Ruiz & Pav leaf extracts in vitro and in vivo by using DPPH and MDA methods in mice models.<\/p>\n

Method<\/strong><\/p>\n

Preparation of Extract Simple<\/strong><\/p>\n

Red betel and avocado leaves were obtained from Aromatic Research Center (Balitro) in Bogor. Samples were cleaned, dried and ground into fine powder. To obtain the extract, the maceration process is done by soaking the fine powder as much as 200 g in 500 mL of solvents. Four different solvents were used in this study, including ethanol, ethyl acetate, hexane and water. The sample solution was stirred every two hours at room temperature to extract the bioactive compounds. After two days, the soaking result was filtered. The residue was again soaked in a fresh volume of solvents and the soaking process was repeated three times until clear filtrates were obtained. The resulting extracts were then evaporated using rotary evaporator at 35\u00b0C, 50 rpm. The concentrated extracts were dried by oven at 40\u00b0C until reached constant dry weight.<\/p>\n

Phytochemical Analysis<\/strong><\/p>\n

Phytochemical analysis was evaluated qualitatively to determine its bioactive compounds in plant leaf extracts of\u00a0 Persea americana<\/em> Mill and Piper crocatum<\/em> Ruiz & Pav leaf at\u00a0 various solvent fractions. Phytochemical analyses were caried out including glycoside, saponins, flavonoids, alkaloids, triterpenoid, steroids, essential oils, and tannins.25<\/sup><\/p>\n

In Vitro Antioxidant Assay with DPPH Method<\/strong><\/p>\n

In this study, antioxidants were determined by DPPH method. Vitamin C was used as standard solution with concentrations of 2.5, 5.0, 10, and 20 \u03bcg\/mL. Moreover, vitamin C were reacted with 0.5 ml DPPH (1 mM in methanol solvent). The solution was homogenized by vortex and was allowed to complete at room temperature for 30 min., then the absorbance of solution was measured using spectrophotometer at 515 nm. The results obtained were given as percentage inhibition. Furthermore, all extract was done following the same method above, at various concentration of 10, 50, 100, 200 \u03bcg\/mL, respectively. The IC50 <\/sub>value represented as the concentration required for 50% inhibition of DPPH was calculated using the graph of inhibition percentage versus the extract concentration in mg\/g (w\/w), by following equation below.<\/p>\n

\"Formula<\/p>\n

The intensity of IC50<\/sub> of antioxidant activity may vary in different medicinal plant as categorized on Table 1.<\/p>\n

Table 1: Category of antioxidant activity strength in vitro against DPPH.<\/strong><\/p>\n\n\n\n\n\n\n\n\n
Intensity of IC50<\/sub><\/strong><\/td>\nValue (\u03bcg\/mL)<\/strong><\/td>\n<\/tr>\n
Very active<\/td>\n<50<\/td>\n<\/tr>\n
Active<\/td>\n50-100<\/td>\n<\/tr>\n
Medium<\/td>\n101-250<\/td>\n<\/tr>\n
Weak<\/td>\n250-500<\/td>\n<\/tr>\n
Inactive<\/td>\n>500<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

In Vivo Antioxidant Assay with Malondialdehyde (MDA) Method<\/strong><\/p>\n

Plasma MDA levels measured by means of a thibabituric acid (TBA) reaction in mice groups before and after treatment. The in vivo test was began by weighing a mouse (190 g – 200 g) and labeled. The extract was administered to the mice daily for 10 days with the dose of 4 mg\/200 g body weight (BW) for the first group, 8 mg\/200 g BW for the second group, and 16 mg\/200g BW for the third group, and a dose of vitamin C was 2.08 mg\/200 g BW for positive control group and 1 ml of water for negative control group. On the tenth day, mice were given maximum physical activity that was in the form of swimming for 20 minutes. Observation was done on all mice activities in water. Furthermore, 2 mL blood sample was taken from each mouse through its tail after centrifugation to separate the blood plasma. Measurement of MDA levels in blood plasma was done by reacting 250 \u03bcl of blood plasma with 100 \u03bcl of 8.1% Sodium Duodecyl Sulfate (SDS) and 750 \u03bcl of 0.5 M HCl, 750 \u03bcl TBA and 125 \u03bcl aquabidest. All substance was vortexed subsequently become homogeneous. The solution was then heated at 90\u00b0C for 15 min., then cooled for 10 min. After cooling in solution, 2.5 ml n-Butanol and 500 \u03bcl aquabidest was added. The absorbance of MDA levels in plasma were measured by fluorometer at 520 nm excitation and 550 nm emission.<\/p>\n

Table 2: In vivo treatment of antioxidant activity with plant extract.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
Mice group <\/strong><\/td>\nFraction<\/strong><\/td>\nDose\u00a0<\/strong>(mg sample\/g mice body wight)<\/strong><\/td>\n<\/tr>\n
A1<\/td>\nPersea americana <\/em>Mill<\/td>\n4 mg\/200 g<\/td>\n<\/tr>\n
A2<\/td>\nPersea americana <\/em>Mill<\/td>\n8 mg\/200 g<\/td>\n<\/tr>\n
A3<\/td>\nPersea americana <\/em>Mill<\/td>\n16 mg\/200 g<\/td>\n<\/tr>\n
B1<\/td>\nPiper crocatum <\/em>Ruiz & Pav<\/td>\n4 mg\/200 g<\/td>\n<\/tr>\n
B2<\/td>\nPiper crocatum <\/em>Ruiz & Pav<\/td>\n8 mg\/200 g<\/td>\n<\/tr>\n
B3<\/td>\nPiper crocatum <\/em>Ruiz & Pav<\/td>\n16 mg\/200 g<\/td>\n<\/tr>\n
D<\/td>\nVitamin C (positive control)<\/td>\n2.08 \u00a0mg\/200 g<\/td>\n<\/tr>\n
E<\/td>\nWater (negative control)<\/td>\n–<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Sample size use in this research was following Federer\u2019s formula:<\/p>\n

(k-1) (n-1) > 15<\/p>\n

(8-1) (n-1) > 15<\/p>\n

n = 3<\/p>\n

k = number of group<\/p>\n

n = number of mice in group<\/p>\n

Results<\/strong><\/p>\n

Phytochemical Analysis and <\/strong>In Vitro Antioxidant Activiti<\/strong>es<\/strong><\/p>\n

The results of phytochemistry analysis of Persea americana<\/em> Mill Piper crocatum <\/em>Ruiz and Puf\u00a0 are shown in Table 3. The in vitro antioxidant activity of Percea americana <\/em>Mill\u00a0 and Piper crocatum<\/em> Ruiz & Pav are showed in Table 4 and Table 5, respectively. Potential antioxidant activity of Persea americana<\/em> Mill leaves extract was tested by comparison with antioxidant activity of vitamin C. The parameter used for antioxidant activity against DPPH radical was IC50<\/sub> which indicated that concentration of compound is required to reduce DPPH radical by 50%. The smaller the value of IC50<\/sub>, the more effective the function of the assayed extracts as antioxidant agents.<\/p>\n

Table 3: Phytochemistry analysis ethanol extract and ethyl acetat of Percea americana <\/em>Mill and Piper crocatum <\/em>Ruiz and Puf.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
Phytochemical<\/strong><\/p>\n

Constituent<\/strong><\/td>\n

\u00a0<\/em><\/strong><\/td>\nPercea americana<\/em><\/strong> Mill extract<\/strong><\/td>\n<\/td>\nPiper crocatum<\/em><\/strong> Ruiz and Puf extract<\/strong><\/td>\n<\/tr>\n
ethanol<\/strong><\/td>\nwater<\/strong><\/td>\nethyl acetate<\/strong><\/td>\nhexane<\/strong><\/td>\nethanol<\/strong><\/td>\nwater<\/strong><\/td>\nethyl acetate<\/strong><\/td>\nhexane<\/strong><\/td>\n<\/tr>\n
Glycosides<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n<\/tr>\n
Saponin<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Flavonoid<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Alkaloid<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n<\/tr>\n
Triterpenoid\/Steroid<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Essential oil<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n<\/tr>\n
Tannin<\/td>\n+<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Table 4: In vitro antioxidant activities of leaf extracts of Persea americana<\/em> Mill in various extraction fractions.<\/strong><\/p>\n\n\n\n\n\n\n\n\n
Extract Fraction<\/strong><\/td>\nConcentration (\u03bcg\/mL)<\/strong><\/td>\n% Inhibition<\/strong><\/td>\nIC50<\/sub> ((\u03bc\/mL)<\/strong><\/td>\n<\/tr>\n
Vitamin C (Positive control)<\/td>\n5<\/td>\n42.5<\/td>\n7.03<\/td>\n<\/tr>\n
Ethanol<\/td>\n50<\/td>\n66.7<\/td>\n35.90<\/td>\n<\/tr>\n
Ethyl Acetate<\/td>\n50<\/td>\n28.9<\/td>\n157.30<\/td>\n<\/tr>\n
Hexane<\/td>\n50<\/td>\n8.0<\/td>\n440.80<\/td>\n<\/tr>\n
Water<\/td>\n50<\/td>\n91.6<\/td>\n29.70<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Table 5: In vitro antioxidant activities of leaf extracts of Piper crocatum <\/em>Ruiz & Pav in various extraction fractions.<\/strong><\/p>\n\n\n\n\n\n\n\n\n
Extract Fraction<\/strong><\/td>\nConcentration (\u03bcg\/mL)<\/strong><\/td>\n% Inhibition<\/strong><\/td>\nIC50<\/sub> ((\u03bc\/mL)<\/strong><\/td>\n<\/tr>\n
Vitamin C (Positive control)<\/td>\n5<\/td>\n42.5<\/td>\n7.03<\/td>\n<\/tr>\n
Ethanol<\/td>\n100<\/td>\n33<\/td>\n185.29<\/td>\n<\/tr>\n
Ethyl Acetate<\/td>\n10<\/td>\n0.8<\/td>\n202.78<\/td>\n<\/tr>\n
Hexane<\/td>\n10<\/td>\n0.1<\/td>\n552.2<\/td>\n<\/tr>\n
Water<\/td>\n10<\/td>\n6.2<\/td>\n81.24<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The results of in vivo assay of Persea americana<\/em> Mill leaf extracts showed MDA levels before treatment was higher than MDA levels after treatment at 4 mg extract concentration (Fig 1). Meanwhile, Piper crocatum<\/em> Ruiz & Pav leaf extracts also showed similar MDA levels before treatment was higher than MDA levels after treatment at 4 mg extract concentration (Fig 2).<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1: In vivo antioxidant activity of Persea americana <\/em>Mill leaf extracts.<\/strong><\/p>\n

 <\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\nFigure 2: In vivo antioxidant activity of Piper crocatum <\/em>Ruiz & Pav leaf extracts.<\/strong><\/p>\n

 <\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Discussions<\/strong><\/p>\n

For both studies of in vitro and in vivo assays, we used water as a negative control and vitamin C as a positive control. These methods were carried out to evaluate the antioxidant potential of Persea americana<\/em> Mill and Piper crocatum<\/em> Ruiz & Pav extracts against free radicals. In vitro antioxidant assays are very beneficial, cost-effective and time saving to investigate the antioxidant potential of Persea americana<\/em> Mill and Piper crocatum<\/em> Ruiz & Pav extracts before getting the extract to the in vivo mouse model for the antioxidant activity by free radical scavenging. Vitamin C as positive control is a water-soluble vitamin with the mechanism works as an antioxidant by stopping the propagation stage (chain-breaking antioxidant) and provides rapid electron transfer inhibiting lipid peroxidation, thus making the potent antioxidant of vitamin C.24<\/sup>\u00a0The differences of IC50<\/sub> value of Persea americana<\/em> Mill extract in water, ethanol, ethyl acetate and hexane are caused by the solvent polarity. Water and ethanol are polar solvents, ethyl acetate is semi polar and hexane is non-polar solvents.26,27\u00a0<\/sup>Identification of major and minor compounds in Persea americana<\/em> Mill extract, ethanol, water and ethyl acetate were found to be most effective in the extraction of different compounds. Meanwhile Piper crocatum<\/em> Ruiz & Pav extract pattern most effective in ethanol and water, but it was moderate in ethyl acetate. However, both Persea americana<\/em> Mill and Piper crocatum<\/em> Ruiz & Pav extracts were found none of constituent compounds in hexane.<\/p>\n

Aqueous fraction of Persea americana<\/em> Mill leaf extract can neutralize free radicals maximally, with IC50<\/sub> value was 29.7, but not as good as antioxidant vitamin C (IC50<\/sub> = 7.03). This is due to the content of vitamin C is pure antioxidant compound that can potentially neutralize the free radicals better than plant extracts. The aqueous fraction of Piper crocatum <\/em>Ruiz & Pav leaf extracts also showed the the best IC50<\/sub> value compared to other solvent extracts. However, it still higher than vitamin C as positive control (IC50<\/sub> = 7.03). Both aqueous plant extracts contained bioactive compounds such as flavonoid, alkoloid polyphenols, glucosides and terpenoids. These compounds contribute to antioxidant activities, but they are not supposed to be pure compounds and still bound to one another with the glycoside group.28<\/sup><\/p>\n

In vivo assay of Persea americana<\/em> Mill and Piper crocatum<\/em> Ruiz & Pav leaf extracts, there were decreases of MDA levels in all groups, except negative control as shown on Figure 1 and Figure 2, respectively. This is because both leaf test extracts potentially as exogenous antioxidants, so it can suppress the free radicals that are formed, after the mice are given the physical burden of swimming for 15 minutes. In the positive control of decreased MDA levels was much better than with MDA level reduction at concentrations of 4 mg, 8 mg and 16 mg groups. The increased plasma MDA levels were elevated in the negative control group after the mice treatment through swimming, suggesting that the endogenous antioxidants in the body of mice were unable to neutralize free radicals. Endogenous and physiological reactive oxygen species (ROS) are largely generated within mitochondria as by-products of respiratory electron transport chain of normal cellular metabolism. Excessive exposure to ROS may interfere the redox homeostasis causes the cell vulnerable to ROS, and ultimately can induce cell damage.<\/p>\n

Conclusion<\/strong><\/p>\n

Persea americana<\/em> Mill and Piper crocatum<\/em> Ruiz & Pav leaf extracts have been successfully determined as antioxidant agents in vitro and in vivo. Persea americana<\/em> Mill in water and ethanol solvents exhibit strong antioxidant properties. Meanwhile, Piper crocatum<\/em> Ruiz & Pav exhibit moderate activity in water and weak antioxidant activity in ethanol. Both plant leaves showed that 8 mg dose was better than the dose of 4 mg and 16 mg, in vivo. The antioxidant systems of exogenous antioxidants could repress the ROS level by regulating the genes expression and related metabolic networks to maintain the redox balance and support cellular component for stress adaption. Thus, the antioxidant remedies using Persea americana<\/em> Mill and Piper crocatum<\/em> Ruiz & Pav leaf extracts offer a promising strategy to prevent and treat the diseases caused by the excessive ROS exposure.<\/p>\n

Acknowledgements<\/strong><\/p>\n

We would like to thank Mr. Winarno and staff in Animal Facility of Faculty of Medicine for providing in vivo mice study. This research was funded by DRPM University of Indonesia.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

There is no conflict of interest.<\/p>\n

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