Antioxidant, Anti-Inflammatory Activities, and Neuroprotective Behaviors of Phyllanthus emblica L. Fruit Extracts

Abstract

Phyllanthus emblica L. is traditionally used as both medicine and food in Taiwan. In this study, we evaluated the antioxidant, anti-inflammatory, and neuroprotection bioactivities of P. emblica fruit. P. emblica fruit extracts had a high content of total phenol and flavonoids, and chlorogenic acids. For antioxidant capacity, 95% ethanol-extracted P. emblica had the best DPPH radical scavenging activity, ferrous ion chelating ability, and reducing power as compared with hot water, 50% ethanol, and commercial extracts, and showed the highest reducing power and lipid peroxidation inhibition. The present results have demonstrated that the P. emblica extracts can protect the oxidative degradation of lipids by inhibiting FeCl₃-ascorbate-mediated lipid peroxidation. For anti-inflammatory activity, P. emblica fruit extracts showed dose-dependent inhibition of nitric oxide in lipopolysaccharide-stimulated RAW264.7 cells and significantly high COX-2 inhibition. For neuroprotection bioactivity, P. emblica extracts had higher percentages of pheochromocytoma cell protection than commercial extracts. Hot water and ethanol extracts showed higher percentages of PC12 cell protection than commercial extracts. P. emblica hydroalcoholic extracts had a neuroprotective effect against oxidative damage, which could be due to their antioxidant and anti-inflammatory activity. P. emblica extracts could be used in daily health beverages, foods, and cosmetic products.

1. Introduction

Phyllanthus emblica L., commonly known as Indian gooseberry, belongs to the family of Euphorbiaceae. It is widely distributed in the subtropical and tropical areas of Southeast Asia. P. emblica has been recognized as a natural source of food and medicine and has potential for the development of novel drug formulations for humans in South and East Asia. Its application is mentioned in many traditional medicinal systems, such as Chinese herbal medicine, Tibetan medicine, and Ayurvedic medicine [1,2]. The whole plant (all parts) can be employed for medicinal purposes, particularly the fruit, which has been used in Ayurveda as a potent rejuvenating agent, the so-called Rasayana. Its fruit has also been used in traditional medicine for the treatment of jaundice, diarrhea, and inflammation. Furthermore, various plant parts have shown antibacterial, antioxidant, antidiabetic, hypolipidemic, antiulcerogenic, gastroprotective, hepatoprotective, and chemopreventive properties. Many reports suggest that the extracts contain tannins, alkaloids, and phenolic compounds along with abundant amounts of vitamin C and superoxide dismutase [3].

The medicinal values of fruits of P. emblica may be due to its phenolic compounds such as gallic acid, catechin, chlorogenic acid, caffeic acid, kaempferol, phyllanemblin, p-coumaric acid, quercetin, and geraniin. The methanolic crude extracts from P. acidus leaves and P. niruri had in-vitro porcine pancreatic lipase (PPL) inhibitory activity as 33.90% and 76.70%, respectively, when using p-nitrophenyl butyrate (pNPB) as a substrate [4]. Similarly, the chromatographic fractionation of the methanol extract of Eucalyptus globulus leaves yielded 2, 2,8-trimethyl-6-formyl-chrom-3-ene 7-O-β-d-glucopyranoside, quercetin 3-O- α-l-4C1 arabinopyranoside-2″ gallate, cornusiin B, ellagitannins, galloyl esters, flavonoids, chromones, terpenoids, and eucalbanin B-like phenolic compounds, which have good antioxidant potential [5]. The inhibitory effect of Eucalyptus globulus leaf extracts on pancreatic lipase determined by turbid-metric assay showed more than 65% inhibition on the enzyme activity using triolein as a substrate [6]. The methanolic and ethanol crude extracts of Tinospora cordiifolia stems also showed the presence of various phenolic acids such as caffeic acid, cinnamic acid, ferulic acid, tannic acid, ellagic acid, and gallic acid [7]. The pancreatic lipase inhibitory activities of the methanolic extracts of Emblica officinalis leaves, Phyllanthus niruri L., and Tinospora crispa plants were measured as 76.3%, 81.4% and 28.9%, respectively, when using palm olein as a substrate [8].

Free radicals may directly cause DNA damage and mutation and result in cancer as well as increase the phosphate peroxidation of cell membranes, thus altering the fluid properties of cell membranes, damaging cellular integrity, or accelerating cell aging [9]. They can continuously damage cells and tissues in the body or lead to decreased energy and organ failure, thus causing diseases such as vascular sclerosis, heart disease, gout, diabetes, arthritis, cataracts, and diseases of the immune system [10]. The human body has a certain number of free radicals as a weapon to prevent and defend against diseases [5]. Studies have found that stress, radiation, trauma, disease, infection, or inflammation can accumulate large amounts of free radicals after activation of the body’s defense system, which may attack healthy cells, trigger apoptosis, or cause cellular aging [11]. Furthermore, oxidative damage plays a key role in the cause and progression of neurodegenerative diseases. Inhibition of oxidative stress represents one of the most effective ways to treat human neurologic diseases.

Natural antioxidants found in vegetables, fruits, and daily beverages, are effective in scavenging free radicals [12,13,14,15,16,17]. A high intake of vegetables and fruits in the diet, in which plant polyphenols composed of flavonoids act as antioxidants for the body, has preventive effects on cancer, diabetes, and cardiovascular diseases [18]. Plant polyphenols have multiple functions, not only as antioxidants but also as reducing agents and in some cases as metal chelators, which may reduce the risk of death from heart-related diseases in older men [19]. They dilate blood vessels and have anti-cancer, anti-inflammatory, antibacterial, immune stimulating, and anti-viral effects [20].

Over the years, the bioactivity and biocompatibility of plant extracts have become increasingly important, and the addition of natural plant extracts to cosmetic products has become more popular and of increasing interest to the public. Many natural plant extracts have been attracting attention for their antioxidant effects. Several studies have shown that antioxidant activities are associated with anti-inflammatory and neuroprotective effects [21,22].

In this work, we used P. emblica for extracting bioactive compounds and studied the antioxidant, anti-inflammatory, and neuroprotection bioactivities including α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging, inhibition of lipid peroxidation activity, reducing power, cyclooxygenase-2 (COX-2) and nitric oxide (NO) inhibition, and pheochromocytoma (PC12) cell protection.

2. Materials and Methods

2.1. Chemicals

All chemicals and solvents used were of analytical grade. Dulbecco’s modified Eagle medium (DMEM) was from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Beit-Haemek, Israel. Soy lecithin was from Wako Pure Chemical Industries (Osaka, Japan). Gallic acid, rutin hydrate, α-diphenyl-β-picrylhydrazyl (DPPH), lipopolysaccharide (LPS), N-(1-naphthyl) ethylenediamine dihydrochloride, sulfanilamide, thiazolyl blue tetrazolium bromide (MTT), L-N^G-nitroarginine methyl ester (L-Name), and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were from Sigma Chemicals (St. Louis, MO, USA). RAW264.7 cells, mouse macrophage cells, PC12 cells, and rat adrenal pheochromocytoma cells were purchased from the Bioresource Collection and Research Centre (BCRC; FIRDI, Hsinchu, Taiwan). A commercial extract (SaberryTM) was identified and authenticated by HG Biomedical Co., ltd. (Taichung, Taiwan).

2.2. Preparation of P. emblica Extracts

The fruits of P. emblica were harvested during the months of March and April 2020, from the local garden in Dahu Township, Miaoli District (Figure 1). The plant material was authenticated as being P. emblica L. (TS02) by the Agricultural Research and Extension Station, Council of Agriculture, Miaoli District. P. emblica fruit without seeds was oven-dried at 45 °C for 48 h, then crushed by using a pulverizer and sieved through a 35-mesh sieve. The powders of P. emblica were placed in dark glass vials. During hot water extraction, P. emblica was kept in a 90 °C hot water bath for 1 h with intermediate mixing. For ethanolic extraction, P. emblica was stirred on a magnetic stirrer in a 1 L screw cap bottle for 12 h at room temperature. The extract was filtered through a filter paper (Toyo Roshi Co., Tokyo, Japan) and the residue was extracted once again with 1 L hot water (100 °C) and ethanol (50% and 95%) as described above. The filtrate was concentrated in a rotary evaporator and dried by lyophilization (Model FDU540, Eyela Co., Tokyo, Japan). The lyophilized powder was stored at −20 °C until use. The yields of hot water, 50% ethanol, and 95% ethanol extracts were 43.7 ± 2.7, 33.0 ± 1.3, and 27.0 ± 1.1%, respectively, on a dry weight basis of P. emblica fruits [21].

2.3. Antioxidant Activities

2.3.1. DPPH Radical Scavenging Activity

DPPH radical scavenging activity was estimated as described by Li et al. (2020) [23]. An aliquot of 0.1 mL samples of trolox was mixed with 0.4 mL of 100 mM Tris-HCl buffer (pH 7.4), then added to 0.5 mL of a DPPH solution (500 µM in ethanol). After shaking vigorously for 20 s, the mixture was left in the dark at room temperature for 20 min. The absorbance of the mixture was measured by spectrometry at 517 nm. The ability to scavenge DPPH radicals was calculated follows:

$$\mathrm{DPPH}\mathrm{scavenging}\mathrm{activity} (\%)=(1-\frac{\mathrm{absorbance}\mathrm{of}\mathrm{sample}\mathrm{at}517\mathrm{nm}}{\mathrm{absorbance}\mathrm{of}\mathrm{control}\mathrm{at}517\mathrm{nm}}\times 100)$$

2.3.2. Reducing Power Activity

The reducing power of the tested samples was determined as described by Le et al. (2021) with some modifications [24]. The reaction mixture, containing an equal volume (0.25 mL) of the samples or standard (ascorbic acid), 0.2 M phosphate buffer (pH 6.6), and 1% potassium ferricyanide was incubated at 50 °C for 20 min. After cooling, the mixture was mixed with 0.25 mL of 10% trichloroacetic acid and then centrifuged at 4500× g for 10 min. The suspension (0.5 mL) was mixed with distilled water (0.5 mL) and a freshly prepared 0.1% ferric chloride solution (0.1 mL). The absorbance was measured at 700 nm by spectrometry. The control was prepared in a similar manner excluding samples. A high absorbance of the reaction mixture indicated high reducing power.

2.3.3. Lipid Peroxidation Inhibition Activity

Lipid peroxidation inhibition of samples was measured as described by Chen et al. (2008) with some modifications [25]. Liposomes were prepared from soybean lecithin by using a chloroform-methanol system and further dried with nitrogen gas. An aliquot of the samples and 20 mM sodium phosphate buffer (165 µL, pH 7.2) were mixed with 300 µL liposome. Liposome peroxidation was induced by FeCl₃-ascorbate. The formation of malondialdehyde-thiobarbituric acid was used as an index of lipid peroxidation by measuring the absorbance at 535 nm by spectrometry. The inhibitory activity against liposome peroxidation was calculated as follows:

$$\mathrm{Inhibition}\mathrm{activity} (\%)=(1-\frac{\mathrm{absorbance}\mathrm{of}\mathrm{sample}\mathrm{at}535\mathrm{nm}}{\mathrm{absorbance}\mathrm{of}\mathrm{control}\mathrm{at}535\mathrm{nm}}\times 100)$$

2.4. Anti-Inflammation Activity and Neuroprotection

2.4.1. NO Inhibition

RAW264.7 cells were cultured in DMEM containing 10% heat-inactivated FBS with 100 units/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO₂. RAW264.7 cells (100 μL) were loaded in a 96-well cell culture microplate (3 × 10⁵ cells/mL) and incubated at 37 °C in 5% CO₂ for 24 h. Samples with or without 1 μg/mL LPS were added and further incubated for 24 h. The production of NO was determined as described by Qureshi et al. (2012) and Li et al. (2020) with some modifications [26,27]. In brief, the supernatant of cell cultures (100 μL/well) was transferred into a fresh 96-well microplate, and 100 μL of Griess reagent (1% sulfanilamide dissolved in 5% H₃PO₄ and 0.1% N-(L-naphthyl)-ethylene diamine dihydrochloride) was added. After incubation for 10 min in the dark at room temperature, the absorbance of the mixture was measured at 540 nm by using an ELISA reader (Fluostar optima, BMG labtech, Ortenberg, Germany). DMEM and L-Name (a known NO synthase inhibitor) were used as the negative and positive controls, respectively. The amount of nitrite in the culture supernatant was calculated from a standard curve of sodium nitrite that was freshly prepared with deionized water. The percentages of NO inhibition were calculated as follows:

$$\mathrm{NO}\mathrm{inhibition}(\%)=\frac{{\left({\mathrm{NO}}_2{}^{-}\right)}_{\mathrm{control}}-{\left({\mathrm{NO}}_2{}^{-}\right)}_{\mathrm{sample}}}{{\left({\mathrm{NO}}_2{}^{-}\right)}_{\mathrm{control}}}\times 100$$

2.4.2. COX-2 Inhibition

All samples were tested for their ability to inhibit COX-2 activity by using a COX-(human recombinant)-inhibitor screening kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions [28]. The kit was provided in 10 K phials performed in Tris-HCl 80 mM, 1% Tween 20, and its purity reached 95%. Enzyme unit (EU) means the quantity of enzyme required to cause the transformation of 1 µmol of substrate per minute, measured at 610 nm. Phials stayed on ice and in a dark place while the experiment was conducted. The enzyme substrate used was AA, the pro-aggregant agent of the previous assays. A chromogenic procedure was established on the oxidation of N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) while the PGG2 reduced to PGH2, as determined by the COX-2 activity. AA was transformed into PGG2 in the platelet aggregation pathway, and this prostacyclin was immediately reduced to PGH2 by COX-2. Thus, TMPD was proportionally oxidized to the enzyme activity. P. emblica fruit extracts dissolved in DMSO were tested at the same concentrations as in prior studies (0.33, 0.83, 1.0, 1.25, and 1.5 mM) and were added to the wells, with 100 EU of pure enzyme and AA, the enzyme substrate. Indomethacin at 0.00257, 0.006, 0.013, 0.019, and 0.02 mM concentrations was the positive control. Absorbance was obtained in a Biotek ELx800 Absorbance Microplate Reader (Izasa Scientific, Madrid, Spain) at a wavelength of 610 nm. The experimental studies were carried out in solutions containing the pure enzyme incubated with the test compounds and AA as the enzyme substrate. Absorbance produced by TMPD was measured at 610 nm in a Biotek ELx800 Absorbance Microplate Reader (Izasa Scientific, Spain). The percentage inhibition of COX-2 was calculated as follows:

$$\mathrm{Cox}-2\mathrm{inhibition}(\%)=\frac{PGc-PGs}{PGc}\times 100$$

where PGc = prostaglandin concentration of 100% initial activity and PGs = prostaglandin concentration of the sample.

2.4.3. PC12 Cell Protection against H₂O₂ Damage

PC12 cells were maintained in DMEM supplemented with 10% heat-inactivated horse serum, 5% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cell lines were incubated at 37 °C in a humidified atmosphere with 5% CO₂, and the medium was changed every other day. The protection effect of the tested samples against H₂O₂-induced damage in PC12 cells was determined as described by Hwang and Yen, (2008) and Rajalakshmi, Vijayakumar, and Praseeth (2019) [29,30]. PC12 cells (100 μL) were seeded in a Corning^®® CellBIND^®® Surface 96-well plate (Corning, NY, USA) at 3 × 10⁵ cells/mL, followed by incubation at 37 °C in 5% CO₂ for 24 h. Freshly prepared hydrogen peroxide (H₂O₂, final concentration 300 µM) and tested samples (10–100 μg/mL) were added for 24h treatment. The activity of the treated PC12 cells was determined by the MTT method.

2.5. Total Flavonoid Content

Total flavonoid content of the sample was determined as described by Wang et al. (2008) [31] with modification. A 2.5 g sample was allocated in a Soxhlet extractor and refluxed with methanol for 12 h at 85 °C. The extractant was evaporated to dryness using a rotary evaporator at 35 °C, then dissolved with methanol for analysis. A 1 mL amount of the extractant was added with 0.3 mL of 5% sodium nitrite (NaNO₂) in a 10 mL volumetric flask, and the mixture was left standing at room temperature for 6 min. A 0.3 mL amount of 10% aluminum nitrate was added to the mixture to stand at room temperature for a further 6 min, then 4 mL of 1 N sodium hydroxide (NaOH) and methanol to volume was added. After incubation for color development at room temperature for 15 min, the absorbance was measured at wavelength 510 nm. Total flavonoid content was expressed as quercetin equivalent (mg QE g⁻¹ dry weight).

2.6. Total Phenolic Content

Total phenolic content was determined by the Folin–Ciocalteu reagent method with the gallic acid standard. Briefly, 20 μL of 10% P. emblica fruit extract solution was mixed with 100 μL Folin–Ciocalteu reagent. Then, 80 μL of 10% sodium carbonate compound was added after 5 min. The mixture was set at room temperature for 1 h. Finally, the absorbance was measured at 765 nm.

2.7. Statistical Analysis

Data are expressed as the mean ± SD (n = 3) and were analyzed by one-way ANOVA, followed by Duncan’s multiple range tests using the SAS 10.0 (SAS institute, Cary, NC, USA). Differences were considered significant at p < 0.05.

3. Results and Discussion

3.1. Antioxidant Activity

DPPH is a popular free radical used to determine antioxidant activity for many crude and purified samples [32]. The DPPH radical accepts an electron or hydrogen radical to become a stable diamagnetic molecule, which shows a characteristic absorption at 517 nm. The data in Figure 2A show the DPPH radical scavenging activity of P. emblica extracts. A lower IC₅₀ value corresponds to high DPPH radical scavenging activity. Among the four extracts (hot water, 50% and 95% ethanol extracts, and commercial), 95% ethanol extract had significantly higher DPPH radical scavenging activity than the other extracts. P. emblica commercial extract had the lowest radical scavenging activity as compared with hot water and the 50% and 95% ethanol extracts. Furthermore, all P. emblica extracts were more potent than Trolox (IC50 348.2 ± 6.6).

The reducing power compounds are electron donors and can act as primary and secondary antioxidants. Samples with reduction potential react with potassium ferricyanide (Fe³⁺) to form potassium ferrocyanide (Fe²⁺), then react with ferric chloride to form a ferric ferrous complex that has an absorption maximum at 700 nm [33]. A low concentration of samples with high absorbance at 700 nm indicates high reducing power activity. The reducing power of P. emblica fruit extracts (Figure 2B) indicated that 95% ethanol extract exhibited the highest reduction potential among the four extracts. The commercial extract had significantly lower reducing power than fresh P. emblica fruit extracts (i.e., hot water and ethanol extracts).

Lipid peroxidation is associated with aging and many diseases, such as atherosclerosis, asthma, Parkinson’s disease, kidney damage, and pre-eclampsia. In this study, we prepared liposome from soybean lecithin to study the inhibition of FeCl₃-ascorbate-mediated lipid peroxidation by P. emblica extracts. A low IC₅₀ value corresponds to high lipid peroxidation inhibition. For the lipid peroxidation inhibition activity of P. emblica extracts, 95% ethanol extract showed significantly higher inhibition of lipid peroxidation than the other extracts and commercial extracts (Figure 2C). The present results have demonstrated that the P. emblica extracts can protect the oxidative degradation of lipids by inhibiting FeCl₃-ascorbate-mediated lipid peroxidation.

The free radical oxidation of polyunsaturated fatty acids in biological systems is known as lipid peroxidation. Phospholipid is a major component of cell membranes, and lipid peroxidation, caused by several reactive oxygen species, such as hydroxyl radical and hydrogen peroxide, leads to cell death [34]. P. emblica extracts showed good activity in DPPH radical scavenging, reducing power and lipid peroxidation inhibition. Lipid peroxidation is highly correlated with antioxidant protection in aging; lipid peroxidation increases with decreasing antioxidant protection in a biological system [35]. The ethanolic extracts of P. emblica demonstrated higher activity for scavenging DPPH radicals, reducing power, and inhibiting lipid peroxidation than the commercial and hot water extracts. This result agrees with Liu et al. (2008) and Luo et al. (2011), who reported that a phenolics compound isolated from P. emblica exhibited strong radical scavenging activity, good potency to chelate Fe²⁺, and good inhibition of lipid peroxidation [3,4]. These phenolics compounds included gallic acid, ellagic acid, mucic acid 1,4-lactone 3-O-gallate, isocorilagin, chebulanin, chebulagic acid, and mallotusinin, which could be the sources of antioxidant properties of ethanolic extracts. Research carried out at the Niwa Institute of Immunology in Japan demonstrated the remarkable scavenging capacity of P. emblica. The results suggested a significant activity of P. emblica against SOD, an enzyme that can help in breaking down potentially harmful reactive oxygen species in cells, which might prevent damage to tissues [36].

3.2. Anti-Inflammatory Activity

The inflammation process involves many complex pathways that lead to infiltration of neutrophils, histamines, serotonin, bradykinins, NO, and prostaglandins. NO is a vital molecule in immuno-signaling pathways. Excess NO production could cause many pathological abnormalities and disorders, such as severe inflammation, sepsis, cardiovascular injury, stroke, and oxidative stress [37]. P. emblica extracts showed dose-dependent NO inhibition in LPS-stimulated RAW264.7 cells (Figure 3). RAW264.7 cell viability estimated by MTT assay was >80% after sample treatment (data not shown). At a 10 µg/mL concentration, all extracts exhibited similar results in NO inhibition. However, at 50 and 100 µg/mL concentration, the 95% ethanolic extract showed significantly higher NO inhibition (up to 49.1%) than other extracts. The commercial extract of P. emblica had significantly lower NO inhibition than hot water and ethanolic extract. Our data support previous reports of the anti-inflammatory activity potential of P. emblica [38].

Cyclooxygenase (COX) enzyme is known to covert arachidonic acid to prostaglandin. Many COX-2 inhibitors, including steroidal and non-steroidal inhibitors, can interfere with this conversion reaction. Non-steroidal COX-2 enzyme inhibitors are used to treat pain, fever, and inflammatory diseases [39]. Three extracts of P. emblica exhibited significantly higher COX-2 inhibition than the commercial extract (Figure 4). At 10 µg/mL concentration, the hot water extracts showed the highest COX-2 inhibition (46.4%) among the three extracts. The 95% ethanol and hot water extracts showed similar COX-2 inhibition (53.4% and 51.0%) at 100 µg/mL concentration. Inhibition of COX-2 may control inflammation in allergy and other inflammatory diseases [40]. According to several traditional medicinal systems, such as Chinese herbal medicine, Tibetan medicine, and Ayurvedic medicine, P. emblica is known for its high antioxidant properties but also for easing tooth pain and inflammation [1]. Our data support the traditional applications of P. emblica for its anti-inflammatory activities by inhibiting NO production in macrophage cells and COX-2 enzyme. P. emblica (50 mg/kg and 250 mg/kg body weight) extracts reduced the elevated levels of these enzymes and inhibited the induction of fibrosis in mice. Thus, this inhibition of cell cycle regulatory enzymes shows that P. emblica might exert anti-tumor properties [41]. In another study, a cytotoxic agent, an acylated glucoside of apigenin, has been reported from P. emblica [42].

3.3. Neuroprotection Effect

Hydrogen peroxide (H₂O₂) is the most prominent reactive oxygen species and is involved in cell death by the apoptosis pathway [43]. The rat adrenal pheochromocytoma cell line (PC12) was designed as a model neuronal-like cell line to investigate neurodegenerative diseases. The PC12 cell protection model has been used in vitro by many researchers to test the ability of drugs for neurodegenerative diseases [44,45]. P. emblica extracts conferred PC12 cells protection against H₂O₂-induced cell death (Figure 5). All extracts of P. emblica showed a dose-dependent and similar protection effect on H₂O₂-induced PC12 cell death. Hot water and ethanol extracts showed higher percentages of PC12 cell protection than commercial extracts. P. emblica hydroalcoholic extracts had neuroprotective effects on kainic acid-induced seizures in rat, which could be due to their antioxidant and anti-inflammatory activity [46]. Our results show good antioxidant and anti-inflammatory activities of P. emblica extracts and also a neuroprotective effect against oxidative damage.

3.4. Total Phenolic and Flavonoid Contents

We expressed total phenolic and flavonoid contents of P. emblica fruit in gallic acid and rutin equivalents (

Table 1. Total phenol and flavonoid content of Phyllanthus emblica L. fruit extracts.

Extract Samples	Total Phenol (mg GA/g Extract)	Total Flavonoid (mg RU/g Extract)
Hot water	196.9 ± 2.1 c	2.40 ± 0.01 c
50 % Ethanol	276.0 ± 0.8 b	3.40 ± 0.01 b
95 % Ethanol	354.5 ± 2.5 a	4.19 ± 0.11 a
Commercial	172.2 ± 4.7 d	4.17 ± 0.11 a

Data are means ± SD of 3 independent experiments. Values in the same column with different superscript letters (^{a, b, c} and ^d) indicate significant difference (p < 0.05) by Duncan’s multiple range test. GA, gallic acid; RU, rutin.

). The 95% ethanol extract of P. emblica had the highest phenol and flavonoid contents among the extracts including the commercial one. Luo et al. (2011) extracted and identified phenolic compounds from P. emblica and obtained several antioxidant compounds including gallic acid, ellagic acid, mucic acid 1,4-lactone 3-O-gallate, isocorilagin, chebulanin, chebulagic acid, and mallotusinin [4]. The total phenolic contents of P. emblica extracts in our study were higher than in previous studies, which might be due to the use of fresh fruits for extraction in our study [3,47].

4. Conclusions

The extracts of P. emblica showed potential antioxidant activities, including scavenging of DPPH radicals, reducing potential and inhibition of lipid peroxidation, as well as high anti-inflammatory activities with NO and COX-2 inhibition, probably because of the high content of total phenols and flavonoids. The neuroprotection bioactivity observed with hot water and ethanol extracts gave higher percentages of PC12 cell protection against oxidation than the commercial extract. The 95% ethanol extract of P. emblica had higher antioxidant and anti-inflammatory activity and neuroprotection than the other extracts, and is recommended for application in daily health drinks or food. For the next step, we may conduct a more in-depth study of the association between P. emblica extracts and vision-protection in Chinese medicine, and identification of the bio-active components by using LC-MS.