key: cord-0028405-u5hazn2d
authors: Abd Elkader, Akwam M.; Labib, Salah; Taha, Taha F.; Althobaiti, Fayez; Aldhahrani, Adil; Salem, Heba M.; Saad, Ahmed; Ibrahim, Faten M.
title: Phytogenic compounds from avocado (Persea americana L.) extracts; antioxidant activity, amylase inhibitory activity, therapeutic potential of type 2 diabetes
date: 2021-11-24
journal: Saudi J Biol Sci
DOI: 10.1016/j.sjbs.2021.11.031
sha: b14fff9ab7086932cb71b559a0c86036c1cc43d8
doc_id: 28405
cord_uid: u5hazn2d

Diabetes is a worldwide public health disease. Currently, the most effective way to treat diabetes is to mitigate postprandial hyperglycemia by inhibiting carbohydrate hydrolysis enzymes in the digestive system. Plant extracts are rich in bioactive compounds, which can be used in diabetes treatment. This study aims to evaluate the polyphenols content in ethanolic extracts of avocado fruit and leaves (Persea americana Mill.). Additionally, their antioxidant activity using DPPH, while the inhibition ability of α-amylase was examined by reacting different amounts of the extracts with α-amylase compared to acarbose as standard inhibitor. The active compounds were detected in the extracts by LC/MS. The obtained results showed that the leaf extract recorded a significant content of total phenolic compounds compared to the fruit extract (178.95 and 145.7 mg GAE /g dry weight, respectively). The total flavonoid values ​​ranged from 32.5 to 70.08 mg QE/g dry weight of fruit and leaves extracts, respectively. Twenty-six phytogenic compounds were detected in leaf and fruit extract by LC/MS. These compounds belong to fatty acids, sterols, triterpenes, phenolic acids, and flavonoids. The antioxidant activity of the extracts is due to the exist of phytogenic compounds, i.e., polyphenols and flavonoids. The antioxidant activity increased in a concentration dependant manner. Avocado fruit extract (1000 µg/mL) scavenged 95% of DPPḢ while leaf extract rummaged 91.03% of free radicals compared with Vit C and BHT. Additionally, higher α-amylase inhibitory activity was observed in fruit extract than the leaf extract, where the fruit and leaf extract (1000 μg/ml) inhibited the enzyme by 92.13% and 88.95%, respectively. The obtained results showed that the ethanolic extracts of avocado could have a significant impact on human health due to their high content of polyphenols.

The use of natural extracts with pharmacological activity has gain great interest because they have therapeutic potential in treatment of different diseases with low side effects. Hyperglycemia increases the production of different active free radicals, which results in diabetic complications, such as oxidative damage of tissue and nephropathy, neuropathy, retinopathy, and memory disorder (Maritim et al., 2003) . Abnormal blood sugar levels are a symptom of diabetes, This is due to the immune system inability to create enough insulin (Petersmann et al., 2018) . Hyperglycemia occurs after feeding because of starch hydrolyzes by a-amylase and a-glucosidase to large amounts of glucose. The intestinal enzymes must be inhibited to treat type 2 diabetes (Wang et al., 2013) . Glucosidase is a one of digestive enzymes that degrade dietetic carbohydrates in simple monosaccharides. Glucosidase inhibitors, such as acarbose, reduce the speed of carbohydrate digestion and delays the absorption of carbohydrates from the digestive tract. Therefore, they can prevent the start of type 2 diabetes by reducing the level of postprandial glucose (Liu et al., 2011) . Avocado (Persea americana Mill.) is subtropical/tropical fruit, a member of Lauraceae and Persea family. It is a Mexican origin. It is extensively cultivated in the Central Region of the United State and consumed worldwide (Melgar et al., 2018) . It has become very popular recently and is often sold as a ''superfood" due to its unique nutritional content, phytochemical composition, and health benefits. (Segovia et al., 2018) . In addition to being used as food, is traditionally used for a variety of medicinal purposes, including lowering blood pressure, lowering blood sugar, antivirals, antidiarrheals, and cardiovascular disease (Cortés-Rojo et al., 2019) . Therefore, these fruits also have analgesic, anti-inflammatory, and antifungal properties (Jesus et al., 2015) . In general, it has been hypothesized that biomolecular oxidation can cause several health problems, including diabetes, pancreatitis, and ageing. (Finkel and Holbrook, 2000) . There is evidence that free radicals are to responsible for these potential threats. (Halliwell and Chirico, 1993) . Free radicals are unstable species because they have unpaired electron pairings with biological macromolecules. Free radicals are well recognized to play a crucial role in extraversion, mainly in organs as brain and pancreas. (Shulman et al., 2004) . The uncontrolled activity of ROS is related to health disorders, such as diabetes, hypertension, cancer, neurological illnesses, gastric ulcers, reperfusion therapy, arthritis, and inflammatory diseases. (Vajragupta et al., 2000) . Secondary metabolites seem to be the most analysed phytochemical group in fruits and vegetables (Domínguez-Avila et al., 2017) . The most abundant phenolic compounds in avocado were phenolic acids, flavonoids and tannins (Tremocoldi et al., 2018) . Avocado wastes have recently rekindled researchers' interest due to their high content of carbohydrates, lipids, protein, fiber, minerals, and other physiologically active components (Lara-Flores et al., 2018) . Organic acids, phenolic alcohol derivatives, flavonoids, procyanidins, terpenes, alkaloids, saponins, acetin, phytosterols, and other polar and non-polar chemicals are illustrations. LC-MS, GC-MS, and FTIR are the most devices which used to detect and quantify these physiologically active compounds (Alkhalaf et al., 2019) . Avocado leaves are a medicinal herb that are commonly utilised in extract formulations for therapeutic purposes as well as in folk medicine as a tea (Isaac, 2020) , This could be because of its diuretic characteristics. (Wright et al., 2007) . Rhamnetin, luteolin, rutin, quercetin, and apigenin are phytochemicals extracted from avocado leaves that can inhibit the progression of oxidative stressrelated disorders (Owolabi et al., 2010) . Avocado wastes are a rich source of several bioactive chemicals with significant nutritional, health, and pharmacological implications, the potential of avocado by-products were discussed in a recent review of (Coman et al., 2020) . Avocado bioactive compounds have been shown to reduce oxidative stress (Melgar et al., 2018) and inflammatory processes (Tremocoldi et al., 2018) , as well as modulate lipids, according to Pahua-Ramos et al., (2012) . This prospective study was designed to investigate the bioactive compounds included phenolic and flavonoids in avocado extracts by LC/MS, detecting their activities;

antioxidant activity, and a-amylase. Aiming to provide more results about biochemical structure of avocado extracts and study of the effect of these extracts as hypoglycemic agent.

Merck Company supplied the ethanol. Sigma-Aldrich Company provided the Folin-Ciocalteau-phenol reagent (FC), 1,1 diphenyl-2picryl-hydrazyl (DPPH), ascorbic acid, and butylated hydroxyl toluene (BHT). Sigma-Aldrich Company supplied a-amylase enzyme and acarbose inhibitor. All the other chemicals were of the highest commercial purity.

Avocado leaves and fruits (500 g) were dried in a vacuum oven (45°C) for 5 h. 100 g of the powder was homogenized in 1000 mL ethanol 70% (1:10, w/v) and stirred for 3 h at room temperature, then filtrated through (Whatman No.1) paper . A rotary evaporator was used to recover the solvent in the filtrate and the obtained free solvent extracts were adjusted to a concentration of 100 mg/mL and stored at 4°C for further analyses.

The total polyphenols (TP) were evaluated according to Saad et al, (2021b) . In brief, 50 mL of fruit and leaves avocado extracts (500 mg/mL) was mixed with 50 mL of diluted Folin-Ciocalteu reagent and 50 mL Na 2 CO 3 (7.5%) in microtiter plate wells, and incubated at room temperature for 60 min, then the absorbance was measured at 760 nm using microtiter plate reader (BioTek Elx808, USA). The total polyphenols content was presented as mg gallic acid equivalent/mL of solutions.

Total flavonoids content (TF) was estimated as per Saad et al, (2021c) . 20 mL of of fruit and leaves avocado extracts (500 mg/ mL) was mixed with 20 mL of sodium nitrite (5%) in microtiter plate wells and 20 mL of ethanolic AlCl3 (10%) was added. The plate was incubated at room temperature for 10 min. 50 mL of NaOH was added to stop the reaction. the absorbance was measured at 450 nm, and TF was expressed as mg quercetin equivalent per mL of each solution.

The free radical scavenging activity of polyphenolic compounds was measured using 1, 1-diphenyl-2-picryl-hydrazil (DPPH ) according to the method of (Wang et al., 2008; El-Saadony et al, 2020 ) DPPH (0.1 mM) was prepared in methanol. Successive concentration from avocado fruit and leaves extracts and standard materials; BHT and Ascorbic Acid were prepared as 100, 250, 500, 750, and 1000 mg/ml in methanol. 1 mL of DPPH Solution of 3 mL of each concentration was added, then vigorously stirred up the mixture and permitted to stand 30 min at room temperature. The same procedure was used to test checks, except for samples of methanol. A spectrophotometer read the absorption at 517 nm (Jasco, serial No. C317961148, Japan). According to following equation DPPH radical scavenging activity was calculated: Radicalscavengingactivity % ð Þ ¼ ðAbs:control À Abs:sampleÞ ðAbs:controlÞ x100

Where A bs control was the absorbance of the control and A bs sample was the absorbance in the presence of the sample of Avocado extracts (fruits and leaves).

The hydrolysis of 1,4 glycosidic bonds in starch and other similar carbohydrates is catalysed by -amylase (EC 3.2.1.1). In particular, a-amylase participates in glucose digestion and is considered a key enzyme that can control postprandial hyperglycemia. The assay's principle is based on the fact that reducing sugars have the ability to reduce a variety of reagents; chemically, they form an aldehyde or ketone in basic solution, and the aldehyde group of glucose converts DNS to its reduced form (3-amino-5nitrosalicylic acid), which results in a change in the amount of light absorbed at 540 nm. The proportion of each extract's -amylase inhibitory activity values was obtained using this method, as follows: The sample (100 lg/mL) solution was prepared using methanol (HPLC analytical grade), filtered using a membrane disc filter (0.2 lm) then subjected to LC-ESI-MS analysis. 10 lL of sample was injected into the UPLC instrument equipped with reverse phase C-18 column. The sample was eluted using gradient elution. The parameters for analysis were carried out using negative ion mode as follows: source temperature 150°C, cone voltage 30 eV, capillary voltage 3 kV, desolvation temperature 440°C, cone gas flow 50 L/h, and desolvation gas flow 900 L/h. The data were detected in 100-1000 m/z mass spectra range and processed using the Maslynx 4.1 software, then identified by comparing its retention time (R t ) and mass spectrum with reported data (Bakr et al., 2021) .

One way ANOVA in SPSS 16 was used to conduct statistical analysis. The results were obtained as a mean standard deviation (SD). The significance of differences between means was assessed at p ‹ 0.05 using post hoc Duncan's test.

The higher polyphenol and flavonoids contents were detected in fruit and leaves ethanolic extracts. It can be seen from the data that the leaves' extract reported significantly more than Fruit extract it was 178.95 and 145.7 mg GAE acid/g DW, respectively. The values of flavonoids contents ranged from 32.5 to 70.08 mg QE /g DW for fruit and leaves ethanolic extracts, respectively. Table 2 shows the values of the a-amylase inhibitory activity affected by avocado fruit and leaves ethanolic extracts compared with acarbose inhibitor, both fruit and leaves extracts recorded lower values as 21.00 ± 0.30, 37.98 ± 0.52, and 51.38 ± 0.21%, and 18.67 ± 0.36, 30.69 ± 0.20 and 42.51 ± 0.51 %, respectively at concentrations 100, 250, and 500 lg/mL. while the avocado fruit extract had more inhibitory effects on a-amylase at high concentrations of (750 and 1000 lg/mL) than the avocado leaves and (18) 

The natural products of plant origin are mainly quarries to find promising reading candidates and play an ordering role in the next drug development program (Sharifi-Rad et al., 2018) . Availability, low cost, and minimal side effects are especially the main key player of all treatments available in rural areas (Mansouri et al., 2015) . In addition, many plants are released from undesirable side effects and provide a rich biological source of biologically active chemical products having a powerful pharmacological behavior (Sharifi-Rad et al., 2018) . Many plants are considered a basic source of powerful antidiabetic medications. In developing countries, particularly medicinal plants deal with diabetes and exceed the burden of traditional drugs to the population (Arumugam et al., 2013) . Recently, it is recommended to treat diseases such as diabetic containing diabetes using medicinal plants (Mansouri et al., 2015) , which including bioactive compounds, flavonoids, terpenoids, saponins, carotenoids, alkaloids, glycolic, etc. act as antidiabetic agents (Durazzo et al., 2018) . Natural products of plant origin are rich in bioactive compounds with antioxidant (Saad et al., 2020a; El-Saadony et al., 2020; Abdel-Moneim et al, 2021) and antimicrobial activity (El-Tarabily et al, 2021) . They are used as food additives for preserving juices (El-Saadony et al, 2021a), meat (Saad et al, 2020b; El-Saadony et al, 2021b) , bakery (Saad et al., 2015; Saad et al, 2021d) , and dairy (El-Saadony et al, pounds (i.e, polyphenols, carotenoids, lignans, coumarin, glycosinological, etc.) , resulting in possible beneficial properties of each plant matrix, which can be represented a first step for biological understanding. The antigram effects resulting from behavioral and beneficial activity plants generally occur by increasing insulin secretion, or due to the ability to improve pancreatic tissue performance carried out by reducing the intestinal absorption of glucose. The adverse effects of these episodes are significant and include subsequent Diabetes mellitus (DM) problems. Because ROS has been linked to the disease's aetiology, it is possible to use antioxidants for the logical technique for managing and expanding its potential burden (Swelum et al., 2020) . It was therefore proven that antioxidants prevent the demise of the pancreatic beta cells through inhibition of automated oxidation reactions and therefore regulate diabetes progression towards complications (Liu et al., 2007) . Synthetic antioxidants, on the other hand, are restricted in their usage due to their carcinogenicity, while plant antioxidants degrade at a much lower level of toxicity. The plants containing a natural antioxidant retain the function of the beta cell and prevent combined Diabetes mellitus (DM) (Aslan et al., 2010) . This study demonstrates the antioxidant and a-amylase inhibitory activity of avocado fruit and leaves extracts. Alpha-amylase is a key enzyme for starch digestion and plays an important role in determining the amount of glucose released. Inhibition of enzyme activity is considered a potential method to control starch digestion and regulate postprandial hyperglycemia in diabetic patients. It is crucial in the creation of nutritional health products as well as the treatment of diabetes. The pathogenesis of diabetes is linked to oxidative free radicals (Baynes and Thorpe, 1999) . LC/MS analysis of the fruit extract of the avocado Persea americana Mill was conducted because of its highest inhibitory effect as antioxidants and its ability to inhibit the activity of the enzyme alpha-amylase the results revealed presence of unsaturated and saturated fatty acids, sterol, triterpenes, flavonoids, and phenolic compounds. Flavonoids have dominated in Lauraceae which Persea americana is a member. Kaempferol, apigenin and isorhamentin glycosides were the main flavaniods in this family (Owolabi et al., 2010) and (Tremocoldi et al., 2018) reported that Persea americana Millis contains Anthocyanins and catechin. (Wang et al., 2020) reported saturated and unsaturated fatty acids i.e., linolenic acid, Palmitoleic acid , linoleic acid, Palmitic acid, Oleic acid, Stearic acid in avocado plants at . Cycloartenol (Peak 15) ; Avenasterol (16) ,Campasterol (18); ɣ-Tocopherol (17) ; a-Tocopherol (20) all these compounds were previously reported in the plant (Ramos-Aguilar et al., 2019) . The presence of these compounds may be a reasonable cause of the hypoglycemic potential observed in this study. It has been shown to have anti-diabetic characteristics by regenerating pancreatic cells and suppressing carbohydrate metabolism enzyme activity (Kwon et al., 2008) ; (Chika and Bello, 2010) . C. Droserifolia aqueous extract is traditionally used to treat hyperglycemia, according to (Abdel Motaal et al., 2020) , Multiple in vitro and in vivo investigations have revealed anti-diabetic potential. The flavonol glycosides accounted for 78 % of the water extract, and there were five main peaks. These 5 flavonol glycosides have been demonstrated to inhibit intestinal enzymes involved in the hydrolysis of polysaccharides to glucose in vitro tests. Furthermore, by inhibiting aldose reductase and reducing oxidative stress, which is linked to the aetiology of diabetes complications, these flavonol glycosides can prevent insulin resistance. Generally, the attributes exhibited of avocado fruit extract are related to its phenols and flavonoids, which are recognized for having antioxidant capabilities. Flavonoids can be a natural phenolic compound of the highest diversity and widely distributed nature compounds (Ward, 1990) . Its wide spectrum chemical and biological activities have been reported by (Prasad et al., 2009) , including free radical scavenging. Flavanol has similar characteristics (Miliauskas et al., 2004) , and it has been found to be an effective hypoglycemic agent (Kim et al., 2004) and had high alpha-glucosidase and alpha-amylase inhibitory effects (Li et al., 2018) .

The general clarification of this study was LC/MS, antioxidants, total phenols, and total ethanol extract flavonoids, from avocado extracts, which made its application as an antidiabetic agent credible. In the treatment of diabetes, it is very important to highly use affinity plant inhibitors to regulate the activity of a-amylase. It promotes the use of a therapeutically significant natural plant because of its availability and relative safety (Persea americana Millis).

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.