A significant by-product of the industrial processing of pistachios: shell skin – RP-HPLC analysis, and antioxidant and enzyme inhibitory activities of the methanol extracts of Pistacia vera L. shell skins cultivated in Gaziantep, Turkey

Abstract

The aim of this study is to evaluate the antioxidant and enzyme inhibitory activities of the methanol extracts of immature and mature shell skins of Pistacia vera L. As well as biological activity tests, phytochemical compositions of the extracts were also investigated. The total phenolic and flavonoid content was determined in addition to the amounts of gallic acid, protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, caffeic acid, (−)-epicatechin, syringic acid, p-coumaric acid, hesperidin, quercetin, kaempferol, and apigenin. The immature shell skin was found to be rich in both phenolic and flavonoid compounds (52.29 mg of gallic acid equivalent (GAE) per g of extract and 16.78 mg of rutin equivalent (RE) per g of extract). In immature shell skins, the amounts of protocatechuic acid (4335 μg per g of extract), p-hydroxybenzoic acid (12 925 μg per g of extract), p-coumaric acid (120 μg per g of extract), quercetin (620 μg per g of extract), and apigenin (190 μg per g of extract) were higher than those of the mature one. In parallel to these findings, immature shell skins exhibited a higher antioxidant activity in all test systems than that of the mature one. The samples did not show any inhibitory activity on butyrylcholinesterase and α-glucosidase. Mature shell skins exhibited considerable inhibitory activity on acetylcholinesterase (2.15 mg of galantamine equivalent (GALAE) per g of extract). The tyrosinase inhibitory activity of the mature shell skin was also found as 3.14 mg kojic acid equivalent (KAE) per g of extract. The immature shell skin remained inactive on this enzyme. The samples also showed remarkable inhibitory activity of α-amylase.

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1. Introduction

With the development of chromatography techniques, scientists have begun to focus on the isolation of biologically active secondary metabolites produced by plants, which are the major sources of bioactive substances. As is well known, the prices of medical services and pharmaceutical preparations are very high and people all over the world do not benefit equally from these services.¹ Therefore, people try to receive their therapeutic requirements from medicinal plants before getting medical help.

Plants synthesize a wide variety of phenolic compounds, such as phenolic acids, flavonoids, vitamins, carotenoids, anthocyanins, alkaloids, etc. Particularly, phenolic acids and flavonoids have remarkable biological properties such as antioxidant, antimicrobial, antiviral, photoprotective, etc.^2,3 Plants and plant components rich in natural antioxidants have been proven to have strong protective activity against the destructive effects of oxidative stress.^4–6

Pistachio (Pistacia vera L.), a member of the Anacardiaceae family, is native to the arid zones of Central and west Asia and distributed throughout the Mediterranean Basin.⁷ According to geographical literature, Turkey is known as the gene center of pistachios. The main pistachio producers in the northern hemisphere are Iran, United States and Turkey. In Turkey, pistachio is cultivated mainly in the Southeast Anatolian region (especially in Gaziantep, Şanlıurfa, Adıyaman, Kahramanmaraş, and Siirt locations). It is mainly cultivated in arid, rocky and sloping lands that are not conducive to the growth of other crops.^8,9

According to the results of several studies, pistachios have been proven to have various groups of valuable phytochemicals such as anthocyanins, flavan-3-ols, proanthocyanidins, flavonols, isoflavons, flavanones, stilbenes and phenolic acids.¹⁰ These phytochemicals have excellent biological activities.¹¹ For example; anthocyanins, which are among the main constituents of the shell skin of pistachios, have been shown to possess antioxidant, anti-inflammatory, anticarcinogenic^12,13 and antiangiogenic activities.¹⁴ Catechins present in the same tissue of pistachios have been shown to decrease the oxidation of low-density lipoproteins (LDL)^15–17 and thus prevent cardiovascular diseases.¹⁸ In addition to these findings, isoflavones have been proven to act as partial agonists on estrogen receptors.¹⁹

Approximately 60–70% of pistachios are consumed as nuts and the rest are used in dessert, cake, ice cream and confectionery industries worldwide. Pistachios cultivated in Turkey are especially preferred on world markets due to their aroma, color and taste. In Turkey, the average annual production volume of pistachios is approximately 100 000 tons. On average, 2500–3000 tons of the pistachios produced in Turkey are exported annually and approximately one million dollars of revenue is earned from this export.^8,9

In Turkey, during the industrial processing of pistachios, approximately 3% of the total production (an average of 3500–4000 tons annually) arises as a waste product that is known as the pistachio shell skin (or external skin) (Fig. 1 and 2). This material can lead to environmental pollution when released into the environment in uncontrolled conditions. Due to its high content of phenolic compounds, the shell skin of pistachios is likely to exhibit excellent biological activities and can be used as an alternative source of biologically active compounds.^8,9

Fig. 1 External and internal skins of P. vera.

Fig. 2 Mature ((A) – pink colored) and immature ((B) – green colored) shell skins of P. vera.

The aim of this study is to evaluate the antioxidant and enzyme inhibitory activities of the methanol extracts of immature and mature shell skins of Pistacia vera L. As well as the biological activity tests, phytochemical compositions of the extracts were also investigated. The total phenolic and flavonoid content was determined in addition to the amounts of gallic acid, protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, caffeic acid, (−)-epicatechin, syringic acid, p-coumaric acid, hesperidin, quercetin, kaempferol, and apigenin. By this study, we hope that the pistachio shell skin, which is an important byproduct of the world’s pistachio industry, can be used as an alternative source in the pharmacology industry for developing and producing new and/or alternative therapeutic agents.

2. Materials and methods

2.1. Chemicals

All standard compounds including phenolics and other standards were supplied from Sigma-Aldrich and their purities were all over 97%.

2.2. Preparation of extracts

Samples (15 g) were macerated with 300 mL of methanol at room temperature for 24 h. Methanol was then removed with a rotary evaporator at 40 °C. The extracts were stored at +4 °C until analyzed. The yields of the methanol extracts from mature and immature shell skins of P. vera were determined as 36.11 and 12.23% (w/w), respectively.

2.3. Phytochemical analyses

2.3.1. Quantification of phenolic compounds using RP-HPLC. Phenolic compounds were evaluated using reversed-phase high performance liquid chromatography (RP-HPLC, Shimadzu Scientific Instruments, Kyoto, Japan). Detection and quantification were carried out with a LC-10ADvp pump, a Diode Array Detector, a CTO-10Avp column heater, an SCL-10Avp system controller, a DGU-14A degasser and an SIL-10ADvp auto sampler (Shimadzu Scientific Instruments, Columbia, MD). Separations were conducted at 30 °C on an Agilent® Eclipse XDB C-18 reversed-phase column (250 mm × 4.6 mm length, 5 μm particle size). The eluates were detected at 278 nm. The mobile phases were A: 3.0% acetic acid in distilled water and B: methanol. For analysis, the samples were dissolved in methanol, and 20 μL of this solution was injected into the column. The elution gradient applied at a flow rate of 0.8 mL min⁻¹ was: 93% A/7% B in 0.1 min, 72% A/28% B in 20 min, 75% A/25% B in 8 min, 70% A/30% B in 7 min and the same gradient for 15 min, 67% A/33% B in 10 min, 58% A/42% B in 2 min, 50% A/50% B in 8 min, 30% A/70% B in 3 min, 20% A/80% B in 2 min and 100% B in 5 min until the end of the run. The phenolic compositions of the extracts were determined according to the method of Sarikurkcu et al.²⁰ Gallic acid, protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, (−)-epicatechin, syringic acid, vanillin, p-coumaric acid, ferulic acid, sinapinic acid, benzoic acid, o-coumaric acid, rutin, hesperidin, rosmarinic acid, eriodictyol, trans-cinnamic acid, quercetin, luteolin, kaempferol and apigenin were used as standards. Identification and quantitative analysis were done by comparison with standards. The amount of each phenolic compound was expressed as micrograms per gram of extract using external calibration curves, which were obtained for each phenolic standard.

2.3.2. Total phenolic and flavonoid content. The total phenolic and flavonoid content of the samples was determined by employing the methods given in the literature.²¹

2.4. Antioxidant activity

2.4.1. DPPH free radical scavenging activity. The effects of the samples on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were estimated according to the procedure in the literature.²²

2.4.2. Reducing power. The reducing power was investigated using cupric ion reducing (CUPRAC)²³ and ferric reducing antioxidant power (FRAP)²⁴ methods, as previously described in the literature.

2.4.3. Metal chelating activity on ferrous ions. Metal chelating activity on ferrous ions was evaluated using the method described by Aktumsek et al.²⁴

2.4.4. Total antioxidant activity using the phosphomolybdenum method. The total antioxidant activities of the samples were evaluated using the phosphomolybdenum method.²¹

2.5. Enzyme inhibitory activity

The enzyme inhibitory activities of the samples were determined using cholinesterase (ChE), α-amylase, α-glucosidase and tyrosinase enzymes by employing the methods given in the literature.²¹

2.6. Statistical analysis

All the assays were carried out in triplicate. The results were expressed as mean values and standard deviations (mean ± SD). Statistical differences between the extracts were analyzed using the Student t-test (α = 0.01). All analyses were carried out using the SPSS v22.0 software.

3. Results and discussion

3.1. Phytochemical composition

The total phenolic and flavonoid compositions of the methanol extracts of immature and mature shell skins of P. vera were evaluated in order to see the correlation between the biological activity potential and phytochemical composition (Table 1). Additionally, the amounts of twelve different phytochemicals were also determined using RP-HPLC analysis (Table 1).

Table 1 Phenolic components, total phenolics and total flavonoids of the mature and immature shell skins of P. vera (mean ± SD)^a and the analytical characteristics used for the determination of the phenolics

No.	Retention time (min)	Phenolic components	Concentration (μg per g of extract)		Analytical characteristics
			Mature shell skin	Immature shell skin	Linear range (mg per L)	R^2	LOD^b (mg per L)	LOQ^c (mg per L)
a Data marked with different superscripts within the same row indicate significant statistical differences (p < 0.01).b LOD, limit of detection.c LOQ, limit of quantification.d nd, not detected.e GAE, gallic acid equivalent.f RE, rutin equivalent.
1	5.2	Gallic acid	13 205 ± 396^a	9115 ± 403^b	0.20–25.0	0.9993	0.075	0.227
2	8.7	Protocatechuic acid	1000 ± 35^b	4335 ± 375^a	0.20–25.0	0.9991	0.086	0.260
3	12.3	(+)-Catechin	nd^d	1240 ± 49	0.90–113	0.9988	0.172	0.522
4	13.5	p-Hydroxybenzoic acid	7400 ± 212^b	12 925 ± 247^a	0.20–25.0	0.9994	0.007	0.020
5	15.1	Chlorogenic acid	nd	nd	0.35–45.0	0.9988	0.080	0.241
6	17.6	Caffeic acid	155 ± 11^a	125 ± 11^a	0.16–21.0	0.9993	0.054	0.162
7	19.1	(−)-Epicatechin	980 ± 42^a	630 ± 42^a	0.50–66.0	0.9990	0.170	0.514
8	19.9	Syringic acid	80 ± 1^a	70 ± 1^b	0.05–12.0	0.9995	0.030	0.090
9	20.8	Vanillin	nd	nd	0.08–10.0	0.9995	0.020	0.060
10	24.5	p-Coumaric acid	70 ± 4^b	120 ± 5^a	0.04–6.0	0.9996	0.066	0.199
11	27.8	Ferulic acid	nd	nd	0.12–17.0	0.9993	0.004	0.011
12	29.2	Sinapic acid	nd	nd	0.12–17.0	0.9993	0.017	0.053
13	33.8	Benzoic acid	nd	nd	0.85–55.0	0.9998	0.111	0.335
14	39.4	o-Coumaric acid	nd	nd	0.24–32.0	0.9988	0.023	0.069
15	44.1	Rutin	nd	nd	0.40–56.0	0.9989	1.113	3.373
16	49.7	Hesperidin	120 ± 3^a	95 ± 3^a	0.43–55.0	0.9992	1.080	3.280
17	54.9	Rosmarinic acid	nd	nd	0.02–7.0	0.9998	0.148	0.447
18	57.3	Eriodictyol	nd	nd	0.33–21.0	0.9998	0.140	0.410
19	65.9	trans-Cinnamic acid	nd	nd	0.02–7.0	0.9998	0.148	0.447
20	71.4	Quercetin	255 ± 11^b	620 ± 11^a	0.40–55.0	0.9999	0.013	0.040
21	74.3	Luteolin	nd	nd	0.13–17.0	0.9999	0.020	0.060
22	76.8	Kaempferol	nd	135 ± 11	0.05–15.0	0.9996	0.021	0.062
23	77.2	Apigenin	145 ± 5^a	190 ± 5^a	0.17–11.0	0.9997	0.034	0.104
Total phenolics (mg of GAE per g of extract)^e			33.65 ± 1.75^b	52.29 ± 0.84^a
Total flavonoids (mg RE per g of extract)^f			3.59 ± 0.07^b	16.78 ± 0.10^a

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As can be seen from the table, the methanol extract of the immature shell skin was found to be rich in both phenolic and flavonoid compounds (52.29 mg GAE per g of extract and 16.78 mg RE per g of extract, respectively). It is quite interesting to point out that the amount of total flavonoids was found to be approximately five folds higher in the immature shell skin extract than in the mature one.

In addition to the qualitative analysis, the amounts of gallic acid, protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, (−)-epicatechin, syringic acid, vanillin, p-coumaric acid, ferulic acid, sinapinic acid, benzoic acid, o-coumaric acid, rutin, hesperidin, rosmarinic acid, eriodictyol, trans-cinnamic acid, quercetin, luteolin, kaempferol and apigenin were also determined quantitatively using RP-HPLC analysis (Fig. 3 and 4). In the immature shell skin extract, the amounts of protocatechuic acid (4335 μg per g of extract), p-hydroxybenzoic acid (12 925 μg per g of extract), p-coumaric acid (120 μg per g of extract), quercetin (620 μg per g of extract), and apigenin (190 μg per g of extract) were higher than those of the mature one (Table 1). Additionally, the immature shell skin contained (+)-catechin and kaempferol at 1240 and 135 μg per g of extract concentrations, respectively. However, these two compounds could not be detected in the mature shell skin. As we understood from this finding, some phytochemicals might be eliminated during the maturation process of the tissue by turning into other compounds, which are not biologically active as the compounds of the immature tissue. On the other hand, gallic acid (13 205 μg per g of extract), caffeic acid (155 μg per g of extract), (−)-epicatechin (980 μg per g of extract), syringic acid (80 μg per g of extract), and hesperidin (120 μg per g of extract) were found at higher concentrations in the mature shell skin extract than in the immature one.

Fig. 3 Chromatographic profile of the chemical standards ((1) gallic acid, (2) protocatechuic acid, (3) (+)-catechin, (4) p-hydroxybenzoic acid, (5) chlorogenic acid, (6) caffeic acid, (7) (−)-epicatechin, (8) syringic acid, (9) vanillin, (10) p-coumaric acid, (11) ferulic acid, (12) sinapinic acid, (13) benzoic acid, (14) o-coumaric acid, (15) rutin, (16) hesperidin, (17) rosmarinic acid, (18) eriodictyol, (19) trans-cinnamic acid, (20) quercetin, (21) luteolin, (22) kaempferol, (23) apigenin).

Fig. 4 HPLC chromatograms of the methanol extracts of the mature (A) and immature (B) shell skins of P. vera.

As can be seen from the biological activity section of this paper, in general, the immature extract exhibited a higher biological activity potential than the mature one. Data obtained from the phytochemical analyses significantly supported the biological activity patterns of the shell skin samples.

As far as our literature survey could ascertain, several studies have been carried out on the phenolic profile of the skin tissue of pistachios. But all of these studies have mainly concerned the internal skin.^7,25,26 Therefore, the phenolic profile data presented for both the immature and mature shell skins (or external skins) of the pistachio could be assumed as the first report in the literature.

3.2. Antioxidant activity

Antioxidant activities of the methanol extracts of the immature and mature shell skins of P. vera were evaluated using five different test systems named as DPPH free radical scavenging, reducing power (CUPRAC and FRAP assays), metal chelating, and phosphomolybdenum assays.

The results obtained from the DPPH free radical scavenging effect assay are presented in Table 2. According to the data presented in the table, the radical scavenging potential of the immature shell skin was found to be superior to that of the mature one (171.35 mg of TE per g of extract). The results obtained from the shell skin samples were found to be different from the statistical point of view (p < 0.01).

Table 2 Radical scavenging (DPPH), reducing power (CUPRAC and FRAP), metal chelating and antioxidant (via the phosphomolybdenum method) activities of the mature and immature shell skins of P. vera (mean ± SD)^a

Assays	Mature shell skin	Immature shell skin
a Data marked with different superscripts within the same row indicate a significant statistical difference (p < 0.01).b TE, trolox equivalent.c EDTAE, disodium edetate equivalent.
DPPH (mg of TE per g of extract)^b	137.16 ± 3.50^b	171.35 ± 6.60^a
CUPRAC (mg of TE per g of extract)^b	175.58 ± 1.91^b	267.93 ± 13.81^a
FRAP (mg of TE per g of extract)^b	140.08 ± 15.35^b	255.23 ± 7.39^a
Chelating effect (mg of EDTAE per g of extract)^c	16.10 ± 0.70^a	17.54 ± 0.30^a
Phosphomolybdenum (mmol TE per g of extract)^b	1.35 ± 0.07^b	2.31 ± 0.01^a

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The DPPH free radical scavenging potential of the gums²⁷ and seeds²⁸ of P. vera have previously been reported elsewhere. None of these reports concentrated on the radical scavenging potential of the shell skin of the pistachio.

The reducing power potentials of the methanol extracts of immature and mature shell skins of P. vera were screened using CUPRAC and FRAP assays (Table 2). As can be seen from the table, the immature shell skin extract showed a higher reducing power than the mature shell skin extract. The activity values of the immature extract were found to be 267.93 and 255.23 mg of TE per g of extract in CUPRAC and FRAP systems, respectively. The ferric reducing antioxidant potential of the immature shell skin was found to be approximately two folds greater than that of the mature one in the FRAP assay. The results of the shell skin samples obtained from these two test systems were found to be different from the statistical point of view (p < 0.01).

The metal chelating activities of the methanol extracts of the mature and immature shell skins of P. vera were also evaluated on ferrous ions (Table 2). Unlike the results of other test systems, both samples showed almost equal activities in this assay (16.10 and 17.54 mg of EDTAE per g of extract, respectively). The metal chelating activities of the samples were found to be similar from the statistical point of view (p > 0.01).

Finally, the antioxidant activities of the samples were also studied using a phosphomolybdenum assay (Table 2). As observed in the results of the previous tests presented here, the phosphomolybdenum assay also demonstrated the superiority of the methanol extract of the immature shell skin for which the antioxidant activity was measured as 2.31 mmol TE per g of extract. The results of the mature and immature shell skins were found to be different from the statistical point of view (p < 0.01).

The antioxidant activity of pistachio nuts has widely been studied by many research groups. According to Tomaino et al.,⁷ pistachio nuts are among rich sources of phenolic compounds and have recently been ranked among the first 50 food products highest in antioxidant potential. On the other hand, some researchers have been particularly focused on the internal skin of the pistachio rather than the nuts, or have compared the skin with the nut in terms of their phytochemical profile and/or biological activity potential, since the internal skin has much more valuable phenolic compounds than the nuts.^7,25 But, the antioxidant activity of the shell skin (or external skin) of the pistachio has not previously been reported. Therefore, the data presented in this section could be assumed as the first report in the literature.

3.3. Enzyme inhibitory activity

Recently, the inhibition of key enzymes is considered as one of the most effective theories for the treatment of Alzheimer’s disease, diabetes mellitus and skin disorders. In this direction, many synthetic inhibitors have been produced (galantamine, acarbose or kojic acid), but they have adverse effects such as gastrointestinal disturbances and liver damage.^29,30 Thus, there is an increasing interest in finding natural enzyme inhibitors from plant materials in order to replace synthetic ones. Hence, the inhibitory activities of the methanol extracts of the mature and immature shell skins of P. vera were evaluated on acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, and tyrosinase (Table 3).

Table 3 Enzyme inhibitory activities of the mature and immature shell skins of P. vera (mean ± SD)^a

Assays	Mature shell skin	Immature shell skin
a Data marked with different superscripts within the same row indicate a significant statistical difference (p < 0.01).b GALAE, galantamine equivalent.c ACE, acarbose equivalent.d KAE, kojic acid equivalent.e na, not active.
Acetyl cholinesterase (mg of GALAE per g of extract)^b	2.15 ± 0.05^a	1.49 ± 0.03^b
Butyryl cholinesterase (mg of GALAE per g of extract)^b	na	na
α-Amylase (mg of ACE per g of extract)^c	3.72 ± 0.15^b	4.91 ± 0.15^a
α-Glucosidase (mg of ACE per g of extract)^c	na^e	na
Tyrosinase (mg of KAE per g of extract)^d	31.14 ± 2.33	na

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As can be seen from the table, the samples did not show any inhibitory activity on butyrylcholinesterase and α-glucosidase. The mature shell skin of P. vera exhibited higher inhibitory activity on acetylcholinesterase than the immature one (2.15 mg of GALAE per g of extract). Tyrosinase inhibitory activity of the mature shell skin was also found as 3.14 mg of KAE per g of extract. However, the immature shell skin remained inactive for this enzyme.

The samples also showed inhibitory activity on α-amylase. According to the data presented in Table 3, the inhibitory activities of the mature and immature shell skins of P. vera were found as 3.72 and 4.91 mg of ACE per g of extract, respectively.

As far as our literature survey could ascertain, the inhibitory activities of P. vera against butyrylcholinesterase, α-amylase, α-glucosidase, and tyrosinase have not previously been reported elsewhere. Therefore, the data presented on these enzymes could be assumed as the first report in the literature.

On the other hand, the acetylcholinesterase inhibitory activity of P. vera hydrolysates obtained by gastrointestinal enzymes has been studied by Li et al.³¹ According to this study, in vitro acetylcholinesterase inhibitory activities of the hydrolysates prepared by pepsin and trypsin digestion were measured as 0.87 mg per mL (IC₅₀). As can be seen from the details of the above-mentioned paper, the target sample is the nut of P. vera and the shell skin was left out of the scope. Therefore, the current study can also be accepted as the first report of the acetylcholinesterase inhibitory activity of the shell skin of P. vera.

4. Conclusions

As can be seen from the results presented here, in general, the immature shell skin of pistachios exhibited a greater activity than the mature one. This finding is also supported by the results of both qualitative and quantitative chromatographic analyses and statistical outputs. Moreover, it could be assumed that some other phytochemicals, which are not detected chromatographically, are likely to contribute to the biological activity of this material. Pistachio nuts with immature shell skins, in particular, are used to produce the world-famous “baklava”, which is a kind of sweet pastry with pistachio nuts. According to the results of our experiments, the immature shell skin of pistachios, arising from the industrial processing of pistachio nuts, exhibited remarkable antioxidant and enzyme inhibitory activity and therefore can be considered as a new source of valuable secondary metabolites, instead of releasing it into the environment under uncontrolled conditions.

Conflict of interest

The authors declare that there are no conflicts of interest.

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