The Hydro-distillation Of The Above-ground Parts Of E. Bonariensis Afforded Golden Yellow EO

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2.3.Chemometric Analysis

The EOs chemical compositions of the major compounds (>3%), reported from different ecospecies of P. Dioscorides and E. bonariensis were constructed in a matrix. These collected data were subjected to agglomerative hierarchical clustering(AHC) and principal component analysis (PCA). The cluster analysis of P.dioscoridis EOs showed that the present studied sample of P.dioscoridis is closely correlated to the Egyptian ecospecies collected from El-Sadat City, and little correlated to that collected from Cairo-Suez desert road, Egypt (Figure 3a). cistanche dosage reddit However, the present sample was different than those purchased from a commercial source in Cairo, Egypt. This means that the commercial samples are not in pure form or may be mixed with other plants.

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The PCA of the P.dioscoridis ecospecies showed that the sample collected from the Cairo-Suez desert road, Egypt is mainly characterized by farnesol, germacrene d-4-ol, and longi-folene (Figure 3b). However, the purchased sample from a commercial source in Cairo, Egypt is characterized by hexadecanoic acid and α-casino.

On the other side, the cluster analysis of E. bonariensis EOs revealed that the present Egyptian sample is closely related to the Venezuelan ecospecies, while it was different than other ecospecies (Figure 4a). While the Indian and Tunisian ecospecies showed a close relation in the composition of the EO. cistanche extract benefits The PCA showed that the present sample of E. bonariensis is characterized by trans-a-Farnesene, O-ocimene, and trans-β-Farnesene (Figure 4b). The sample collected from Alexandria, Egypt, showed a close correlation with a-bergamotene, limone, and u-curcumin, while the Indian ecospecies is characterized by β-eudesmol, caryophyllene oxide,allo-aromadendrene, and carvacrol.

The observed variation among the present samples and other reported ones revealed the profitable information derived from the EOs analysis, which could be a useful tool in chemotaxonomy [27].

2.4.Anti-Aging Activity

The EOs from P. dioscoridis,E. bonariensis, and the mixture of the two EOs (1:1)have a strong inhibitory activity of collagenase, elastase, hyaluronidase, and tyrosinase (Figure 5). All the EO treatments exhibited potent inhibition of collagenase enzyme with ICso of 1.85,2.90,and 1.73 ug/mL for P.dioscoridis, E.bonariensis, and the mixture, respectively. Furthermore, the three EO treatments strongly inhibit the elastase enzyme with respective values of ICso of 14.63，16.52 and 1101 μg/mL. Furthermore， strong suppression of hyaluronidase was demonstrated via the three EO treatments based upon the respective observed values of ICso of 17.18,15.16, and 13.54 ug/mL. By the same, the three tested displayed strong tyrosinase enzyme inhibition with ICso values at 19.52,18.93, and 15.81 ug/mL, respectively. All the results were constructed based upon comparing with the polyphenolic compound, epigallocatechin gallate (EGCG), as a standard ant-aging reference [44] that exhibit inhibition of collagenase, elastase, hyaluronidase, and tyrosinase with IC5o of 1.56，10.29，12.71，and 14.37 μg/mL.

cistanche can anti-aging

In the matrix of extracellular, the elastin and hyaluronan degradation were principally correlated with the two respective proteolytic enzymes, elastase, and hyaluronidase that cause the main reasons for aging of the skin such as wrinkles, sagging. Moreover, tyrosinase caused the regulation of the synthesis of melanin in human melanocytes, leading to skin ailments. In the present study, the anti-aging of the 1:1 mixture of the two EOs was evaluated to study the synergetic effects of the combination of the two EOs. Results revealed that the EO of P.dioscoridis, E.bonariensis,and the mixture of the two EOs (1:1) have strong anti-aging activity. These results might be attributed to the chemical components of these oils. The anti-aging activity was directly correlated with antioxidant potentiality [45]. The main constituents in both EOs, sesquiterpenes,were described to play a significant role as antiox-idants, anti-inflammatory agents,and thus anti-aging[45]. Tu and Tawata[45] reported that EO of the leaves of Alpinia zerumbet exhibit antioxidant and anti-aging activities due to the high concentration of terpenoids, especially sesquiterpenes. Furthermore, the monoter-penes were documented as active anti-aging agents in EO of Juniperus communis [46] and Origanum vulgare [47]. cistanche genghis khan These reports concludled that the increase of free radical scavenging constituents in EOs lead to an increase in their anti-aging activity. Based upon this fact, the high concentrations of terpenes especially the oxygenated sesqui- and monoterpenes caused increasing in the anti-aging activity cof EOs of these two plants. All these reported data deduced the role of synergetic effects between the components of the EOs. This fact of the role of synergetic effect was obvious ir our results in which the mixture of the two EOs (1:1) exhibited better activity than the individual EO of each plant.In the mixture of the two EOs, the raising of concentration of oxygenated terpenes as well the synergetic effects between the components caused increasing of the inhibition potentiality.

2.5. Cytotoxic Activity of EOs of P. dioscoridis and E. bonariensis

The cytotoxicity of EOs of the above-ground parts of the two plants,P. Dioscorides and E. bonariensis, as well as a mixture of the two EO:(1:1) against the three cancer cell lines, breast adenocarcinoma cells (MCF-7),lung cancer cells(A-549),and hepatocellular carcinoma cells (HepG2) are shown in Figure 6. The results exhibited that the EO of P. dioscoridis have a significant inhibition of the two cancer cells, MCF-7 and A-549, with ICso of 37.3 and 22.3 uM, respectively (Figure 6A,B), without any activity against HepG2. While, the EO of E.bonariensis showed inhibitory potentiality only against HepG2 with ICso of 25.6 uM (Figure 6C), with negative results against MCF-7 and A-549. The 1:1 mixture of the two EOs did not exhibit any activity against the three cancer cells.

The significant activities of the two EOs might be attributed to the chemical composi-tion in which the synergetic effect of the conpounds contributes to this activity [48]. The sesquiterpenes in both forms, oxygenated and hydrocarbons, represented very effective compounds as anticancer leaders[49,50].Several reports deduced that the increasing of sesquiterpene contents in EOs caused increasiing in anticancer activity [51,52]. For example, caryophyllene with high concentration in EOs was reported as a known potential cytotoxic agent, especially against the growth of breast adenocarcinoma cells (MCF-7) [53,54] The present data revealed that these two EOs are selective against the tested cancer cells. This selectivity was in full agreement with several documented results of EOs derived from other plants. For example,EO derived from Sideritis perfoliata, Satureia thymbra, Salvia officinalis, Laurus nobilis, and Pistacia Palestina were found to have selective inhibitory effects against, amelanotic melanoma (C32), renal celladenocarcinoma(ACHN), hormone-dependent prostatecarcinoma (LNCaP),and breast cancer (MCF-7)[55]. Moreover,EOs extracted from the three plants, Satureja montana, Coriandrum sativum, and Ocimum basilicum, an, were found to have selective cytotoxic activity against HeLa, MDA-MB-453, K562, and MRC-5[56]. The disappearance of the mixtures of the two EOs (1:1)might be ascribed to the negative synergetic effects of each EO upon the other and this phenomenon was reported in some reports. Haroun and Al-Kayali [57] found that the different extracts of Thymbra spicata showed positive synergetic effects via combination with some reference antibiotics against some strains of bacteria alnd a while negative synergetic effects against other strains.

3.Materials and Methods

3.1.Plant Materials Collection and Preparation

The above-ground parts of P.dioscoridis and E.bonariensis were collected from two populations along the Cairo-Alexandria desert road, Egypt in November 2019. From each population, the healthy and fresh plant samples were clipped from three individuals and pooled as composite samples (two per each plant; P.dioscoridis and E.bonariensis). The two plants were authenticated according to Tackholm [58] and Boulos [25]. Voucher specimens (CZ-D-x908-019&CZ-L-x909-019)have been deposited in the herbarium of the National Research Center, Egypt. cistanche life extension The above-ground parts were dried in the shade, ground into a fine powder, and packed in paper bags till further analysis [13]. 3.2.Extraction of EOs

The air-dried powder of the above-ground parts of the P.dioscoridis, and E. bonariensis, (200 gm,each) were subjected separately to Clevenger-type apparatuses using round flask (2.5L)comtaining water(1.5L) for hydro-distillation for 3h. The oily layer of each plant was isolated separately by n-hexane, then dried using anhydrous NagSO4 (0.5 g), and finally stored in glass vials in the freezer till further analysis via GC-MS. This extraction of the EO of each plant was repeated as duplicates.

3.3. Gas Chromatography-Mass Spectroscopy(GC-MIS) Analysis and Chemical Components Investigations

The four EOs samples (two samples for each plant) were analyzed via Gas Chromatography-Mass Spectroscopy (GC-MS) at National Research Center, Egypt [8]The adjustment of the GC/MS instrument specifications has occurred as the following conditions: TRACE GC Ultra Gas Chromatographs (THERMO ScientificTM Corporate Waltham, MA, USA), lined with a Thermo Scientific ISQTM EC single quadrupole mass spectrometer. The GC-MS system was equipped with a TR-5 MS column with a dimension of 30 m × 0.32 mm i.d.， 0.25 μm film thickness. Helium as carrier gas at a flow rate of 1.0 mL/min with a split ratio of 1:10 using the following temperature program: 60℃ for 1 min; rising at 4.0 ℃/min to 240℃ and held for 1 min was used for the analyses. Both injector and detector were held at 210 ℃. An aliquot of 1 uL of diluted samples in hexane (1:10, w/v) was always injected. Mass spectra were recorded by electron ionization (EI) at 70 eV, using a spectral range of m/z 40-450. The chemical constituent of the EOs under investigation was characterized by Auto-mated Mass spectral Deconvolution and Identification (AMDIS) software(ww.amdis.net, accessed on 2 January 2020),retention indexes (relative to n-alkanes Ca-C22), comparison of the mass spectrum with authentics (if available),and Wiley spectral library collection and NSIT library database (Gaithersburg,MD, USA; Wiley,Hoboken, N, USA).

3.4.Anti-Aging Activity of the EOs

3.4.1.Anti-Collagenase Assay

The anti-collagenase assay of the two studied plants EOs as well as the 1:1 mixture were performed according to Thring, et al. [59] with minor modifications for use in a microplate reader. The assay was performed in 50 mM tricine buffer (pH7.5) with 400 mM NaCl and 10 mM CaClz. Collagenase from Clostridium histolyticum (ChC-EC.3.4.23) was dissolved in a buffer for use at an initial concentration of 0.8 units/mL according to the supplier's activity data. The synthetic substrate N-[3-(2-furyl) acryll-Leu-Gly-Pro-Ala (FALGPA) was dissolved in tricine buffer to 2mM. Two studied EOs and the mixture of EOs of the two plants (1:1,w/w),separately,were incubated with the enzyme in a buffer for 15 min before adding substrate to start the reaction. Absorbance at 490 nm was measured using a Microplate reader (TECAN, Group Ltd.,Mannedorf, Switzerland). Epigallocatechin gallate (EGCG) was used as a positive control.

3.4.2.Anti-Elastase Assay

For the anti-elastase inhibitory assay the two studied plants EOs as well as the 1:1 mixture, this assay was performed according to Kim, et al. [60] with minor modifications. Briefly;Porcine pancreatic elastase, was dissolved to make a 3.33 mg/mL stock solution in sterile water. The substrate, N-succinyl-Ala-Ala-Ala-p-nitroanilide (AAPVN) was dissolved in buffer at 1.6 mM. The test EOs were incubated with the enzyme for 15 min before adding substrate to begin the reaction. The final reaction mixture （250 μL total volume） contained buffer，0.8 mM AAAPVN，1 μg/mL PE and 25 μg test sample. cistanche nz The studied EOs and a mixture of EOs of the two plants(1:1, w/w), separately, were incubated. EGCG was used as a positive control. Absorbance values at 400 nm were measured in 96 well microtitre plates using a Microplate reader (TECAN, Inc.). The percentage inhibition for this assay is calculated.

3.4.3.Anti-Tyrosinase Assay

Assays of tyrosinase inhibition of the two plants EOs, as well as the 1:1 mixture, were carried out via measuring of L-DOPA chrome formation according to the described protocol of Batubara,et al.[61]. Briefly,the two EOsand a mixture of them (1:1,w/w),separately, were dissolved in a solvent with three certain concentrations （10，100， and 250 μg/mL）. The assays were performed by insertion of the following components: (a) phosphate buffer （120 μL，20mM，pH6.8），（b）20μL sample，and （c）20μLmushroom tyrosinase（500U/mL in 20 mM phosphate buffer)in 96-well plates. After 15 min of incubation at 25 ℃, the intiation of reaction was occurred by insertion of 20 uL L-tyrosine solution (0.85 mM) for every well and followed by incubation for 10 min at room temperature. The activity of the enzyme was monitored at 475 nm using a Microplate reader (TECAN, Inc.). EGCG was used as a positive control. The calculation of the tyrosinase inhibition % was performed via the following equation: where A is the absorbance of the control withthe enzyme,Bis the absorbance of the control without the enzyme, C is the absorbance of the test sample with the enzyme, and D is the absorbance of the test sample without the enzyme.

3.4.4.Anti-Hyaluronidase Assay

The fluorimetric Morgan-Elson assay method was performed according to Reissig, et al. 【62】 that modified by Takahashi， et al.【63】. In a brief description， a 5 μL of tested EOs and a mixture of EOs of the two plants (1:1, w/w), separately, were incubated for 10 min at 37°C with bovine hyaluronidase （1.50 U） in 100 μL of 20 mM sodium phosphate buffer solution (pH7.0), sodium chloride (7 mM), in addition to 0.01% bovine serum albumin (BSA).The assay reaction was initiated via adding the hyaluronic acid sodium salt （100 μL）from rooster comb（0.03% in 300 mM sodium phosphate，pH5.35）to the incubation mixture, then the mixture was incubated at 37℃ for 45 min. The precipitation of undigested hyaluronic acid was carried out by 1mL acidic solution of albumin, involving 0.1% BSA in sodium acetate (24 mM) and acetic acid (79mM, pH3.75).The mixture was stoped by allowing it for 10 min at room temperature,and fluorescence was detected using a Tecan Infinite microplate reader at 545 nm excitation and 612 nm emission EGCG was used as a positive control. The percentage of the collagenase, elastase, and hyaluronidase inhibition was calcu-lated via the following equation: where S: the corrected absorbance of the samples containing elastase inhibitor (the enzyme activity in the presence of the samples);and C: the corrected absorbance of controls (the enzyme activity in the absence of the samples).

The ICso, the concentration required to inhibit 50% of the enzyme under the assay con-ditions, was estimated from graphic plots of tlhe dose-response curve for each concentration using Graphpad Prism software (San Diego, CA, USA). 3.5.

Cytotoxicity of the Two EOs

Cytotoxic activity of the P.dioscoridis and E.bonariensis EOs and a mixture of them (1:1, w/w), separately were carried out against the three human cancer cells, breast adenocarci-noma cells (MCF-7),lung cancer cells(A-549),and hepatocellular carcinoma cells (HepG2), using sulforhodamine B (SRB) protocol.

3.5.1.Cell Culture

The three cancer cell lines, breast adenocarcinoma cells (MCF-7),lung cancer cells(A-549), and hepatocellular carcinoma cells (HepG2) were obtained from VACCERA,Mokatam, Giza, Egypt. Cells were maintained in DMEM media supplemented with 100mg/mL of streptomycin,100 units/mL of penicillin,and 10% of heat-inactivated fetal bovine serum in humidified,5%(v/v)CO2 atmosphere at 37°C. 3.5.2. Cytotoxicity Assay

Cell viability was assessed by SRB assay. Aliquots of 100 μL cell suspension （5 × 10³ cells） were in 96-well plates and incubated in complete media for 24h.Cells were treated with another aliquot of 100 uL media containing EOs and a mixture of them (1:1 w/w), separately, at various concentrations ranging from (0.01,1,10,and 100 ug/mL). After72h of drug exposure,cells were fixed by replacing media with 150 uL of 10% TCA and incubated at 4℃ for 1h. The TCA solution was removed, and the cells were washed 5 times with distilled water. Aliquots of 70 uL SRB solution (0.4% w/s) were added and incubated in a dark place at room temperature for 10 min. Plates were washed 3times with 1% acetic acid and allowed to air-dry overnight. Then，150 μL of TRIS （10 mM） was added to dissolve the protein-bound SRB stain; the absorbance was measured at 540 nm using a BMG LABTECH@-FLUOstar Omega microplate reader(Ortenberg, Germany)[64,65].

3.6.Data Treatment

The data of the anti-aging activity of various enzymes were presented in three replica-tions and subjected to one-way ANOVA followed by Duncan's test using CoStat version 6.311(CoHort,Monterey,CA,USA,http://www.cohort.com).

A matrix of the concentration of a total of 30 major chemical compounds (>3%)identified in the EO of five P. dioscoridis ecospecies was constructed, these samples were (1)present sample; (2) purchased from a market in Cairo, Egypt; (3)collected from El-Sadat City, Egypt; (4) collected from Cairo-Suez desert road, Egypt dring April; and (5) collected from Turkey. While for E.bonariensis, a matrix of 27 major chemical compounds (>3%)represented six samples was designed, these samples were (1) present sample;(2) collected from Alexandria,Egypt;(3)collected from Monastir, Tunisia;(4)collected from Venezuela;(5)collected from Vietnam; and (6)collected from India. The matrices were subjected to principal component analysis (PCA) and agglomerative hierarchical clustering(AHC) via XLSTAT statistical computer software package (version 2018, Addinsoft Inc., New York, NY,USA).

4. Conclusions

Herein, the GC-MS analysis of EOs of the above-ground parts of P. dioscoridis and E. bonariensis, revealed the identification of 29 and 35 compounds, respectively. Sesquiterpenes were characterized as the main components of EOs derived from the two plants. The major components of EO of P. dioscoridis were a-maliene, berkheyaradulen, dehydro-cyclolongifolene oxide, aromadendrene oxide-2,and β-muurolene. While, trans-x-farnesene, O-ocimene,and o-maaliene represented the abundant constituents of E. bonariensis EO.The observed variation in the EOs composition among the studied ecospecies and that reported support the changing of the taxa names. EOof P. dioscoridis exhibited cytotoxicity against the two cancer cells, MCF-7 and A-549, while the EO of the E. bonariensis showed activity only against HepG2. The EOs of P. dioscoridis and E. bonariensis as well as the mixture of them (1:1), exhibited significant anti-aging activity in which the mixture (1:1)>P. Dioscorides >E. bonariensis. All these data deduced the studied EOs of these two plants may be used as antiaging and anticancer leading agents.

This article is extracted from Plants 2021, 10, 667. https://doi.org/10.3390/plants10040667 https://www.mdpi.com/journal/plants