*Article* **Biomolecular Evaluation of** *Lavandula stoechas* **L. for Nootropic Activity**

**Aamir Mushtaq 1,2, Rukhsana Anwar 1, Umar Farooq Gohar 3, Mobasher Ahmad 1,2, Romina Alina Marc (Vlaic) 4,\*, Crina Carmen Mure¸san 4, Marius Irimie 5,\* and Elena Bobescu <sup>5</sup>**

	- Tel.: +40-740379436 (R.A.M. & M.I.)

**Abstract:** *Lavandula Stoechas* L. is widely known for its pharmacological properties. This study was performed to identify its biomolecules, which are responsible for enhancement of memory. *L. stoechas* aqueous extract was first purified by liquid column chromatography. The purified fractions were analyzed for in vitro anti-cholinesterase activity. The fraction that produced the best anti-cholinesterase activity was named an active fraction of *L. stoechas* (AfL.s). This was then subjected to GC–MS for identifications of biomolecules present in it. GC–MS indicated the presence of phenethylamine and α-tocopherol in AfL.s. Different doses of AfL.s were orally administered (for seven days) to scopolamine-induced hyper-amnesic albino mice and then behavioral studies were performed on mice for two days. After that, animals were sacrificed and their brains were isolated to perform the biochemical assay. Results of behavioral studies indicated that AfL.s improved the inflexion ratio in mice, which indicated improvement in retention behavior. Similarly, AfL.s significantly (*p* < 0.001) reduced acetylcholinesterase and malondialdehyde contents of mice brain, but on the other hand, it improved the level of choline acetyltransferase, catalase, superoxide dismutase, and glutathione. It was found that that high doses of AfL.s (≥400 mg/Kg/p.o.) produced hyper-activity, hyperstimulation, ataxia, seizures, and ultimate death in mice. Its LD50 was calculated as 325 mg/Kg/p.o. The study concludes that α-tocopherol and phenethylamine (a primary amine) present in *L. stoechas* enhance memory in animal models.

**Keywords:** phenethylamine; *L. stoechas*; acetylcholine; choline acetyltransferase; AChE; aromatic amine; enhancement of memory

#### **1. Introduction**

*Lavandula stoechas* L. (Lamiaceae) is an aromatic and medicinal plant of the Mediterranean region. It was the most popular folk remedy for the management of digestive disorders, kidney disease, diabetes mellitus, hyperlipidemia, cough, asthma, headache, and flu-like symptoms [1,2]. It is also known as "broom of the brain" due to its extensive use in the treatment of migraines, epilepsy, and memory related disorders [3,4]. The plant is rich in camphor, erythrodiol, eucalyptol, fenchone, *lavanol*, longipene-2-ene, longipene-2-ene monoacetate, lupeol, luteolin, myrtenol, oleanolic acid, pinocarvyl acetate, terpineol, ursolic acid, vergatic acid, vitexin, α-amyrin, β-sitosterol, and a variety of aromatic compounds [5,6]. Linalool present in *L. stoechas* has a sedative effect [7] and it acts on different brain receptors to modify behavioral patterns, along with creating a sense of

**Citation:** Mushtaq, A.; Anwar, R.; Gohar, U.F.; Ahmad, M.; Marc (Vlaic), R.A.; Mure¸san, C.C.; Irimie, M.; Bobescu, E. Biomolecular Evaluation of *Lavandula stoechas* L. for Nootropic Activity. *Plants* **2021**, *10*, 1259. https://doi.org/10.3390/plants 10061259

Academic Editor: Antonella Smeriglio

Received: 30 April 2021 Accepted: 16 June 2021 Published: 21 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

calmness and wellbeing. Camphor, present in *L. stoechas,* has stimulating effects on the brain [8]. Lavender oils extracted from *L. stoechas* are famous for their spicy fragrances and are used in aromatherapy to treat anxiety, depression, sleeplessness, headaches, and migraine [9]. Moreover, *L. stoechas* showed antispasmodic [8], sedative, anti-epileptic, antibacterial [10], antifungal [11], anti-leishmaniasis [5], anti-inflammatory [12], cytotoxic [13] and anti-diabetic [14] properties.

Keeping in mind the traditional uses of *L. stoechas*, as a neurotonic and memory enhancer, as part of our studies on the methanolic extract of *L. stoechas* [15], our current study was designed to report the active ingredient of *L. stoechas* aqueous extract responsible for nootropic activity.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Drugs*

The following chemicals used were of analytical grade, and procured from Sigma Aldrich, MS Traders, Lahore Pakistan: 2,2-*diphenyl*-1-picrylhydrazyl (DPPH) (95%), 5,5 dithiobis-*2*-nitrobenzoic acid (*DTNB*) (99%), 4,4-dithiodipyridine (98%), Folin–Ciocalteu reagent (FCR), trichloroacetic acid (TCA) (98%), thiobarbituric acid (TBA) (99%), tannic acid (99.9%), superoxide dismutase (SOD), sodium dodecyl sulfate, sodium carbonate (99%), rutin, reduced nicotinamide adenine dinucleotide (NADH), potassium dichromate (99.5%), potassium acetate (99%), phenyl methanesulfonate (PMS), nitro blue tetrazolium (NBT), *n*-butanol (99.8%), *n*-hexane (99%), methanol (99.8%), hydrogen peroxide (H2O2) (85%), gallic acid, ethanol, chloroform (99%), carboxy methyl cellulose (CMC), ascorbic acid (99%), aluminum chloride (99.9%), acetyl thiocholine iodide, silica gel-60, acetyl coenzyme-A, acetylcholine esterase, choline chloride (98%), EDTA, fast blue B salt, neostigmine sulfate, β-naphthyl acetate, and acetic acid. Piracetam was gifted from Jiangxi Yuehua Pharmaceutical, China, and scopolamine was obtained from Merck Pharmaceutical Pvt. Ltd. (Kenilworth, NJ, USA).

#### *2.2. Extraction and Fractionation by Column Chromatography*

*L. stoechas* L. (aerial parts) was purchased from a local market in Lahore, Pakistan. It was then identified by a botanist from the department of Botany, Government College University (GCU), Lahore. The specimen was preserved in the herbarium of the GCU and was assigned a voucher number: GC.Herb.Bot.3386. The extraction was conducted in methanol, by using simple maceration, as described in our previous study [15]. Then, fractional extraction was conducted by using different solvents, i.e. *n*-hexane, chloroform, ethyl acetate, and *n*-butanol. Finally, the aqueous layer left behind was obtained and the solvent was evaporated to obtain a semi-solid aqueous extract of *L. stoechas.* Column chromatography was used for the fractionation of the aqueous extract of *L. stoechas*. An appropriate-sized glass column was packed with slurry, which was made by dissolving almost 10 g of silica gel-60 in the same solvent as used for the mobile phase. Standard protocols were followed for the packing and running of the column. *L. stoechas* aqueous extract was dissolved in a small amount of solvent; that sample mixture was loaded in a column via a pipette. The space above the sample in the column was filled with solvent and the stopper was opened to obtain the separated fraction in a flask below the outlet of the column [16]. This way, different solvent systems were used for the separation of different constituents in the crude extract. Separated fractions were collected in the test tubes and labeled for further tests.

#### *2.3. Anti-Cholinesterase Activity (In Vitro Assay)*

In vitro anti-cholinesterase activity was performed on all fractions obtained through column chromatography by using an (NA-FB) microwell plate assay. The solution was prepared by dissolving β-naphthyl acetate (0.25 mg) in methanol (1 mL); 50 μL of this was mixed with 10 μL of plant extract. Then, 200 μL of acetylcholine esterase solution (3.33 U/mL) was poured in the reaction mixture by keeping the temperature of the mixture

at 4 ◦C. It was incubated for 40 min at the same temperature and then 2.5 mg of fast blue B salt was dissolved in 1 mL of distilled water. Out of which, 10 μL was dropped into the incubated reaction mixture; a change in the solution color was observed. β-naphthyl acetate was used as a substrate, while fast blue B salt was used as a color reagent. The principle applies that β-naphthyl acetate is hydrolyzed into naphthol acetic acid by AChE. Naphthol then reacts with fast blue B salt, imparting a purple color to the mixture. No change in color of the solution indicates a strong anti-cholinesterase activity of the fraction, while a dark purple color indicates no inhibition of AChE [17].

#### *2.4. GC–MS Analysis*

The bioactive compounds present in the final fraction of *L. stoechas* were detected by performing gas chromatography mass spectroscopy (GC–MS) analysis by using GC– MS equipment (Agilent 6890N). The TR-5-MS capillary non-polar standard column, with dimensions of 30 Mts, an internal diameter of 0.25 mm, and film thickness of 0.25 μm, was used. Helium gas (99.99%) was used as carrier gas and mobile phase was run with a flow rate of 1 mL/min. The starting temperature of the oven was 40 ◦C, which was raised to 250 ◦C @ 10 ◦C/min. The sample was dissolved in methanol and an aliquot of 2 μL was injected by keeping the temperature of the injector and detector fixed at 250 ◦C and 280 ◦C, respectively, while the ion source temperature was fixed at 200 ◦C. Identification of the compound was by molecular mass and the structure of the compound by interpretation of the GC–MS standard library.

#### *2.5. Behavioral and Biochemical Studies*

Behavioral studies were performed by using elevated plus maze, light/dark test, and hole-board test models, using standard protocols. Biochemical studies were performed to assess the level of acetylcholinesterase (AChE), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in the brain homogenates of mice, of all groups, using standard protocols. Detailed procedures of these tests are described in a previously published paper in this series [15].

#### *2.6. Animals*

All studies were performed on male Swiss albino mice, which were provided standard living conditions, as described in a previous manuscript [15], while female albino mice (20–25 g) were used for toxicity studies.

#### *2.7. Study Design for Behavioral/Biochemical Studies*

For behavioral/biochemical studies, mice were divided into seven groups (*n* = 6) and were treated accordingly, as shown in Table 1. While ChAT was performed on five groups (*n* = 6) of mice, which were treated accordingly, as shown in Table 2.

#### **Table 1.** Study design for behavioral and biochemical studies.


Doses were prepared by suspending AfL.s in CMC (5%) and by dissolving piracetam and scopolamine in normal saline. Then, behavioral studies were performed on days 7 and 8, and animals were sacrificed for the performance of biochemical studies on the eigth day after completing behavioral trials.


**Table 2.** Study design for the assessment of choline acetyltransferase (ChAT) activity.

Two hours after administration of the last dose, the animals were sacrificed by using chloroform to get their brain. Then brain homogenates were formed according to standard procedure [15] and ChAT activity was determined by the above-described spectroscopic method.

#### *2.8. Choline Acetyltransferase Activity (ChAT)*

The reagent was prepared by mixing 10 μL of each sodium phosphate buffer (0.5 M, pH 7.2), sodium chloride (3 M), neostigmine sulfate (7.6 × <sup>10</sup>−<sup>4</sup> M), acetyl coenzyme-A (6.2 × <sup>10</sup>−<sup>3</sup> M prepared in 0.01N HCl), EDTA (1.1 × <sup>10</sup>−<sup>3</sup> M), choline chloride (1 M), and sodium chloride (3 M), and incubated at 37 ◦C for 5–10 min. Brain homogenate (100 μL) was mixed in the smallest volume of the reagent and the final volume was 0.2 mL. It was then boiled in a water bath for 2 min after incubating at 37 ◦C for 25 min. Then, oxygen-free distilled water was added to the mixture, which was then centrifuged at high speed to separate out the denatured protein. Then, 10 μL of 4,4-dithiodipyridine (10−<sup>3</sup> M) was added to 0.5 mL of the supernatant and absorbance was read at 324 nm against the blank in a UV-visible spectrophotometer [18].

#### *2.9. Acute Toxicity Studies*

Acute toxicity study was performed on female albino mice according to OECD guidelines 423 2001. Initially, the pilot study was done on a small number of mice to find the dose range at which animals started to die. It was found that active fractions of *L. stoechas* (AfL.s) showed no death up to 300 mg/Kg/p.o. and showed 100% mortality at 500 mg/Kg/p.o. Thus, animals were divided into six groups (*n* = 5). Group I was normal control; animals in Groups II to VI were administered with AfL.s in wide, spaced doses, in ascending order (300, 350, 400, 450, and 500 mg/Kg/p.o., respectively). They were then observed for 24 h to find the number of mortalities. Finally, LD50 was calculated by using the following formula:

$$\text{LD}\_{50} = \text{Least} \, \text{Lethal Dose} - \Sigma \text{ (a} \times \text{b)} / \text{n}$$

The animals who survived in different groups after administration of acute toxic dose were observed for two weeks for the assessment of physical and behavioral changes. Ataxia, blanching, convulsions, cyanosis, depression, hyperactivity, hypnosis, irritability, jumping, loss of traction, muscle spasm, piloerection, redness, rigidity, salivation, secretions, sedation, stimulation and straub reaction were the parameters that were observed after acute doses administration.

#### *2.10. Statistical Analysis*

The data are expressed as mean ± SEM. Student's *t*-test analysis was applied on data with paired comparisons, and multiple comparisons were made by ANOVA followed by Dunnett's test by using GraphPad Prism software version 7. Value of *p* < 0.05 was marked as significant.

#### **3. Results**

#### *3.1. Fractionation by Column Chromatography*

*L. stoechas* aqueous extract was fractionated further by column chromatography. In total, 55 fractions were obtained by using different solvent systems (based on polarity). The fractions were evaluated by thin layer chromatography (TLC), were combined, and again passed from the column; finally, there were 15 fractions (Figure 1).

**Figure 1.** General scheme of fractionation by column chromatography.

Anti-Cholinesterase Activity (In Vitro)

In vitro testing indicated that fraction no. 6 showed the best enzyme inhibition among all the fractions, as expressed in Table 3. Fraction no. 6 was named an active fraction of *L. stoechas* (AfL.s) and was tested for chemical analysis by GC–MS. Furthermore, invivo studies (behavioral and biochemical) were performed on mice to find the memory enhancing effects of AfL.s.


**Table 3.** Anti-cholinesterase activity (in vitro) shown by different fractions of *L. stoechas*.

Fraction no. 6 (F-6) possessed strong anti-cholinesterase activity, so it was selected for further chemical and in-vivo studies.

#### *3.2. GC–MS Analysis of AfL.s*

GC–MS analysis indicated that AfL.s contained the phenethylamine group of compounds and cholestan-7-one. The details are shown in Table 4 and spectrum is given in Figure S1.


**Table 4.** Compounds detected in AfL.s by GC–MS.

*3.3. Behavioral Studies (Effect of AfL.s on EPM, Light/Dark Test, and Hole-Board Paradigm in Mice)*

Results of behavioral studies indicated that animals treated with AfL.s significantly (*p* < 0.001) reduced the initial transfer latencies (ITL) and retention transfer latencies (RTL) in comparison to the amnesic control group (Figure 2). Similarly, inflexion ratio (IR) calculated from ITL and RTL values, indicated that active fraction-treated mice showed maximum IR value (0.17 ± 0.04) in comparison to scopolamine-treated mice (−0.19 ± 0.04). Thus, higher IR values indicated significant (*p* < 0.001) improvement of memory in AfL.s-treated mice.

**Figure 2.** *Cont.*

**Figure 2.** *Cont.*

**Figure 2.** Effect of AfL.s on (**A**) initial transfer latency; (**B**) retention transfer latency; (**C**) inflexion ratio in the elevated plus maze paradigm; (**D**) time spent (sec) in the light compartment on day 1; (**E**) time spent (sec) in the dark compartment on day 1; (**F**) time spent (sec) in the light compartment on day 2; (**G**) time spent (sec) in the dark compartment on day 2; (**H**) number of hole pokings by mice on day 1, and (**I**) number of hole pokings by mice on day 2. Data are presented as mean ± SEM (*n* = 6) and one-way ANOVA (Dunnett's test) was applied by comparing G-II to G-I (presented by "a" on the bar). All other groups were compared to G-II (presented by "b" on the bar). The signs *ns,* \*, \*\* and \*\*\* presented the *p* values as ≥0.05, ≤0.05, ≤0.01, and ≤0.001, respectively).

Results of the light/dark test indicated that AfL.s-treated mice spent most of the time in the dark portion of the apparatus, on the first and second day of observation, in comparison to the amnesic control group. This finding is based on the principle that animals in the amnesic control group forgot to find the dark area of the apparatus while the standard control and AfL.s-treated mice retained their memory of exploration. Thus, it is clear that AfL.s significantly (*p* < 0.001) improved the memory in AfL.s-treated mice (Figure 2).

Similarly, results of hole-board paradigm indicated that amnesic control animals significantly reduced the number of hole-pokings while standard control and AfL.s-treated mice retained their memory of exploration and showed a significantly (*p* < 0.001) increased number of hole-pokings on both the first and second day of observation (Figure 2).

#### *3.4. Biochemical Studies (Effect of AfL.s on Levels of AChE, MDA, SOD, CAT, and GSH in Mice Brains)*

Biochemical studies indicated that the level of acetylcholinesterase (AChE) was significantly (*p* < 0.001) reduced in group-II (scopolamine treated) animals, while group-VII animals (AfL.s 18 mg/Kg/p.o.) showed maximum inhibition of AChE among all the groups (Figure 3A). Similarly, the level of MDA is reduced significantly (*p* < 0.001) in AfL.s-treated mice as compared to the amnesic control group (which showed the highest MDA level) (Figure 3B). It was observed that the levels of SOD, CAT, and GSH increased significantly in group-V (AfL.s 9 mg/Kg/p.o.) among all groups (Figure 3C–E). This observation leads to the fact that AfL.s possesses strong antioxidant activity when used in low doses.

**Figure 3.** *Cont.*

**Figure 3.** Effect of AfL.s on concentration of (**A**) acetylcholinesterase (AChE); (**B**) MDA; (**C**) SOD; (**D**) CAT; and (**E**) GSH in brain homogenate. Data are presented as mean ± SEM (*n* = 6) and one-way ANOVA (Dunnett's test) was applied by comparing G-II to G-I (presented by "a" on bar). All other groups were compared to G-II (presented by "b" on bar). The signs *ns*, \*, \*\* and \*\*\* presented the *p* values as ≥0.05, ≤0.05, ≤0.01, and ≤0.001, respectively).

#### *3.5. Effect of AfL.s on ChAT Activity*

The level of ChAT was observed, 11.85 ± 0.92, 7.59 ± 0.76, 18.13 ± 1.23, 16.70 ± 1.16, 12.10 ± 1.45, 11.63 ± 0.66, 10.84 ± 1.22, and 7.08 ± 0.68 μmol/min/mg, from group-I to V, respectively. Thus it is clear that animals treated only with AfL.s (18 mg/Kg/p.o.) for seven consecutive days showed the best elevation in ChAT levels as compared to the normal control group, with the level of significance as (*p* < 0.01), as shown in Figure 4.

**Figure 4.** Effect of AfL.s on concentration of ChAT in mice brains. Data are presented as mean ± SEM (*n* = 6) and one-way ANOVA (Dunnett's test) was applied by comparing all groups with G-I. The signs *ns*, \*, \*\* and \*\*\* presented the *p* values as ≥ 0.05, ≤ 0.05, ≤ 0.01, and ≤ 0.001 respectively.

#### *3.6. Acute Toxicity Study*

Acute toxicity study performed on six groups (*n* = 5) of female albino mice indicated that AfL.s produced no death up to a dose of 350 mg/Kg/p.o. Animals began to die when the dose increased above 350 mg/Kg/p.o. All the animals died when they were administered with a single oral dose 500 mg/Kg of AfL.s. All the details and calculations for median lethal dose LD50 are provided in (Table 5). LD50 for AfL.s was calculated as 325 mg/Kg/p.o. The animals who survived after the administration of an acute single dose of AfL.s 450 mg/Kg/p.o. were kept in observations for two weeks for the assessment of behavioral and physiological changes. It was observed that animals positively exhibited stimulation, straub reaction. salivation, rigidity, piloerection, other secretions, muscle spasm, loss of traction, jumping, irritability, hyperactivity, convulsions, blanching, and ataxia, while, sedation, redness, ptosis, hypnosis, depression, and cyanosis were not observed in the mice (Table 6).

(**E**)


**Table 5.** Calculation of median lethal dose LD50 of AfL.s.

Σ(a × b)/*n* = 375/5; LD50 = Least lethal Dose − Σ(a × b)/*n*; 400 − 375/5 = 325 mg/Kg.



"-"Sign indicates absence of effect while "+" sign indicates the presence of effect.

#### **4. Discussion**

Aromatic plants of the Mediterranean region have advanced medicinal values. Alcoholic and hydro-alcoholic extracts of many aromatic plants of Asteraceae, Apiaceae, and Lamiaceae families have been used extensively for their wide variety of pharmacological activities. Genus Lavandula contains as much as 39 species, including two economically well renowned species—*L. stoechas* and *L. angustifolia*. Hydroalcoholic extracts of *L. stoechas* contain catechic tannins, flavonoids, coumarins, sterols, mucilages, and leucoanthocyanins [19]. Different compounds, such as α-amyrin, β-sitosterol, α-amyrin acetate, oleanolic acid, vergatic acid, ursolic acid, erythrodiol, lupeol, luteolin, vitexin, acacetin, lavanol, 7-methoxy coumarin, and two longipinane derivatives (longipin-2-ene-7β,9α-diol-1-one-9-monoacetate and longipin-2-ene-7β,9α-diol-1-one) have been isolated from areal parts of *L. stoechas* [20]. *L. stoechas* exhibits a wide array of pharmacological activities; research shows that it boosts memory in albino mice [15]. This is an original research work specifically designed to report the biomolecules of *L. stoechas,* which boosts memory in mice brains. In this study, the *L. stoechas* aqueous extract was purified, and chemical characterization of AfL.s by GC–MS indicated the presence of two main compounds

(phenethylamine and α-tocopherol) (Figure S1, Table 4). Past studies have scientifically proven the anticholinesterase activity of phenethylamine [21] and the antioxidant potential of cholestan-7-one, usually named α-tocopherol [22].

Phenethylamine has been scientifically proven as a brain neuromodulator and a strong inhibitor of AChE [23]. Phenethylamine is composed of an aromatic ring to which a side chain of two carbons having amine at the terminal position is attached. Substitution of alkyl groups at different positions on the phenyl ring would attribute to its strong neuromodulative and psychoactive activities. Thus, overall cognition and brain performance is enhanced by phenethylamine [21,24].

The results of the behavioral studies concluded that AfL.s significantly (*p* < 0.001) enhanced the retention power and learning capacity of the mice brains. Similarly, treatment of animal with AfL.s showed significant (*p* < 0.001) reduction in the level of AChE (Figure 3A). Inhibiting AChE improves cholinergic transmission and relieves the patient of memory loss [25].

On the other hand, the level of choline acetyltransferase (ChAT) was elevated (Figure 4) in mice brains. Both of these findings strongly suggest that the level of acetylcholine (ACh) increased in mice brains by dual mechanisms. This effect would be due to the action of phenethylamine on trace amine-associated receptors (TAARs), which are abundantly present in the brain, pituitary glands, kidney, liver, and stomach [26]. It has been proposed that phenethylamine binds with G-protein (either Gs or Gq subunit) coupled receptors (TAARs) and enhances memory and cognition [27,28].

The possible antioxidant mechanism of AfL.s is due to the presence of α-tocopherol, which not only promotes the glutathione level in the brain, but also causes attenuation of reactive oxygen species [22]. Furthermore, α-tocopherol is responsible for the formation of the stable and inert tocopheroxyl radical, by reacting with lipid peroxyl radical (LOO˙), as shown in Equation [29].

#### LOO˙ + α-tocopherol-OH → LOOH + α-tocopherol-O˙

Loss of memory may take place, either due to reduction in the level of acetylcholine [30] or by deposition of the β-amyloid protein [31] in the cerebral cortex and hippocampus of the brain [32]. Moreover, severe oxidative damage to neuronal circuits in the brain is another leading cause of memory loss [33,34], which is exhibited in scopolamine-treated mice [35]. The findings of the current study indicate that scopolamine-treated mice showed marked elevation of AChE (Figure 3A) and reduction in ChAT (Figure 4). Oxidative stress induced by scopolamine is the main factor behind this enzyme disturbance [36]. The treatment of mice with AfL.s not only reduced the level of AChE (Figure 3A), but also significantly (*p* < 0.001) boosted the level of ChAT (Figure 4) in mice brains. It is proposed that elevation in ChAT levels by AfL.s is caused by the antioxidant action of α-tocopherol on the brain. Similarly, phenethylamine present in AfL.s is responsible for the enzymemediated release of ACh in the brain [37]. The results also clearly indicate that animals only treated with AfL.s, without prior or subsequent administration of scopolamine, produced the highest elevation of ChAT levels in the brain (Figure 4).

Acute toxicity study indicated that AfL.s is toxic when used in high doses (≥400 mg/Kg/p.o.), which produces hyperactivity, hyperstimulation, ataxia, seizures, and ultimate death (Table 6). This toxicity is due to the primary toxic effect of high doses of phenethylamine, which is responsible for headaches, confusions, hallucination, seizures, and ultimately death in human beings [38]. Restlessness, diarrhea, headache, aggression, and tremors are mild side effects, which may be observed with the overdoses of phenethylamine [39].

Past studies have reported that high toxic doses (125–200 mg/Kg/i.p.) of phenethylamine produced very severe seizures and ultimate deaths due to cardiac arrest and overstimulation of the brain [40]. The LD50 for AfL.s was calculated as 325 mg/Kg/p.o. (Table 5), which indicated that it had a broad therapeutic index.

#### **5. Conclusions**

Two principle compounds—α-tocopherol and phenethylamine, present in *L. stoechas* are responsible for the attenuation of dementia. α-tocopherol reduces oxidative stress of the free radicals in mice brains while phenethylamine enhances the level of acetylcholine in the hippocampus of mice brains. Thus, it is concluded that *L. stoechas* L. can be used as a memory enhancer. Further studies are needed to elaborate on its detailed mechanisms, in regards to enhancement of memory and toxicity profile.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/plants10061259/s1, Figure S1: Spectrum of analysis of AfL.s by GC-MS.

**Author Contributions:** Conceptualization, A.M. and R.A.; methodology, M.A.; software, U.F.G.; validation, R.A.M. and C.C.M.; formal analysis, A.M.; investigation, M.I.; resources, A.M.; data curation, E.B. and A.M.; writing—original draft preparation, A.M.; visualization, M.I. and E.B.; project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Government College University Lahore.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All of the data are available and can be produced upon request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Aromatic Profile Variation of Essential Oil from Dried Makwhaen Fruit and Related Species**

**Trid Sriwichai 1,†, Jiratchaya Wisetkomolmat 1,2,†, Tonapha Pusadee 2,3, Korawan Sringarm 2,4, Kiattisak Duangmal 5,6, Shashanka K. Prasad 7, Bajaree Chuttong 2,8,\* and Sarana Rose Sommano 1,2,\***


**Abstract:** The aim of this research is to evaluate the relationship between genotype, phenotype, and chemical profiles of essential oil obtained from available *Zanthoxylum* spp. Three specimens of makhwaen (MK) distributed in Northern Thailand were genetically and morphologically compared with other *Zanthoxylum* spices, known locally as huajiao (HJ) and makwoung (MKO), respectively. HJ was taxonomically confirmed as *Z. armatum* while MKO and MK were identified as *Z. rhetsa* and *Z. myriacanthum*. Genetic sequencing distributed these species into three groups accordingly to their confirmed species. Essential oil of the dried fruits from these samples was extracted and analyzed for their chemical and physical properties. Cluster analysis of their volatile compositions separated MKO and MK apart from HJ with L-limonene, terpinen-4-ol, *β*-phellandrene, and *β*-philandrene. By using odor attributes, the essential oil of MKO and MK were closely related possessing fruity, woody, and citrus aromas, while the HJ was distinctive. Overall, the phenotypic characteristic can be used to elucidate the species among makhwaen fruits of different sources. The volatile profiling was nonetheless dependent on the genotypes but makwoung and makhwaen showed similar profiles.

**Keywords:** aromatic plant; chemical profiles; huajiao; spicy plant; taxonomical description; volatile compositions

#### **1. Introduction**

Plants of the *Zanthoxylum* spp. (Rutaceae) contain oil glands that yield high amounts of essential oil with distinctive aroma [1]. Their fruits are known as spices for ethnic food particular in Asia such as those of *Z*. *piperitum* [2], *Z*. *armatum* [3], *Z*. *fagara* [4], and the essential oils extracted from the fruits and leaves are used as food additives and functional ingredients in food and pharmaceutical industries. Commonly known as makhwaen or makhan, *Z*. *myriacanthum* is grown extensively in many areas of northern Thailand viz., Pong district of Payao, Song Khwae district of Nan and in many high-altitude areas of

**Citation:** Sriwichai, T.;

Wisetkomolmat, J.; Pusadee, T.; Sringarm, K.; Duangmal, K.; Prasad, S.K.; Chuttong, B.; Sommano, S.R. Aromatic Profile Variation of Essential Oil from Dried Makwhaen Fruit and Related Species. *Plants* **2021**, *10*, 803. https://doi.org/10.3390/ plants10040803

Academic Editor: Marc (Vlaic) Romina Alina

Received: 24 March 2021 Accepted: 16 April 2021 Published: 19 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Chiang Mai [5]. Previous studies described that *Z*. *myriacanthum* essential oil gives a unique citrus top-note followed by a woody and spice aromatic profile [4–6]. The analysis of the volatile compositions of *Z*. *myriacanthum* has illustrated that the main compounds are comprised of sabinene, terpinene-4-ol, and L-limonene [5,6]. Moreover, essential oils of plants in this genus also possess biological activities such as antimicrobial properties [7] antioxidant activity [8] and anti-inflammatory [9] thereby are used for medicinal purposes. With an increasing commercial need for exotic ingredients, there is, therefore demands for high-quality raw material of essential oil for food and perfumery industries. To the perfumery industry claim, the complaint made from the raw material purchaser asserted that plant morphological characteristics such as tree structure, sizes, and color of the berry clusters were variable in different sourcing regions which made the final quality of essential oil unsteady (Mrs. Anne Saget pers. Comm.). Moreover, the complexity within the species remains ambiguous as such *Z*. *myriacanthum* is often misidentified as *Z*. *limonella* [10]. Thus, there is urge commercially to truly describe plant species.

Genetic and environmental variables—i.e., growing condition, light intensity, day length, temperature, altitude, as well as their interactions—could generally influence the quantity and quality of the essential oils [11,12]. Identification of plant species and variety in the same genus can be accomplished by taxonomic description and chemical compositions [13]. However, only the use of these phenomena may not be enough to accurately describe the species. Studies on the essential oil containing plants revealed that chemical compositions and characteristics of essential oils from plants within the same genus are diverse such as those belonging to *Ocimum* spp. [14] and *Zanthoxylum* spp. [15]. The use of DNA fingerprints can therefore accomplish for the reliable identification of plant species [16].

Internal transcribed spacer DNA barcode (ITS2) detects nuclear marker of the rDNA region in nuclear genome that is useful for directly detecting reticulate phenomena. This technique has been reported to be an efficient barcode locus for plant identification [17,18] and classification by many plant species such as Indian *Berberis* [19], timber species of the mahogany family [20], and *Dendrobium* species [21]. In addition to the ITS region, RAPD analysis is an alternate method for estimating genetic diversity and relatedness in plant populations, cultivars and germplasm accessions, especially in non-model plant species. By using the markers, the technique is able to amplify DNA from dispersed polymorphic loci and thereby indirectly distinguishes small differences within the gene sequences. To draw accurate conclusion on genetic relations of plants species, it is therefore vital to combine these techniques. There is no research work to-date that fully describe genotyping differences among raw materials for makhwaen essential oil production in relation to their physical properties and aromatic profiles as compared to those of other *Zanthoxylum* species. The aim of this research, therefore, is to descriptively establish profile specification of raw materials used in makhwaen essential oil extraction industry.

#### **2. Results and Discussion**

#### *2.1. Morphological Confirmation*

The morphological descriptions of the specimens of the *Zanthoxylum* spp. known locally as huajiao (HJ), makwoung (MKO), and makwhaen (MK1-3) were documented using plant structure, thorn, leaf type, floral structures, and fresh fruit color [22]. From our data in Supplementary Table S1, the plant structure of HJ was of shrub and was different from that of MKO and MK1-3 (tree-like structure). Thorns of all specimens were either initiate on the trunk or branches. The same compound leaf type was observed in the MKO and MK1-3 (even-pinnately) which were different from the HJ (odd-pinnately compound leaf). The floral compositions were different in every species; the HJ consisted of the flower with six to nine petals, while the MKO was with four petals and MK1-3 were with five petals. Within a similar pattern, the number of anthers was different in every species, four to eight anthers for HJ, three or four anthers for MKO, and five anthers for MK1-3. The color of fresh fruit was red in HJ and MKO while MK1-3 gave greenish-red color

characteristics. Fruit sizes varied from 2–3 mm of the MK1-3 to 4–5 mm of the HJ and the MKO was 5–7 mm, respectively. The three species gave brown fruit when dried with crack revealing the inner seeds. According to these specific characteristics, the scientific names of *Z*. *armatum, Z*. *rhetsa*, and *Z*. *myriacanthum* are given to HJ, MKO, and MK specimens [22,23]. To describe the verity within the same species, floral and fruit characteristics of MK1-3 were compared (Supplementary Table S2). The result confirmed that the MKs were those of *Z*. *myriacanthum* as the sepals and petals were pentamerous and male flower organs composed five stamens.

According to the results from UPGMA analysis of plant characteristics and seven samples of *Zanthoxylum* spp., MK1-3 were far distinctive from MKO and HJ. As it could be seen, MKO and MK1-3 were both tree plants while HJ was a shrub. Nonetheless, MK1-3 were detached from MKO by their floral characteristics (Figure S1).

#### *2.2. ITS Sequencing Analysis*

The aligned lengths of the ITS region (including both ITS1 and ITS2 regions) ranged from 596 bp for MK (*Z. myriacanthum*) to 600 bp for HJ (*Z. armatum*). Among the five MK sampling (two specimens from Mae Tang district: MK1-1 and MK1-2, one from Mae Rim: MK2 and two from Nan: MK3-1 and MK3-2), the ITS sequences were completely identical whereas 39 single nucleotide polymorphism were found among MK, MKO, and HJ samples. The phylogenetic relationship analysis was investigated based on the total ITS region sequences. The dendrogram showed three major clades (Figure 1A), the first formed among five MK samples from the three regions, the second consisted of MKO while the last is HJ. ITS sequence is an efficient tool for genetic identification among species however, very low efficiency for evaluation of genetic variation within the species.

**Figure 1.** The dendrogram of *Zanthoxylum* spp. in North of Thailand; HJ (huajiao), MKO (makwoung), MK1 (makhwaen from Mae Tang district), MK2 (makhwaen from Mae Rim district), and MK3 (makhwaen from Song Kwae district) derived by UPGMA from the similarity matrix of the ITS sequence data (**A**) and from the similarity matrix based on 37 DNA bands obtained from five RAPD markers (**B**).

#### *2.3. RAPD Analysis*

Only S6, S7, S9, OPA01, and OPA04 primers gave responses with the DNA thus they were used for the calculation of the unweighted pair group method with arithmetic mean (UPGMA). Result was illustrated as a dendrogram in which the samples were split into three groups: group 1—HJ, group 2—MKO, and group 3 consisting of MK1-3, as shown in Figure 1B. The dendrogram illustrated that MK1-3 were clustered as closely related species while HJ and MKO were genetically identified as separated species. Indeed, the RAPD makers revealed slight genetic variables between the *Z. myriacanthum* samples from different geographical regions. The result was correspondent with our taxonomic data described previously. In addition to the ITS sequencing, the RAPD technique was successful to determine the genetic variation of the Zanthoxylum spp. as well as many plants of this kind including *Z*. *hamiltonianum, Z*. *nitidum, Z*. *oxyphyllum, Z*. *rhesta, Z*. *armatum*, and *Z*. *schinifolium* [24–26].

#### *2.4. Essential Oil Analysis*

Essential oils were extracted from dried fruits of makhwaen samples from three areas (MK1, MK2, and MK3), huajiao (HJ) and makwoung (MKO) using hydro-distillation. The extraction yield varied by mean of species differentiation i.e., MK1-3 (~7%), followed by HJ (~5%) and MKO (~2%). Thirty-five volatile compounds were detected using GC-MS (Table 1). Essential oil of the MKO contained the major content of linalool (7.35 <sup>μ</sup>g·mL<sup>−</sup>1) following by <sup>β</sup>-thujone (1.03 <sup>μ</sup>g·mL−1) and sabinene (0.44 <sup>μ</sup>g·mL−1), respectively. Sabinene was the key dominant substance in the *Zanthoxylum* species analyzed, except for the essential oil of the MKO. This is in agreement with other works done with plants belongs to the *Zanthoxylum* species—i.e., *Z*. *xanthoxyloïdes, Z*. *leprieurii* [27], and *Z*. *rhoifolium* [28] with sabinene and limonene that represented woody and citrus aromas [29].

The chemical profiles of the essential oils from makhwaen fruits collected from different locations were variable. The major components of all samples could be described as following sequence: MK1; limonene (4.05 <sup>μ</sup>g·mL−1), sabinene (3.20 <sup>μ</sup>g·mL−1) and <sup>L</sup>−phellandrene (1.47 <sup>μ</sup>g·mL<sup>−</sup>1), MK2; sabinene (2.55 <sup>μ</sup>g·mL<sup>−</sup>1), terpinen-4-ol (2.05 <sup>μ</sup>g·mL<sup>−</sup>1) and *<sup>β</sup>*-phellandrene (1.85 <sup>μ</sup>g·mL<sup>−</sup>1), MK3; limonene (6.89 <sup>μ</sup>g·mL<sup>−</sup>1), sabinene (3.00 <sup>μ</sup>g·mL<sup>−</sup>1) and *<sup>β</sup>*-ocimen (1.47 <sup>μ</sup>g·mL−1), HJ; sabinene (4.56 <sup>μ</sup>g·mL−1), terpinen-4-ol (4.31 <sup>μ</sup>g·mL−1) and <sup>γ</sup>-terpene (1.08 <sup>μ</sup>g·mL<sup>−</sup>1). To this extend, geographical or environmental factors would play an important role in the chemical composition of the volatiles [12]. The variations due to growing locations of aromatic crops were fully described in chamomile (*Matricaria recutita* L.) [30], *Satureja kitaibelii* [31] and *Myrsine leuconeura* [32]. In the *Zanthoxylum* spp., plants growing at different altitudes yielded essential oil with alternating principal volatiles (limonene, sabinene, and linalool) viz., *Z. armatum* [3,33,34] and *Z*. *alatum* [35]. Our results agree with this as plant samples taken for this experiment were grown at different altitudes.

The relationships between the chemical components and the *Zanthoxylum* species were analysed using the PCA in Figure 2. The PCA revealed that HJ was distinctive from the other *Zanthoxylum* spp. and MKO could not be detached from MK (PC1 40.78% and PC2 20.49%). According to the bi-plot (Figure 2b), L-linalool was principal in the HJ while L-limonene, terpinen-4-ol, and *β*-phellandrene were among the major components found in MK and MKO. By interpreting the volatile substances according to their descriptors using a heatmap, it was found that HJ was also separated from other species (Figure 3) with different aromatic profile patterns.

Demands of high-quality essential oil from raw material of unique plant taxa for food and perfumery production has ramped up recently. Essential oil compositions could assist in genetic analysis of plant species thus the generic term of chemotypes is well perceived [36,37]. Based on our result of the chemometric analyses, L-linalool was separated from the others and projected with the HJ similar with the result form the RAPD analysis. Therefore, it could be used indirectly as a marker for characterization of the *Zanthoxylum* species. More importantly, the heat mapping of the odor descriptors also convinced that of all the analyzed species, HJ represents the citrusy-floral aroma which is its unique aroma identity. This has been described as the generic perception of Sichuan pepper aroma [38]. Besides, the volatile compositions, non-volatiles such as alkylamides and polyphenols are known as specific chemotypes of the *Zanthoxylum* spp. These compounds offer spice flavor with tingling and numbing sensations [37–39].


**Table 1.** Chemical profiles of makhwaen, huajiao, and makwoung essential oils

RIcal: Calculated retention index. RIRef: Retention index from the referent [5]. # Values are calculated as a reference to internal standard toluene (0.003% *<sup>w</sup>*·*v*<sup>−</sup>1). Makhwaen fruit, huajiao and makwhoung essential oil were analyzed by GC-MS (MK1, MK2, MK3, HJ, and MKO). ND: not detected.

**Figure 2.** Principal component analysis (PCA) illustrating the relationships among the *Zanthoxylum* species (**a**) and bi-plot factor analysis of the chemical components of the *Zanthoxylum* essential oils (**b**). Abbreviations; HJ (huajiao), MKO (makwoung), MK1 (makhwaen from Mae Tang district), MK2 (makhwaen from Mae Rim district), and MK3 (makhwaen from Song Kwae district).

**Figure 3.** Heatmap relationship of the odor descriptors representing the volatile composition of the *Zanthoxylum* essential oils. Abbreviations; HJ (huajiao), MKO (makwoung), MK1 (makhwaen from Mae Tang district), MK2 (makhwaen from Mae Rim district), and MK3 (makhwaen from Song Kwae district).

#### *2.5. FTIR Analysis*

Fourier transform infrared spectroscopy (FTIR) spectrum patterns have been adopted to expose authentically volatile composition of plant essential oils such as those of lavender (*Lavandula officinalis*), pepper-mint (*Mentha piperita*), green doulas (*Pseudotsuga menziesii*), fir (*Abies alba*), and chicory (*Cichorium intybus*) [40,41]. The spectrum patterns of their EOs responded to the wavenumber ranges 2800–2300 and 1800–1000 cm−1) representing of free O-H bond valence and carboxylic acid broadband absorption. Our results illustrated that the oil samples were dominated by overtones and different combinations of C-H reflection and shine occurring between 500–4000 cm−<sup>1</sup> and aromatic ring at ~1600 cm−1. FTIR spectrum scans of the three *Zanthoxylum* species essential oil (MK1-3, HJ, and MKO) absorbed light at a wavenumber range of 1722–798 cm−<sup>1</sup> and 2967–2926 cm<sup>−</sup>1, respectively, therefore illustrating similar light transmission. EO of the HJ on the other hand showed distinct spectrum characteristics from other samples (Figure 4 and Table 2). This distinction

was in parallel with the odor descriptions above analyzed where HJ was indicated to have a sweet and floral scent.

**Figure 4.** Fourier transform infrared spectrophotometer (FTIR) spectra of the essential oils from five different *Zanthoxylum* species. The insertion is the inset evidence of the peaks between 500–4000 cm<sup>−</sup>1: (—) MK1, (—) MK2, (—) MK3, (—) HJ, and (—) MKO. Abbreviations; huajiao (HJ), makwoung (MKO), MK1 (makhwaen from Mae Tang district), MK2 (makhwaen from Mae Rim district), and MK3 (makhwaen from Song Kwae district).


**Table 2.** Wavenumbers and functional groups of *Zanthoxylum* spp. essential oils.

Abbreviations: huajiao (HJ), makwoung (MKO), and makhwaen (MK1-3).

#### **3. Materials and Methods**

#### *3.1. Plant Materials*

Three plant specimens of the *Zanthozylum* spp. locally known as makhwaen were collected from the local orchards in three areas: (MK1) Papea, Mae Tang district, Chiang Mai province (19◦7 27 N, 98◦42 14 E); (MK2) Pong Yang, Mae Rim district, Chiang Mai province (18◦53 24 N, 98◦49 53 E); (MK3) Yod, Song Kwae district, Nan province (19◦22 37 N, 100◦35 49 E) in September 2018. Huajiao (HJ) was harvested from Ban Rak Thai, Mok Champae, Muang district, Mae Hong Sorn province (19◦32 32 N, 97◦53 35 E) in September 2018. Makwoung specimen (MKO) was sampled from Phichai, Muang Lampang District, Lampang province (18◦22 11 N, 99◦35 44 E) in September 2018 (Table S3). Based on the samples from harvest, all samples can be divided into two groups: (i) young leaves for DNA analysis and (ii) fruits for essential oil analysis.

The morphological appearances of leaves, flower, and fruit were recorded [19,41]. Their fruits correspondent to all specimen samples were also collected for the essential quality assessment at the mature stage and subjected to the initial drying process as described in the previous report [5]. A taxonomical confirmation has been done by comparison of the taxonomical descriptions with those of the literature data [22] and also confirmed by a botanist. The sample specimens were deposited at Queen Sirikit Botanic Garden (QSBG, Mae Rim, Chiang Mai, Thailand) and the accession numbers of Trid01-05C were assigned.

#### *3.2. Morphology Relationship within Species of Zanthoxylum* spp.

Collected data of the part of Plant for classification were analyzed. Those characters were assigned and scored as plant structure: shrub = 0, tree = 1; thorn: not have thorn = 0, thorn on tree = 1; compound leaf type: odd-pinnate = 0, even-pinnate = 1; number of petals: four petals = 0, five petals = 1, more than six petals = 2; number of anthers: four anthers = 0, five anthers = 1, more than six anthers = 2; fresh fruit color: red = 0, greenish-red = 1, and dry fruit color: brown = 0, no brown = 1. These data were analyzed using cluster analysis (Dendrogram and PCA-biplot) via XLstat, version 2016.

#### *3.3. ITS and RAPD Analysis*

#### 3.3.1. DNA Extraction

For the extraction of DNA, the DT-S DNA extraction kit (Kurabo, Osaka, Japan) was used with modification of the CTAB extraction procedure. Young leaf tissue of three *Zanthozylum* spp. from five samples (0.5 g) were ground to powder using a mortar and pestle in the presence of liquid nitrogen and transferred to a 1.5 mL polypropylene centrifuge tubes and follow the steps of the DNA extraction kit. Tissue lysis-buffer (MDT) 200 μL and proteinase K (EDT) 20 μL were combined and mixed. After that, the centrifuge tubes were incubated by using the incubator at a temperature of 55 ◦C for an hour. At this stage, the centrifuge tubes were flipped every 15 min. Then, these tubes were centrifuged at 10,000× *g*. When the process was completed, the supernatant (~200 μL) was moved to a new centrifuge tubes and 180 μL lysis buffer (LDT) was added. Later, these new tubes were centrifuged with vortex for 15 s. before they were incubated at a temperature of 70 ◦C for 10 min. A solution was moved into the new cartridge tubes and west tubes, then these tubes were aerated. After that, 75 μL wash buffer (WDT) was added into the tubes. These tubes were aerated repeatedly three times to elute DNA. Then, the cartridge tubes were moved into the collection tubes. At this stage, 50 μL elution buffer (CDT) was added and left for 30 min. After that, they were aerated repeatedly for two times. Finally, the centrifuge tubes were tested and stored at a temperature of −20 ◦C.

After extraction, total DNA was quantified using a nano-drop spectrophotometer (NanoDropTM 1000 Spectrophotometer, Thermo Fisher Scientific, Bath, UK). For requantification, the extracted DNA was run on 1.5% agarose gel electrophoresis using <sup>1</sup>× TBE buffer at 5–8 V·mL−<sup>1</sup> for 30 min and visualized under BLook LED transilluminator (Genedirex, Taoyuan, Taiwan) by staining with MaestroSafe TM (Maestrogen, Las Vegas, NV, USA). The DNA solution was diluted with sterile distilled water (DI) to a concentration of 10 ng·μL−<sup>1</sup> for PCR analysis and kept at −<sup>20</sup> ◦C until use [42].

#### 3.3.2. ITS Sequence

The ITS2 sequences were amplified using the following pair of universal primers, ITS5- ITS4 (including both ITS1 and ITS2 regions), ITS5 GGAAGTAAAAGTCGTAACAAGG and ITS4 TCCTCCGCTTATTGATATGC. Each 50 μL reaction contained 5 μL 10× PCR buffer, 2.5 μL 2.5 mm MgCl2, 0.4 μL 0.2 mm deoxyribonucleotides (dNTP), 5 μL of each primer (10 ng·μL<sup>−</sup>1), 0.4 <sup>μ</sup>L 0.5 U Taq DNA polymerase (HIMEDIA, Mumbai, India), 40 <sup>μ</sup><sup>L</sup> sterile distilled water, and 5 <sup>μ</sup>L genomic DNA (50 ng·μL<sup>−</sup>1). The amplification consisted of <sup>94</sup> ◦C·2 min<sup>−</sup>1, followed by 40 cycles of 94 ◦C·45 s<sup>−</sup>1, 50 ◦C·45 s<sup>−</sup>1, and 72 ◦C·1 min<sup>−</sup>1, and ending with 72 ◦C for 5 min for the final extension. Amplified products were genotyped using 1.5% agarose gel electrophoresis. Then they were staining with MaestroSafeTM Nucleic Acid Stains (MAESTROGEN, Hsinchu, Taiwan) and visualized under UV transilluminator (BioDoc-It2 imaging systems, Analytik Jena, Thuringia, Germany) before samples were sent to sequencing at Macrogen, Inc. (Seoul, South Korea).

#### 3.3.3. RAPD-PCR Protocols

For RAPD analysis of the genomic DNA, 10-base primers from Operon Technologies (Alameda, GA, USA) and UBC (University of British Columbia, Canada) were chosen (Table S4). A total of nine primers from previous studies were screened [43–46]. The polymerase chain reaction (PCR) was adjusted to 10 μL−<sup>1</sup> containing 8 μL−<sup>1</sup> of OnePCRTM Plus (Genedirex, Taoyuan, Taiwan), 1 μL−<sup>1</sup> of 1 μm RAPD primer and 1 μL−<sup>1</sup> of 10 ng genomic DNA. All the reactions were carried out on a Flexcycler2 thermal cycler (Analytik Jena, Thuringia, Germany) using the following profile: 1 cycle, 94 ◦C, 4 min; 40 cycles, 95 ◦C, 30 s; 37 ◦C, 30 s; 72 ◦C, 60 s; 1 cycle, 72 ◦C, 10 min. The sample was separated in a 1.5% agarose gel in 1× TBE buffer. The samples were run at 70 V for 120 min. The gels were then visualized using the BLooK LED transilluminator (Genedirex, Taoyuan, Taiwan).

#### *3.4. Dendrogram Analysis*

The banding pattern for each primer was scored as diallelic (1 = band present, 0 = band absent), and stored in an Excel (Microsoft) spreadsheet file in the form of a binary matrix. To determine the genetic differentiation between the five samples accessions, 10 RAPD markers were analyzed using the statistical package XLSTAT version 2016 software. The coefficients of genetic similarity for all the pair-wise comparisons were computed using Jaccard's coefficient of similarity and then the distance matrix was subjected to cluster analysis by using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) to produce a dendrogram.

#### *3.5. Essential Oil Analysis*

The essential oil was extracted by hydro-distillation for 4 h, from 100 g of dried fruits in 600 mL of DI water in a 2 L flask Clevenger-type apparatus (MTopo®, heating mantle, Korea). The oil was dried over anhydrous sodium sulphate (Merck Co., Darmstadt, Germany) and was kept at 4 ◦C until analysis (usually within three days). The extraction was repeated twice and yield (mean value) was reported as a percentage of essential oil from dry plant material [33].

Gas chromatography-mass spectrometry (GC–MS) analysis was performed on Bruker-Scion 436 GC (Bruker, Hamburg, Germany) a Rxi 5Sil MS (30 m × 0.25 mm; 0.25 μm film thickness) (Restek, Bellefonte, PA, USA). Essential oil samples (2 μL at the dilution of 1%, *<sup>v</sup>*·*v*−1, in dichloromethane (RCI Labscan, Bangkok, Thailand) with a presence of 0.003% *<sup>w</sup>*·*v*−<sup>1</sup> toluene (RCI Labscan, Bangkok, Thailand) as an internal standard) were injected in a split mode (1:20). Temperature program includes oven temperature held for 2 min at 60 ◦C and was enhanced to 150 ◦C with 3 ◦C min−1. Then, temperature enhancement was programmed up to 270 ◦C at 5 ◦C min−<sup>1</sup> and held at this temperature

for 15 min. Other operating conditions include carrier gas was Helium with a flow rate of 1.1 mL min−1; injector and detector temperatures were 300 ◦C, and split ratio, 1:50. Mass spectra (MS), 50–500 (*m*·*z*−1) were taken at 70 eV. The mass spectra and retention indices of essential oil components were identified by comparison to MS computer library (NIST 05.L and NIST 98.L. Homologous series of C8–C20 n–alkanes (Sigma–Aldrich, Steineheim, Germany) were used for identification of all constituents by calculation of the retention indexes (RI). The compounds were confirmed by their RI as well as those from the literature [14]. The amount in <sup>μ</sup>g·mL−<sup>1</sup> of essential oil was calculated as relative to that of internal standard.

#### *3.6. Fourier Transforms Infrared Spectrophotometer (FTIR) Analysis*

The FTIR spectrometer used was Bruker model ALPHA II, Diamond ATR (Hamburg, Germany) and operating at the basic of 500–4000 cm−<sup>1</sup> wavenumbers for averaging 47 scans per spectrum [40].

#### *3.7. Statistical Analysis*

The data were statistically analyzed using a comparison of the means of yield for essential oils evaluated by Tukey Multiple Comparison's test at a 95% confidential level. A principal component analysis (PCA) was used to identify the main sources of systematic variation in the chemical compounds data using XLstat software version 2016 [5]. The amount of each volatiles was combined according to their descriptors as described in Sriwichai et al. [6] which then was used to explicate the odor profile of the essential oil. Heatmap was generated with Biovinci software (BioTuring Inc., San Diego, CA, USA).

#### **4. Conclusions**

Even though a large number of secondary metabolites interfere with DNA sequencing, morphological description is adequate for the differentiation of plant belonging to the *Zanthoxylum* genus. The locally known makhwaen were taxonomically and genetically confirmed as *Z*. *myriacanthum*. From the principal component evaluation, huajiao essential oil was described to have different aroma characteristic as compared to the rest of *Zanthoxylum* spp. analyzed. The essential oils of makwoung and makhwaens from Nan and Chiang Mai were similar in terms of quantity and characteristics of the chemical compositions. For example, limonene and sabinene represent the aroma of citrus and woody. In summary, for sourcing of the raw material, phenotypical characteristic can be used to distinguish the species. Furthermore, the chemical profile of the essential oil depends upon the genotypes which closer similarity was with makwoung and makhwaen, whereas huajiao represented the unique chemotype of citrus-floral aroma.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/plants10040803/s1, Figure S1: The dendrogram of *Zanthoxylum* spp. in North of Thailand; huajiao (HJ), makwhoung (MKO), MK1 (makhwaen from Mae Tang district), MK2 (makhwaen from Mae Rim district) and MK3 (makhwaen from Song Kwae district) derived by UPGMA from the similarity matrix based on seven morphology data (plant structure, thorn, compound leaf type, petals, anthers, fresh and dry fruit color); Table S1: Plant characteristics for taxonomical identification of collected *Zanthoxylum* spps. used in this experiment; Table S2: Floral and fruit characteristics of makhwaen collected from different locations (MK1-3); Table S3: Study site the sample collections; Table S4: Sequence of RAPD primers.

**Author Contributions:** Conceptualization, T.P. and S.R.S.; Methodology, T.P. and S.R.S.; Validation, T.S. and J.W.; Formal analysis, T.S. and J.W.; Investigation, K.D.; Data curation, T.S. and J.W.; Writing original draft preparation, T.S., J.W., and S.R.S.; Writing—review and editing, S.R.S., K.D., S.K.P., and B.C.; Visualization, T.S. and J.W.; Supervision, T.P. and S.R.S.; Funding acquisition, K.S., B.C. and K.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research work was partially supported by Chiang Mai University.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We would like to acknowledge the external supports from Cosmo Ingredients, Mougins, France. We also appreciate the generosity of Anne Marie Saget and Wei Raksa during course of experimentation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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