*Article* **Antidiarrheal and Cardio-Depressant Effects of** *Himalaiella heteromalla* **(D.Don) Raab-Straube: In Vitro, In Vivo, and In Silico Studies**

**Fatima Saqib 1,\*, Faisal Usman 1, Shehneela Malik 1, Naheed Bano 2, Najm Ur-Rahman 3, Muhammad Riaz 3, Romina Alina Marc (Vlaic) 4,\* and Crina Carmen Mure¸san <sup>4</sup>**


**Abstract:** *Himalaiella heteromalla* (D.Don) Raab-Straube is a commonly used remedy against various diseases. Crude extract and fractions of *H. heteromalla* were investigated for a gastrointestinal, bronchodilator, cardiovascular, and anti-inflammatory activities. *H. heteromalla* crude extract (Hh.Cr) relaxed spontaneous contractions and K+ (80 mM)-induced contraction in jejunum tissue dosedependently. The relaxation of K+ (80 mM) indicates the presence of Ca++ channel blocking (CCB) effect, which was further confirmed by constructing calcium response curves (CRCs) as they caused rightward parallel shift of CRCs in a manner comparable to verapamil, so the spasmolytic effect of Hh.Cr was due to its CCB activity. Application of Hh.Cr on CCh (1 μM) and K+ (80 mM)-induced contraction in tracheal preparation resulted in complete relaxation, showing its bronchodilator effect mediated through Ca++ channels and cholinergic antagonist activity. Application of Hh.Cr on aortic preparations exhibited vasorelaxant activity through angiotensin and α-adrenergic receptors blockage. It also showed the cardio suppressant effect with negative chronotropic and inotropic response in paired atrium preparation. Similar effects were observed in in vivo models, i.e., decreased propulsive movement, wet feces, and inhibition of edema formation.

**Keywords:** antidiarrheal; calcium ion channel; cardio-depressant; *Himalaiella heteromalla*

#### **1. Introduction**

*Himalaiella heteromalla* (D.Don) Raab-Straube (*Asteraceae*), commonly known as Batula, is found in low-temperature regions of Asia, Europe, and North America [1]. *Himalaiella heteromalla* is a rich source of chlorojanerin, arctigenin [2] glycosides, alkaloids, terpenoids, saponins, flavonoids, sesquiterpene lactones, and arctiin [3]. Gao et al. [4] and Kang et al. [5] reported arctigenin and its glycoside, arctiin, have anti-inflammatory activities by inhibiting iNOS and exerting vasodilation effect, while Hayashi et al. [6] reported the anti-viral activity against influenza A virus.

Traditionally, *H. heteromalla* is used in herbal products to treat fever, menstruation, circulation, pain, and rheumatic arthritis [7]. It is used in wounds, cuts, and fever [8]. The leaf paste and mustard oil mixture are used for wounds and leukoderma. It has carminative property, used for coeliac diseases [9,10]. It is used to remedy burning parts of the body, menstrual problems, piles, psoriasis, rheumatoid arthritis, cardiotonic cough with cold, and altitude sickness, and provide anticancer and anti-fatigue actions [11]. It is used as an

**Citation:** Saqib, F.; Usman, F.; Malik, S.; Bano, N.; Ur-Rahman, N.; Riaz, M.; Marc (Vlaic), R.A.; Mure¸san, C.C. Antidiarrheal and Cardio-Depressant Effects of *Himalaiella heteromalla* (D.Don) Raab-Straube: In Vitro, In Vivo, and In Silico Studies. *Plants* **2022**, *11*, 78. https://doi.org/ 10.3390/plants11010078

Academic Editor: Antonella Smeriglio

Received: 7 December 2021 Accepted: 20 December 2021 Published: 27 December 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/).

anti-inflammatory and prevents ischemic stroke [3]. Therefore, *H. heteromalla* was investigated in in vitro, in vivo, and in silico models as a possible tool to treat gastrointestinal, cardiovascular, respiratory, and inflammatory ailments.

#### **2. Results**

#### *2.1. Phytochemical Analysis of Himalaiella heteromalla*

The preliminary phytochemical analysis of Hh.Cr confirmed the presence of glycosides, saponins, alkaloids, and flavonoids.

#### *2.2. HPLC Separation of Phenolic Acids and Flavonoids*

The separation factor and resolution of all separated compounds were >1.0 and >1.5, respectively. The reproducibility of separate components was also good with RSD < 2% (run to run) and 2.7% (day to day) [12]. The HPLC chromatograms for the identified phenolic and flavonoid compounds are shown in Figure 1, and Table 1 presents the phenolic and flavonoid compounds identified in Hh.Cr. The most abundant phenolic compounds were gallic acid (184.98 μg/g), hydroxybenzoic acid (6.8 μg/g), and vanillic acid (8.1 μg/g); while the identified flavonoid compound was catechin (160.37 μg/g).

**Figure 1.** HPLC chromatogram of (**A**) standard phenolic compounds (**B**) Hh.Cr.

**Table 1.** Phenolic and flavonoid compounds of Hh.Cr.


#### *2.3. Effect on Jejunum Preparations*

The Hh.Cr and its ethyl acetate (Hh.Ea) fraction showed the relaxant effect on exposure to the rhythmic contraction of jejunum preparations in organ bath within concentration range 0.01 to 0.3 mg/mL with EC50 0.06 mg/mL (95% CI: 0.045–0.080 mg/mL; n = 5) and 0.01 to 0.1 mg/mL with EC50 0.032 mg/mL (95% CI: 0.021–0.51 mg/mL; n = 5), respectively similar to verapamil with EC50 0.42 μM (95% CI: 0.22–1.27), whereas aqueous fraction *H. heteromalla* failed to complete relaxation of spontaneous contractions of jejunum preparations. Hh.Cr and Hh.Ea also caused a complete relaxation of K+ (80 mM) induced spastic contractions at 1 mg/mL with EC50 0.13 mg/mL (95% CI: 0.088–0.220 mg/mL; n = 5) and 0.3 mg/mL with EC50 0.06 mg/mL (95% CI: 0.045–0.0.089 mg/mL; n = 5), respectively similar to verapamil with EC50 0.251 μM (95% CI: 0.082–0.784). Furthermore, Hh.Cr showed a rightward shift of calcium CRCs which confirm the presence calcium ion channel blockade activity in Hh.Cr, similar to verapamil (Figures 2 and 3).

**Figure 2.** (**A**) Control spontaneous contraction. Effect of (**B**) crude extract (Hh.Cr), (**C**) Ethyl acetate fraction (Ea.Hh), (**D**) Aqueous fraction (Ea.Aq), and (**E**) verapamil on spontaneous. Effect of (**F**) crude extract (Hh.Cr), (**G**) Ethyl acetate fraction (Ea.Hh), (**H**) Aqueous fraction (Ea.Aq), and (**I**) verapamil on K+ Induced Contraction on rabbit jejunum preparations.

**Figure 3.** Effect of crude extract (Hh.Cr) Ethyl acetate fraction (Ea.Hh) and aqueous fraction (Ea.Aq) on (**A**) spontaneous contraction and (**B**) K<sup>+</sup> (80 mM)-induce Contraction on rabbit jejunum preparations. (**C**) Effect of verapamil on spontaneous and K+ induced contraction on rabbit jejunum preparations. Dose–response curves of Ca++ in the presence and absence of (**D**) Hh.Cr (**E**) verapamil in the isolated rabbit jejunum preparations. Values are expressed as mean ± SEM.

#### *2.4. Effect on Tracheal Preparations*

The Hh.Cr and its ethyl acetate (Hh.Ea) fraction showed the relaxant effect on tracheal preparations, when exposed K+ (80 mM) and CCh (1 μM)-induced contractions. Hh.Cr and its Hh.Ea fraction relaxed the K+ (80 mM) induce contractions at 0.3 mg/mL with EC50 0.19 mg/mL (95% CI: 0.099–0.452; n = 5) and 0.1 mg/mL with EC50 0.042 mg/mL (95% CI: 0.024–0.072 mg/mL; n = 5), respectively. Hh.Cr and its Hh.Ea fraction also relaxed the CCh (1 μM) induce contractions at 1 mg/mL with EC50 0.23 mg/mL (95% CI: 0.158–0.357 mg/mL; n = 5) and 0.3 mg/mL with EC50 0.155 mg/mL (95% CI: 0.076–0.302 mg/mL; n = 5), respectively. Similarly, verapamil also caused relaxation of K+ (80 mM) and CCh (1 μM) induced contractions with respective EC50 0.82 μM (95% CI: 0.82–0.82 μM) and EC50 values of 2.35 μM (95% CI: 02.32–2.39 μM). The aqueous fraction (Hh.Aq) exerted partially relaxation of K<sup>+</sup> (80 mM) and CCh (1 μM)-induced contractions on tracheal preparation (Figure 4).

**Figure 4.** Effect of (**A**) crude extract (Hh.Cr) (**B**) Ethyl acetate fraction (Ea.Hh) (**C**) Aqueous fraction (Ea.Aq) on K<sup>+</sup> Induce Contraction and Effect of (**D**) crude extract (Hh.Cr) (**E**) Ethyl acetate fraction (Ea.Hh) (**F**) Aqueous fraction (Ea.Aq) on CCh-induced contraction on rabbit tracheal preparations. Effect of crude extract (Hh.Cr) ethyl acetate fraction (Ea.Hh) and aqueous fraction (Ea.Aq) of on (**G**) K+ (80 mM) Induce Contraction and (**H**) CCh-induced contraction on tracheal preparations. (**I**) Effect of verapamil on CCh1 μM and K<sup>+</sup> induced contraction on rabbit tracheal preparations. Values are expressed as mean ± SEM.

#### *2.5. Effect on Aortic Preparations*

The crude extract (Hh.Cr) and its ethyl acetate (Hh.Ea) fraction showed the relaxant effect on aortic preparations, when exposed K<sup>+</sup> (80 mM) and PE (1 μM) induced contractions. Hh.Cr and its Hh.Ea fraction relaxed the K<sup>+</sup> (80 mM) induce contractions at 3 mg/mL with EC50 2.88 mg/mL (95% CI: 2.106–4.156 mg/mL; n = 5) and 1 mg/mL with EC50 0.148 mg/mL (95% CI: 0.09491–0.2332 mg/mL; n = 5), respectively. Hh.Cr and its Hh.Ea fraction also relaxed the PE (1 μM) induce contractions at 5 mg/mL with EC50 15.53 mg/mL (95% CI: 7.965 to 62.27 mg/mL; n = 5) and 3 mg/mL with EC50 4.2 mg/mL (95% CI: 2.991 to 6.670 mg/mL; n = 5), respectively. Similarly, verapamil also caused relaxation of K+(80 mM) and PE (1 μM) induced contractions with respective EC50 1.054 μM (95% CI: 0.45–5.68) and 0.764 μM (95% CI: 0.33–68.8). The aqueous fraction of (Hh.Aq) partially exerted relaxation of K+(80 mM) and PE (1 μM) induced contractions on aortic preparation (Figure 5).

**Figure 5.** Effect of (**A**) crude extract (Hh.Cr), (**B**) Ethyl acetate fraction (Ea.Hh), (**C**) Aqueous fraction (Ea.Aq), and (**D**) verapamil on K+ induced contraction and effect of (**E**) crude extract (Hh.Cr), (**F**) Ethyl acetate fraction (Ea.Hh), (**G**) Aqueous fraction (Ea.Aq), and (**H**) verapamil on PE 1 μM Induced Contraction on rabbit aorta preparations. Effect of crude extract (Hh.Cr) Ethyl acetate fraction (Ea.Hh) and Aqueous fraction (Ea.Aq) on (**I**) K+ (80 mM) Induced Contraction and (**J**) PE 1 μM Induced Contraction on aortic jejunum preparations. (**K**) Effect of verapamil on PE 1 μM and K<sup>+</sup> Induced Contraction on rabbit tracheal preparations. Values are expressed as mean ± SEM.

#### *2.6. Effect on Atria Preparations*

The crude extract (Hh.Cr) and its ethyl acetate (Hh.Ea) fraction caused the negative chronotropic effect (i.e., decrease in heart rate) and negative inotropic effect (i.e., force of contraction) on atrium preparation [12]. Hh.Cr and its Hh.Ea fraction showed negative inotropic effect within concentration range 0.01–5.0 mg/mL with EC50 0. 9 mg/mL (95% CI: 0.375–1.356 mg/mL; n = 3) and 0.01–3.0 mg/mL with EC50 0.7 mg/mL (95% CI: 0.265–0.586 mg/mL; n = 3), respectively. Hh.Cr and its Hh.Ea fraction showed the negative chronotropic effect within concentration range 0.01–5.0 mg/mL with the EC50 0.5 mg/mL (95% CI: 0.406–0.680 mg/mL; n = 3) and 0.01–3.0 mg/mL with the EC50 value calculated to be 0.4 mg/mL (95% CI: 0.106–0.050 mg/mL; n = 3), respectively. Similarly, verapamil also showed negative inotropic and chronotropic effect with concentration range 0.01–1.0 mg/mL with EC50 value of 0.053 μM (95% CI: 0.034–0.084 μM; n = 3) and 0.01–0.3 mg/mL with EC50 0.037 μM (95% CI: 0.024–0.045 μM; n = 3). The aqueous fraction (Hh.Aq) exerted partially negative inotropic and chronotropic effect on atrium preparation with in concentration range 3–10 mg/mL with EC50 1.02 mg/mL (95% CI: 0.485–0.856 mg/mL; n = 3) and 3–10 mg/mL with 1.14 mg/mL (95% CI: 0.575–1.756 mg/mL; n = 3), respectively (Figure 6).

**Figure 6.** (**A**) Spontaneous contraction. Effect of (**B**) crude extract (Hh.Cr), (**C**) Ethyl acetate fraction (Ea.Hh), and (**D**) Aqueous fraction (Ea.Aq) on spontaneous contraction rabbit paired atrium preparations. (**E**) Effect of verapamil on spontaneous contraction rabbit paired atrium preparations. Effect of crude extract (Hh.Cr) Ethyl acetate fraction (Ea.Hh) and Aqueous fraction (Ea.Aq) on (**F**) K+(80mM)-induced contraction. (**G**) force of contraction. (**H**) Effect of verapamil on the force of contraction and heart rate on rabbit atrium preparations. Values are expressed as mean ± SEM.

#### *2.7. Antiperistalsis Activity*

The crude extract (Hh.Cr) showed a significant antiperistalsis response in mice with less distance traveled by charcoal meal as compared to control (33 ± 2.3%). The group was treated with 400 mg/kg of Hh.Cr and CCh (3 mg/kg) and peristaltic movements were significantly decreased by 1.2 ± 0.37 and 8.6 ± 1.8, respectively (Figure 7).

**Figure 7.** GI Charcoal meal transit (antiperistalsis) activity, castor oil-induced diarrhea activity, and carrageenan induce inflammation. Values are expressed as Mean ± SEM, and data was analyzed One way ANOVA or Two way ANOVA; \* *p* < 0.05, \*\* *p* < 0.005, \*\*\* *p* < 0.0005 and \*\*\*\* *p* < 0.0001.

#### *2.8. Antidiarrheal Activity*

The crude extract (Hh.Cr) showed a significant antidiarrheal response in rats with fewer wet fecal masses than control (15.60 ± 1.4). The group was treated with 400 mg/kg of Hh.Cr and loperamide (3 mg/kg) showed highly significant anti-diarrheal effect 1.2 ± 0.37 and 0.8 ± 0.37, respectively (Figure 7).

#### *2.9. Anti-Inflammatory Activity*

The crude extract (Hh.Cr) showed a significant anti-inflammatory response in rats with inhibition of edematous volume of hind paw as compared to control (2.93 ± 0.2 mL) at maximum duration. The Hh.Cr inhibited the paw edema at 100 mg/kg as 0.96 ± 0.08 mL, 1.10 ± 0.01 mL, 1.16 ± 0.01 mL, 1.12 ± 0.015 mL, and 1.23± 0.01 mL at 0, 1, 2, 3, and 4 h duration, respectively, whereas at dose 300 mg/kg, it showed maximum inhibition, i.e., 0.87 ± 0.01 mL, 0.86 ± 0.01 mL, 0.86 ± 0.03 mL, 0.91 ± 0.01 mL, 1.04 ± 0.001 mL, and 1.15 ± 0.02 mL at 0, 1, 2, 3, and 4 h duration, respectively. Hh.Cr inhibited the edematous volume as similar to the aspirin, i.e., 1.05 ± 0.02 mL (Figure 7).

#### *2.10. In Silico Studies*

The docking calculations are beneficial to predict ligand pose within the binding site of the target protein. The involvement of physical energies terms (i.e., solvation energy) with suitable force field make docking calculation of compounds more acceptable with accuracy (Table 2) [12–14].


**2.**Bindingenergies(kcal/mol)ofcompoundswithMuscarinic-3(MM3,PDBID:4U14),Cyclooxygenase-2(COX-2,PDBID:5IKQ)


**Table 2.** *Cont.*



**Table 2.** *Cont.*

*Molecular docking for Muscarinic M3 receptor:* The selected compounds were studied against muscarinic M3 (MM3, PDB ID: 4U14)) for antispasmodic activity (Table 2, Figure 8). Arctiin was predicted with the lowest binding energy (ΔGbind: −60.79 kcal/mol) with hydrophobic energies ΔGvdW (−55.32 kcal/mol) and ΔGLipo (−42.47 kcal/mol) major contributors to the ligand binding energy. It formed the two π-donor hydrogen interaction with residue Trp525 and hydrophobic interactions (π-π Stacked Bond: Trp503; π-π T shaped Bond: Tyr148) within the pocket of MM3. Besides these, it also formed the π-Sulfur interaction with residue Cys532. Arctigenin second to arctiin also found potent with have ligand binding energy (ΔGbind: −46.56 kcal/mol) mainly contributed with hydrogen bond interaction (ΔGHbond: −0.68 kcal/mol) and hydrophobic interaction (ΔGvdW: −34.45 kcal/mol and ΔGLipo: −30.61 kcal/mol). It formed hydrophobic π-π T-shaped interaction with residue Trp503 and Trp525 within the protein cleft. Moreover, catechin has the lowest binding energy (ΔGbind: 52.22 kcal/mol) with ΔGvdW (−34.12 kcal/mol) and ΔGLipo (−13.93 kcal/mol) and formed π-π T shaped interaction with residue Tyr506. The ranking orders of ligands with COX-2 is given below: arctiin > arctigenin > catechin > chlorojanerin > cynaropicrin.

*Molecular docking for cyclooxygenase-2 enzyme*: The selected compounds were studied against cyclooxygenase-2 enzyme (COX-2, PDB ID:5IKQ) for anti-inflammatory activity (Table 2, Figure 8). Arctiin was predicted with the lowest binding energy (ΔGbind: −41.01 kcal/mol) among the selected compounds. As mentioned earlier, Van der Waals (ΔGvdW) and lipophilic interactions (ΔGLipo) are significant contributors to the ligand binding energy. It was observed that the binding energies value of ΔGvdW was −36.96 kcal/mol, and ΔGLipo was −23.77 kcal/mol, whereas hydrogen bond (ΔGHbond) energy contribution was −2.56 kcal/mol. It also formed hydrophobic interactions π-π T shaped interaction with Tyr11. Arctigenin second to arctiin in docking score was found with potential hydrophobic interactions within hydrophobic clefts of COX-2. The ligand binding energy of arctigenin (ΔGbind: −27.91 kcal/mol) was driven mainly by these hydrophobic interaction energies; Δ GvdW (−35.44 kcal/mol) and ΔGLipo (−31.09 kcal/mol) and formed hydrophobic π–σ interaction with Val117. The ranking orders of ligands with COX-2 are given below: arctiin > arctigenin > cynaropicrin > catechin.

*Molecular docking for lipoxygenase-5 enzyme:* The selected compounds were studied against lipoxygenase-2 enzyme (LOX-5, PDB ID: 6N2W) for anti-inflammatory activity (Table 2, Figure 8). The contribution of hydrophobic interactions in ligand binding energy was more abundant within pockets of LOX-5. Arctigenin has higher ligand binding energy (ΔGbind: −42.94 kcal/mol) but ranks third in the docking score. The ligand binding energy contributed with hydrogen bond interaction (ΔGHbond: −3.06 kcal/mol) and hydrophobic interaction (ΔGvdW: −29.61 kcal/mol and ΔGLipo: −18.99 kcal/mol). It also formed hydrophobic interactions (π-π Stacked Bond: His372) within the pocket of LOX-5. Arctiin ranked at first in position docking score with ligand binding energy (ΔGbind: −30.76 kcal/mol) which mainly contributed from hydrophobic interaction energies ΔGvdW (−46.53 kcal/mol) and ΔGLipo (−16.17 kcal/mol), whereas hydrogen bond (ΔGHbond) energy contribution was −2.11 kcal/mol. Besides hydrophobic interaction, arctigenin and arctiin also formed electrostatic charge interaction with residue Arg596 and Ile673 within the cleft of COX-2, respectively. Catechin have ligand binding energy (ΔGbind: −30.81 kcal/mol) mainly contributed with hydrogen bond interaction (ΔGHbond: −2.38 kcal/mol) and hydrophobic interaction (ΔGvdW: −32.68 kcal/mol and ΔGLipo: −15.10 kcal/mol). Catechin formed the π–donor hydrogen interaction with residue His372 and hydrophobic interactions (π-π Stacked Bond: His367; π-π T shaped Bond: His372, Trp599) within the pocket of LOX-5. The ranking order of ligands with COX-2 is given below: arctiin > catechin > arctigenin > cynaropicrin > chlorojanerin

**Figure 8.** Molecular docking of selected compounds against muscarinic receptor, cyclooxygenase-2, and lipoxygenase 5.

#### **3. Discussion**

*Himalaiella heteromalla* has a potential pharmacological role in the management of various diseases. This research was employed to investigate its pharmacological characteristics. The presence of alkaloids, glycosides, triterpenoids, flavonoids, saponins, sesquiterpene, which play a vital role in the pharmacological potential of *Himalaiella heteromalla*. The HPLC results indicate the presence of gallic acid, catechin, HB acid, and vanillin acid. Gallic acid (3,4,5trihydroxybenzoic acid), a natural polyphenol product, has anti-oxidant, anti-inflammatory, antimicrobial, and radical scavenging activities. Gallic acid is used as a spasmolytic effect on smooth muscle isolated jejunum tissues and trachea by calcium channel blocking activity [15]. Gallic acid is used as an antispasmodic in diarrhea [16]. Gallic acid possesses an anti-inflammatory effect [17]. Catechin abundant flavonoid present in plants, it reported for several gastrointestinal, respiratory, and inflammatory disorders [18–20]. Vanilla acid and HB acid are polyphenolics used for gastrointestinal, respiratory, and cardiovascular disorders by spasmolytic effects on isolated tissues of the jejunum, trachea, and aorta [20]. The water content in *H. heteromalla* play a vital role in the biological activities, so it is more important to measure the water content in *H. heteromalla* therefore, infra red radiation can be used to measure water content determination [21].

*Himalaiella heteromalla* crude extract (Hh.Cr) was studied on isolated jejunum preparations to elaborate the mechanism of *H. heteromalla* in gastrointestinal diseases. It is reported that jejunum preparations have rhythmic contractions due to the influx of calcium ions and potassium ions through their respective ions channels. *H. heteromalla* crude extract and its fraction ethyl acetate exerted spasmolytic response in dose concentration when exposed to spontaneous contraction of jejunum preparations [22]. Thus, *H. heteromalla* crude extract and its fraction ethyl acetate showed the antispasmodic response by suppressing rhythmic contractions in jejunum preparations. These results indicate that *H. heteromalla* crude extract and its fraction ethyl acetate decrease or blockade the cytoplasmic free Ca++ ions through the blockade of voltage-dependent calcium ion channels. As a result, activation of calmodulin and other contractile proteins, i.e., actin and myosin, does not occur [23]. *H. heteromalla* crude extract and its fraction ethyl acetate and aqueous were exposed to K<sup>+</sup> (80 mM)-induced contractions on jejunum preparations, *H. heteromalla* crude extract and its fraction ethyl acetate relaxed the K<sup>+</sup> (80 mM)-induced contractions in dose concentration manner in a tissue organ bath. It was previously reported that K+ (80 mM) induces contractions to cause cell depolarization by the influx of calcium ions into the cell through the voltage-gated L-type calcium ion channel [24]. Similar to verapamil, any substance inhibited K<sup>+</sup> (80 mM)-induced contractions were considered calcium channel blockers (CCB). Thus, *H. heteromalla* crude extract and its fraction ethyl acetate blockade the calcium influx into the cell by alternating or binding with voltage-dependent calcium channels. Furthermore, calcium concentration–response curves (CRCs) were constructed on pretreated Hh.Cr jejunum preparations to confirm the calcium channel blockade activity of Hh.Cr in a tissue organ bath. The results showed that partial blockade with the rightward parallels dose–response curves at low doses while completely blocking the dose–response curves at 0.3 mg/m. Thus, *Himalaiella heteromalla* exhibited a strong calcium antagonistic effect [25].

To evaluate another possible mechanism of *H. heteromalla* crude extract on the gastrointestinal tract, Hh.Cr was studied in antiperistalsis and antidiarrheal in vivo models. *H. heteromalla* crude extract showed the antispasmodic response by inhibiting the traveling of charcoal meal in antiperistalsis activity. *H. heteromalla* crude extract also inhibited diarrheal response in castor oil-induced diarrhea. It decreased the wet feces by inhibiting the electrolyte and water imbalance that may cause diarrhea in rats [26].

*H. heteromalla* crude extract and its fractions ethyl acetate and aqueous were tested for possible bronchodilator activity against CCh (1 μM) and K<sup>+</sup> (80 mM)-induced contractions on tracheal preparations. The results showed that *H. heteromalla* crude extract and its fractions ethyl acetate exhibited relaxant response against CCh (1 μM) and K<sup>+</sup> (80 mM)- induced contractions, but a partial relaxant effect was observed by the aqueous fraction. However, EC50 of *H. heteromalla* crude extract and its fractions ethyl acetate against K+ (80 mM) induced contractions that were more minor than CCh-induced contractions, similar to that of verapamil. CCh is a cholinergic agonist which causes smooth muscle contraction through activation of muscarinic receptors. Hence, *H. heteromalla* crude extract and its fractions ethyl acetate showed bronchodilator response was found due to Ca++ ion channel and muscarinic receptor blockade. Nowadays, Ca++ channel blockers and muscarinic antagonists are used to treat the relief from respiratory diseases such as asthma [27,28].

*H. heteromalla* crude extract and its fractions ethyl acetate and aqueous were tested for possible vasorelaxant activity against PE (1 μM) and K+ (80 mM)-induced contractions on aortic preparations. The results showed that *H. heteromalla* crude extract and its fractions ethyl acetate exhibited a relaxant response against PE (1 μM) and K<sup>+</sup> (80 mM)-induced contractions, similar to verapamil. *H. heteromalla* aqueous fraction partially relaxed the PE and K<sup>+</sup> (80 mM) induced contractions. Relaxation of the PE (PE) and K<sup>+</sup> (80 mM) induced contractions indicates a blockade of intracellular Ca++ influx by blocking Ca++ channels. Ca++ channel blockers are essential drugs used clinically to manage angina and hypertension [29,30].

*H. heteromalla* crude extract and its fractions ethyl acetate and aqueous were tested on paired atrium for possible effects on force and rate of atrial contractions. *H. heteromalla* crude extract and its fractions ethyl acetate and aqueous showed cardio suppressant response via blocking calcium channels, hence Hh.Cr and its fractions were found with adverse inotropic and chronotropic effects on the paired atrium [31].

*Himalaiella heteromalla* crude extract was tested for anti-inflammatory activity. It was found that Hh.Cr blocked the release of inflammatory mediators in rat paw edema and other models. It is reported that carrageenan acetic acid and formalin release inflammatory mediators such as bradykinin, histamine, TNF, IL-1b, IL-6, PEG2, and TNF were blocked by crude extract of crude extract *Himalaiella heteromalla.* The reduction in inflammatory mediators by carrageenan, inducing the rat's paw edema model to show that Hh.Cr inhibits factors that cause inflammation and swelling. On the other side, pain sensation is a significant indicator in the inflammation process, which Hh blocked Cr, so that it exhibited analgesic activity. This anti-inflammatory result was compared with standard drug analgesic aspirin, reducing all models' inflammation and pain sensations. The comparative results in between aspirin and Hh.Cr showed that Hh.Cr exhibited thde same potential as aspirin to reduce the pain and inflammation via blockading inflammatory mediators [32].

Molecular docking is a helpful tool to predict the possible mechanism of actions of the selected compounds of various pharmacological studies—the present study correlated and defined antispasmodic and anti-inflammatory activities of *Himalaiella heteromalla.* The five compounds of *H. heteromalla* were studied for cyclooxygenase 2, lipoxygenase 5, and muscarinic M3 receptor. The docking calculations of these compounds indicate the presence of antispasmodic and anti-inflammatory activities, which were previously proven in experimental studies. Arctiin and arctigenin were more potent compounds responsible for these activities. These results conclude that these compounds interact with cyclooxygenase 2, lipoxygenase 5, and muscarinic M3 receptor to exert the activity. As mentioned earlier, Gao et al. [4] reported that arctigenin and arctiin have anti-inflammatory and vasodilation properties and help treat acute lung injury, local edema, brain trauma, and colitis. These studies support the potent results of arctigenin and arctiin in silico studies against major inflammatory proteins COX-2 and LOX-5.

The pretreatment of various chemical, physical, physicochemical, and biological methods have been suggested to improve enzymatic hydrolysis; these techniques can be improved by the activities of extract [33–35]. In addition, the pretreatment and pyrolysis process require a high amount of external heat for (1) drying the washed biomass, (2) biomass torrefaction, and (3) biomass pyrolysis. Biomass drying and pyrolysis require a high amount of external heat for drying and torrefaction [36,37]. The plant also contains potassium (K), calcium (Ca), sodium (Na), and magnesium (Mg), which will significantly affect the behaviors of extract activity. It is essential to remove the metals by adopting different methods [38].

#### **4. Materials and Methods**

#### *4.1. Extract Preparation*

*Himalaiella heteromalla* (D.Don) Raab-Straube was collected from hilly areas of Islamabad and identified by Dr. Zafarullah Zafar, taxonomist, Institute of Pure and Applied Biology, and submitted with voucher no: http://www.theplantlist.org/tpl1.1/record/gcc-138921 dated 18 June 2018. Plant material was ground through a herbal grinder for coarse powder, then powder (1 kg) was macerated in methanol aqueous (70:30) for maceration in

an amber color glass bottle for three days at room temperature and periodically shaken 3–4 times a day. The solvent was filtered to remove plant debris with muslin cloth and Whattman-1 filter paper. This procedure was replicated thrice, and the filtrate obtained by all steps was combined and processed in a rotary evaporator (BUCHI) under reduced pressure at 36 ± 2 ◦C to obtain a brownish colored semi-solid (Hh.Cr) and stored at −20 ◦C in an airtight jar with a percentage yield of 12%. The Hh.Cr (20 g) was subjected to solvent– solvent extraction with ethyl acetate and distilled water to produce an ethyl-acetate fraction (Hh.Ea) and aqueous fraction (Hh.Aq) with approximately 5.5% and 40% yield, respectively. *H. heteromalla* crude extract (Hh.Cr) and its fractions were moderately soluble in aqueous. All dilutions were prepared fresh on the day of the experiment.

#### *4.2. Animal Housing*

Both sexes of albino mice (weight: 20–30 g), rats (weight: 150–200 g), and rabbits (weight: 1–1.8 kg) were used in this study and kept under controlled housing conditions with a temperature of 23 ± 3 ◦C in the animal house of the Faculty of Pharmacy, Bahauddin Zakariya University, Multan. Before the experiment, animals were deprived of food overnight but had free access to water. For in vitro experimentation, rabbits were sacrificed following a blow, while mice and rats were killed by cervical dislocation. All the experimentations were performed under rules specified by the Institute of Laboratory Animal Resources, Commission on Life Sciences (NRC, 1996) endorsed by the Ethical Committee of Bahauddin Zakariya University, Multan.

#### *4.3. Chemicals*

All the chemicals used in this study have high purity with research-grade quality. Acetylcholine (Ach), aspirin, carbamylcholine chloride HCl, Carbachol (CCh), verapamil HCl, phenylephrine (PE) were purchased from Sigma Chemical Company, St. Louis, MO, USA. While Potassium dihydrogen phosphate, magnesium chloride, sodium bicarbonate, sodium chloride, magnesium sulfate, sodium dihydrogen phosphate, calcium chloride, potassium chloride, ethylenediaminetetraacetic acid (EDTA), glucose were purchased from Merck, Dermstadat Germany. Furthermore, loperamide, and dicyclomine were supplied by Sigma Chemical company, St. Louis, MO, USA).

#### *4.4. Qualitative Phytochemical Detection*

The qualitative phytochemical investigation of *H. heteromalla* was performed to identify alkaloids, glycosides, anthraquinones, terpenes, saponins, flavonoids, and phenols.

#### *4.5. HPLC Separation of Phenolic Acids and Flavonoids*

The phenolic acids and flavonoids components in *Himalaiella heteromalla* were quantified by developing a binary gradient solvent system to run in Chromera HPLC system (Perkin Elmer, Houston, TX, USA) consisting of Felexer Binary Liquid chromatography (LC) pump coupled with UV/Vis LC Detector (Shelton, CT, USA) which was operated with the help of a software. HPLC system consisted of a C-18 column (250 × 4.6 mm internal diameter) with a thickness of 5 μM film. The mobile phase consisted of solvent A (methanol (30): acetonitrile (70)) and solvent B (0.5% glacial acetic acid in double-distilled water), mobile phase run at flow rate 0.08 mL/min, and data was recorded at 275 nm of UV spectra. The peaks and retention times of phenolic acids and flavonoids of *H. heteromalla* were matched with external standards to quantify the components [12]. The resolution and separation factor was used to determine HPLC separation efficiency.

#### *4.6. In Vitro Experiments*

The physiological response of tissues was recorded with isotonic and force-displacement isometric transducers amplified with acquisition system Power Lab (AD Instruments, Bella Vista, NSW, Australia) coupled with a computer having Lab chart Pro. The effect was taken as percent change on the part of test substance recorded instantly preceding a dose of test substance [22].

#### 4.6.1. Isolated Rabbit Jejunum Preparation

The jejunum was dissected from a rabbit; the adhesive fatty tissues were carefully removed, and then ~2 cm long piece of jejunum was prepared. This tissue was hung in an organ bath containing Tyrode's solution with a continuous supply of carbogen (95% O2 + 5% CO2) at 37 ◦C and equilibrated for 30 min. Acetylcholine (1 μM) was added to spontaneous rhythmic contractions of jejunum for control response and washed it. The Hh.Cr was added cumulatively for antispasmodic effect. The spontaneous contractions jejunum preparation was exposed to K+ (80 mM) induced contraction for estimation of CCB activity [39].

The extract was exposed to calcium concentration response curves (CRCs) for further confirmation. The jejunum preparation was stabilized in Tyrode's solution, subsequently replaced with calcium-free Tyrode's solution with EDTA (0.1 mM) to remove calcium from tissue. Afterward, with an incubation duration of 40 min, two superimposable control calcium CRCs were constructed in an organ bath, then tissue was incubated with the plant exact for one h, and calcium CRCs were obtained and compared to control. The calcium CRCs were recorded in the presence of different concentrations of plant extract.

#### 4.6.2. Isolated Rabbit Tracheal Preparations

The trachea was dissected from a rabbit for bronchodilator activity, and the 2 mm tracheal ring tissue was prepared. A longitudinal incision was made opposite the smooth muscle layer to form a strip. This tracheal preparation was hung in an organ bath containing Krebs's solution with a continuous supply of carbogen at 37 ◦C. Preload tension (1 g) was applied and allowed to equilibrate for 60 min prior to the dose of any drug. The tracheal preparation was exposed to CCh (1 μM), and K<sup>+</sup> (80 mM) induced contraction for bronchodilator activity in a cumulative manner.

#### 4.6.3. Isolated Rabbit Paired Atria Preparations:

The heart was dissected from a rabbit for cardiac activity, and the ventricles were carefully removed to isolate paired atria. This atrium preparation was hung in an organ bath containing Krebs's solution with a continuous supply of carbogen at 34 ◦C. Then, 1 g preload tension was applied and allowed to equilibrate for 30 min prior to the dose of any drug. The isolated atrium preparation was exposed for possible cardiac effects in a cumulative fashion, and changes in rate and force of contractions were observed.

#### 4.6.4. Isolated Rabbit Aorta Preparations

For vasorelaxant activity, the thoracic aorta was dissected from a rabbit, carefully removed the adhesive fatty tissues, and prepared 2–3 mm aortic rings. This aortic preparation was hung in an organ bath containing Krebs's solution with a continuous supply of carbogen. Preload tension (2 g) was applied and allowed to equilibrate for 60 min prior to the dose of any drug. The isolated aortic preparation was exposed to *H. heteromalla* for possible vasorelaxant effects in a cumulative manner. Further to define the possible mechanism, *H. heteromalla* was challenged to PE (1 μM), and K+ (80 mM) induced contraction for the possible activity of *H. heteromalla* in a cumulative manner.

#### *4.7. In Vivo Activities*

#### 4.7.1. Antiperistalsis Activity

Antiperistalsis activity was performed according to the method prescribed byWahid et al. [12]. Mice (25) of either sex were divided into 5 groups, i.e., control (0.9% normal saline), standard drug (CCh10 mg/kg), and *H. heteromalla* doses (100, 200, and 400 mg/kg). After 15 min of administering the test or standard material orally, each animal received 0.3 mL of the charcoal meal (10% gum acacia, 20 starch, and 10% vegetable charcoal) in distilled water. Thirty minutes later, mice were

killed, and the abdomen was incised to excise the whole small intestine. The distance from the pylorus region was measured to the front of the charcoal meal.

#### 4.7.2. Antidiarrheal Activity

The antidiarrheal activity was performed according to the method prescribed by Wahid et al. [12] with modifications. Mice (20) of either sex were divided into five groups, i.e., negative control (0.9% normal saline), standard drug (loperamide 10 mg/kg), and *H. heteromalla* doses (100, 200, and 400 mg/kg). After 30 min of dose administration (p.o.), animals received the castor oil (10 mL/kg p.o) and were observed for six hours in cages with a white paper surface with adsorbent properties. The percent inhibition of wet fecal was calculated.

#### 4.7.3. Carrageenan-Induced Rat's Hind Paw Edema Method

The anti-inflammatory activity was performed [32,40] on 25 rats of either sex divided into 5 groups, i.e., control (0.9% normal saline) standard drug (aspirin 10 mg/kg), and *H. heteromalla* doses (100, 200, and 400 mg/kg). After 30 min of dose administration (p.o), edema was induced by injecting 1% carrageenan in the right hind paw's sub-planter region and measuring the edema size at up to 4 h through a plethysmometer. The percentage of edema inhibition was calculated.

#### *4.8. In Silico Studies*

In silico studies were performed according to the method previously reported by Wahid et al. [12] and Sirous et al. [13].

*Ligand Preparation:* The 2D structures of HPLC quantified phytocompounds were retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov accessed date 20 March 2019) and treated in the LigPrep module of Maestro (Schrodinger suite 2015) to ionization, minimization, and optimization of ligands. The Epik tool of this module was used to generate the ionization state of ligands at cellular pH (7.4 ± 0.5) and applied the OPLS3e force field through the module for minimization and optimization of ligands that produce the lowest energy conformer of ligands.

*Protein Preparation:* For molecular docking, the highest resolution X-ray structures of proteins were downloaded from The Protein Databank (RCSB PDB) (https://www.rcsb.org accessed date 20 March 2019) and subjected to Protein preparation wizard of Maestro (Schrodinger suite 2015). This module processed the protein by adding hydrogen atoms to protein structure, removal of solvents (water) molecules, assigning bond orders, creating disulfide bonds, filling missing side chains and loops, and generating protonation state using Epik tool of protein structures for ligands at the cellular level pH (7.4 ± 0.5). After processing protein structures, these structures were optimized using PROPKA under pH 7.0, and the OPLS3e force field was utilized to perform restrained minimization for energy minimization and geometry optimization of protein structure.

*Molecular Docking and Receptor grid generation:* The active sites of protein structures for molecular docking were defined in the Receptor Grid Generation module of Maestro (Schrodinger suite 2015). A cubic grid box of each protein was defined with the help of a literature survey and with a selection of previously bonded ligands of proteins. The length of the grid box was adjusted to the length of 16 Å. The potential of nonpolar parts of the receptor was decreased to scaling factor 1.0 Å on Van der Waals radius of nonpolar atoms of protein having partial atomic charge cut-off 0.25 Å.

For molecular docking, the prepared ligands and protein structures were subjected to extra precision (XP) mode of Ligand Docking (Glide) module of Maestro (Schrodinger suite 2015) using pre-generated grid file for receptor. Additionally, 0.80 Å scaling factor was adjusted for Van der Waals radii with a partial charge cut-off of 0.15 Å. The docking results were subjected to the Prime MM-GBSA module to calculate the binding energies of ligands with protein structure using the VSGB solvation model with OPLS3e force field.

*Inhibition Constant (Ki):* The inhibition constant was determined from the binding free energy of ligand previously generated from Prime MM-GBSA, according to the following equation [12]:

$$
\Delta \mathbf{G} = -\mathbf{R} \mathbf{T} (\ln \mathbf{K\_i}) \text{ or } \mathbf{K\_i} = \mathbf{e}^{(-\Delta \mathbf{G}/\mathbf{R} \mathbf{T})}.
$$

where <sup>Δ</sup>G is binding free energy of ligand, R is gas constant (cal·mol−1·K<sup>−</sup>1), and T is room temperature (298 Kelvin).

#### *4.9. Statistical Analysis*

The data were expressed as the mean ± standard error of the mean (S.E.M.) and median effective concentration (EC50) with a 95% confidence interval (CI). One-way and two-way ANOVA tests were applied for in vivo experiments. All graphs and data were analyzed with the help of Graph pad prism software (San Diego, CA, USA).

#### **5. Conclusions**

*Himalaiella heteromalla* exhibited a more spasmolytic effect in ethyl acetate fraction and caused complete relaxation on isolated jejunum, trachea, aorta, and paired atria, supported with in silico studies. *H. heteromalla* proved various disease management-related activities. Further studies could be taken on *Himalaiella heteromalla* for drug discovery for the welfare of human beings.

**Author Contributions:** F.S. and S.M. planned the project and worked on statistical analysis of data and results interpretation; S.M. performed the experiments and F.U. worked on in-silico studies; R.A.M., C.C.M., N.B., N.U.-R. and M.R. drafted the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by FDI-0013.

**Institutional Review Board Statement:** Ethical approval was attained from Ethical Committee of Bahauddin Zakariya University, Multan (EC/25-MPHIL-S2018) dated 26 March 2018. Researchers agreed using the approved informed consent documented before their enrolment into study.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The author(s) are very thankful Chairman Department of Pharmacology; Imran for Provision of animal house and lab related facilities.

**Conflicts of Interest:** Authors announce there are no conflict of interest concerning the publication of this research article.

#### **References**


## *Article* **Chemical Composition and Assessment of Antimicrobial Activity of Lavender Essential Oil and Some By-Products**

**Alexandru Ciocarlan 1, Lucian Lupascu 1, Aculina Aricu 1, Ion Dragalin 1, Violeta Popescu 1, Elisabeta-Irina Geana 2,3, Roxana Elena Ionete 2, Nicoleta Vornicu 3, Octavian G. Duliu 4,5, Gergana Hristozova 5,6 and Inga Zinicovscaia 1,5,7,\***


**Abstract:** The producers of essential oils from the Republic of Moldova care about the quality of their products and at the same time, try to capitalize on the waste from processing. The purpose of the present study was to analyze the chemical composition of lavender (*Lavanda angustifolia* L.) essential oil and some by-products derived from its production (residual water, residual herbs), as well as to assess their "in vitro" antimicrobial activity. The gas chromatography-mass spectrometry analysis of essential oils produced by seven industrial manufacturers led to the identification of 41 constituents that meant 96.80–99.79% of the total. The main constituents are monoterpenes (84.08–92.55%), followed by sesquiterpenes (3.30–13.45%), and some aliphatic compounds (1.42–3.90%). The high-performance liquid chromatography analysis allowed the quantification of known triterpenes, ursolic, and oleanolic acids, in freshly dried lavender plants and in the residual by-products after hydrodistillation of the essential oil. The lavender essential oil showed good antibacterial activity against *Bacillus subtilis*, *Pseudomonas fluorescens*, *Xanthomonas campestris*, *Erwinia carotovora* at 300 μg/mL concentration, and *Erwinia amylovora*, *Candida utilis* at 150 μg/mL concentration, respectively. Lavender plant material but also the residual water and ethanolic extracts from the solid waste residue showed high antimicrobial activity against *Aspergillus niger*, *Alternaria alternata*, *Penicillium chrysogenum*, *Bacillus* sp., and *Pseudomonas aeroginosa* strains, at 0.75–6.0 μg/mL, 0.08–0.125 μg/mL, and 0.05–4.0 μg/mL, respectively.

**Keywords:** *Lavandula angustifolia* L.; essential oil; by-products; terpenic compounds; chromatographic analyses; antimicrobial activity; statistical data analysis

#### **1. Introduction**

*Lavandula angustifolia* Mill. (syn. *Lavandula vera* DC, syn. *Lavandula officinalis* Chaix ex Vill., syn. *Lavandula spica* L.) is a perennial evergreen shrub of the family *Lamiaceae*, native to the Mediterranean region. Nowadays, this species is naturalized almost all over Europe, North Africa, United States, and Australia [1]. *L.angustifolia* (Lavander) is one of the most valuable medicinal and aromatic plants traditionally used to treat pain, parasitic infections,

**Citation:** Ciocarlan, A.; Lupascu, L.; Aricu, A.; Dragalin, I.; Popescu, V.; Geana, E.-I.; Ionete, R.E.; Vornicu, N.; Duliu, O.G.; Hristozova, G.; et al. Chemical Composition and Assessment of Antimicrobial Activity of Lavender Essential Oil and Some By-Products. *Plants* **2021**, *10*, 1829. https://doi.org/10.3390/ plants10091829

Academic Editors: Marc (Vlaic) Romina Alina, Crina Muresan, Andruta Muresan and Juei-Tang Cheng

Received: 29 July 2021 Accepted: 31 August 2021 Published: 3 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

burns, insect bites, cramps, and muscle spasms [2]. In addition to its application in herbal treatment, lavender is also cultivated for the essential oils used in aromatherapy and the cosmetic, food, and flavour industries [3–5].

This is possible due to the presence of a set of biologically active substances, especially in essential oil, which possesses a multidirectional therapeutic activity being used in the treatment of gastrointestinal, cardiovascular, respiratory, and urinary infections [6]. Scientific studies reported anti-inflammatory [7], antioxidant [8,9], sedative [10], cytotoxic [11,12], analgesic [7], antimicrobial [6,13,14], and anticonvulsive [15] properties of *L. angustifolia* essential oil. Literature data reveal a huge variation in terms of *L. angustifolia* essential oil content, with values ranging between 0.5 and 6.25% in the case of essential oil obtained from fresh and dry inflorescences [16]. The main constituents of *L. angustifolia* essential oil are linalool, linalyl acetate, 1,8-cineole, borneol, camphor, lavandulyl acetate, *β*-caryophyllene, *β*-ocimene, *α*-fenchone, terpinen-4-ol, caryophyllene oxide, limonene, pinenes, geranyl acetate, *β*-farnesene, santalene, lavandulol, camphene, geraniol, and *α*-terpineol [8,11,13,14,17–26]. The content of oxygenated monoterpenes prevails in *L. angustifolia* essential oil and varies between 36.33 and 92.90% [16].

The therapeutic effects of *L. angustifolia* are also determined by secondary metabolites such as oleanolic and ursolic acids, together with other pentacyclic triterpenes. [27,28]. It has been proven experimentally that both compounds in pure forms, as well as their synthetic derivatives, show multiple biological activities [29–38].

Some by-products, e.g., pomace or solid residues, that resulted after hydrodistillation of essential oil-producing plants could be considered as a source of biologically active compounds such as ursolic and oleanolic acids. In addition, residual distillation waters have various applications due to their aromatic and antimicrobial properties [39–43].

Antibiotic resistance is becoming one of the main problems of modern medicine since it substantially reduces the effectiveness of antibacterial treatments and is linked to increased patient mortality. As a result, known antibacterial preparations cease to be safe and effective against infections caused by resistant bacteria, leading to increasingly serious cases, including hospital-acquired complications. This requires the discovery of new classes of antibiotics or optimization and a combination of known compounds. However, microorganisms will likely evolve resistance in time and further research and development may be hard to sustain by the pharmaceutical companies. For this reason, studies are being conducted to identify effective remedies against multidrug-resistant strains. Preference is given to natural products among which are the essential oils [44], including lavender [45], or their combination with antibiotics [46]. Still, information about the antimicrobial activity of residual water and ethanolic extracts is very scanty and is mainly related to Lavander hydrosol, which is produced synthetically [47].

The aim of this study was to (i) evaluate the chemical composition of lavender essential oil and some of the waste by-products produced industrially in the Republic of Moldova using different chromatographic techniques; (ii) assess the in vitro antimicrobial activity of extracted compounds; and (iii) distinguish, using statistical analysis, between different lavender oils produced in different regions of the Republic of Moldova (Northern, Central, and Southern), based on the terpenic and aliphatic compounds.

#### **2. Results**

#### *2.1. GC-MS Analysis Results*

A total of 41 constituents of lavender essential oil were identified by means of gas chromatography-mass spectrometry (GC-MS) analysis (Table 1).

It must be mentioned that the essential oil with the richest content was made by producer P1, which is the largest and operates a stationary modern factory. By contrast, producers P2 to P7 use mobile installations and process raw plant material directly in the field, in modernized or artisanal installations, and this may influence the chemical composition of essential oils and resulting by-products.


**Table 1.** Phytochemical (terpenic and aliphatic compounds) composition of lavender essential oil of Moldovan origin.

\*RT: Retention time; P 1–7: Producers.

According to the GC-MS data, the chemical composition of lavender essential oil produced in Moldova consisted mainly of terpenic and aliphatic compounds and their content varied within the limits indicated in Table 2.

The GC-MS analysis of extracts from residual waters (RW) showed that they contained only several hydrophilic components (see Section 3.3) and represented about 0.3–0.5% of the volume.

#### *2.2. RP-HPLC Analysis Results*

The content of triterpenic oleanolic acid (OA) and ursolic acid (UA) was established in freshly dried lavender plants and in dried solid residues (after hydrodistillation) via RP-HPLC analysis.


**Table 2.** Chemical composition of lavender essential oil.

The results were expressed as mg/g for extracts and mg/100 g for the ratio plant material/solid residue (Tables 3 and 4). It was observed that fresh plants had a much higher content of OA and UA.



**Table 4.** The OA and UA content of lavender by-product (solid waste residue), (DW).


The lower content of OA and UA in solid residues can be explained by their loss and derivatization/degradation during hydrodistillation in an aqueous medium at elevated temperatures (Table 4). The latter seemed more relevant since neither OA nor UA was found in residual water extracts (see Section 3.3).

#### *2.3. Microbial Inhibition Assessment Results*

The microbial activity assessment of lavender essential oil extracts from lavender plant material (LPM), lavender by-products (residual water (RW), and solid waste residue (SR)) was performed by serial dilution methods against several non-pathogenic Gram-positive and Gram-negative bacteria strains and fungi species, including phytopathogenic ones (e.g., *Xanthomonas campestris, Erwinia amylovora*, and *Erwinia carotovora*).

The results of the lavender essential oil antibacterial and antifungal activity tests are presented in Table 5.


**Table 5.** The antimicrobial activity of lavender essential oil.

MBC: Minimal bactericidal concentration; MFC: Minimal fungicidal concentration.

The same method was applied for residual waters, ethanolic extracts from solid residues, and freshly dried lavender plant materials (Table 6).

**Table 6.** The antimicrobial activity of residual water and ethanolic extracts from lavender plants.


RSD (μg/mL): <sup>a</sup> ±0.001 <sup>b</sup> ±0.0002.

All of the samples were preliminarily tested for their in vitro antimicrobial activity and antifungal effect against pure cultures of three species of fungi (*Aspergillus niger*, *Alternaria alternate, Penicillium chrysogenum*) and against Gram-positive (*Bacillus* sp.) and Gram-negative bacteria (*Pseudomonas aeruginosa*). Microorganisms were provided by the American Type Culture Collection (ATCC, USA). Caspofungin and Kanamycin were used as performance standards for testing the antifungal and antibacterial activities. The minimum inhibitory concentration values (MIC) for all the samples and standards are summarized in Table 6.

#### **3. Discussion**

#### *3.1. Chemical Composition of Lavender Essential Oils*

The essential oil manufactured by producer P1, destined for export, had the following physico-chemical properties: Density (20 ◦C)—0.8920 g/mL; refractive index (n20D)—1.4660, and optical rotation (*α*20D)— −7.0◦.

The most multitudinous group of terpenic compounds are monoterpenes, which include C10-hidrocarbones (8.72–15.32%) and their oxygenated derivatives (69.0–83.83%). The main constituents of this group which determine the quality and genuineness of lavender essential oil, according to the International Standard [48], are (%): 1,8-cineol (eucalyptol) (<1.0), (*E*)-ocimene (4.0–10.0), (*Z*)-ocimene (1.5–6.0), linalool (25.0–38.0), camphor (<0.5), terpin-1-en-4-ol (2.0–6.0), *α*-terpineol (<1.0), linalyl acetate (25.0–45.0), and lavandulyl acetate (>2.0) (Tables 1 and 2).

The content of sesquiterpene hydrocarbons and their oxygenated derivatives is reported to be within the limits of 3.09–12.83% and 0.19–1.26%, respectively. According to the same source [48], the most important sesquiterpenes are: *β*-caryophyllene (4.78%), (*E*)-*β*-farnesene (1.52%), and caryophyllene oxide (0.36%) (Tables 1 and 2).

Aliphatic compounds are of lesser concentration (1.42–3.90%) and in [48] are mentioned: 1-octen-3-ol (0.33%) and octan-3-one (<2.0%) (Tables 1 and 2).

#### *3.2. Chemical Composition of Lavender Plant Material*

For the selective extraction of ursolic and oleanolic triterpene acids from the lavender plant materials (LPM), the extraction yield varied between 8.83–9.94%, with the OA content between 13.43–19.09 mg/g and UA content between 33.28–60.82 mg/g. The content of OA and UA in dry (DW) LPM was in the range of 133.11–168.57 mg/100 g, and respectively 329.83–537.00 mg/100 g DW LPM (Table 3).

Moreover, the experimental results showed that the sum of isomeric OA and UA in LPM was about 5% of the DW, in a 1:3.7 ratio, confirming that lavender is a valuable source of natural OA and UA triterpene acids.

#### *3.3. Chemical Composition of Lavender by-Products*

The GC-MS analysis of etheric extracts of residual water (RW) proved that they contain hydrophilic monoterpenic compounds such as 1,8-cineol (eucalyptol, 6.31%), linalool oxide (3.08%), linalool (78.05%), terpin-1-en-4-ol (1.92%), and *α*-terpineol (10.64%).

HPLC quantification of UA and OA indicated that RWs did not contain OA and UA triterpene acids.

In the case of solid waste residues (SR), the average extraction yield was about 3.91%, with the OA content between 27.48–39.37 mg/g and UA content between 80.82–135.56 mg/g (Table 4). The isomeric OA and UA in DW SR ranged between 113.47–144.98 and 313.95–499.15 mg/100 g, respectively (Table 4), with their amount accounting to about 1% of DW, in a 1:3.1 ratio, indicating that lavender by-products are a promising source of OA and UA triterpene acids.

Our results are consistent with other literature data reporting DW of lavender SR values between 136.0–259.7 and 346.3–648.4 mg/100 g [49].

#### *3.4. Antimicrobial Assessments*

Phytopathogenic bacteria can cause various diseases of agricultural plants, especially the genera Erwinia and Xanthomonas. For example, *Erwinia amylovora*, the Gramnegative bacterium of the Enterobacteriaceae family, is the causative agent of fire blight, a devastating plant disease that affects a wide range of species of the family Rosaceae and is a major global threat to commercial apple and pear production. [50]. Another species, *E. carotovora*, causes bacterial soft rot in economically important crops, such as potatoes, tomatoes, and cucumbers. In the case of potatoes, the soft rot of the stem and tubers occurs even after harvest, thus considerably reducing the yield [51]. *Xanthomonas campestris* pv. *vesicatoria* is a biotrophic Gram-negative bacterium and is the agent that causes bacterial leaf scorch on tomatoes (*Solanum lycopersicum* L.) and peppers (*Capsicum annuum*), a disease that is present worldwide. Symptoms of bacterial infection include defoliation and chlorotic necrotic lesions on leaves, stems, fruits, and flowers, which subsequently lead to reduced fruit yield [52].

The species *Bacillus subtilis* and *Pseudomonas fluorescens* do not cause any disease to plants but were selected as reference bacteria from the Gram-positive and Gram-negative groups. They are also very suitable as test objects for evaluating the antibacterial activity of the lavender extract. *Candida utilis* and *Saccharomyces cerevisiae* are also non-pathogenic but were used as representatives of the yeast-fungus group for evaluating the antifungal activity of the extract.

It should be mentioned that there is a lack of information about any antimicrobial effects of lavender essential oil on *E. carotovora*, *E. amylovora*, and *C. utilis*.

The in vitro assessment of lavender essential oil of Moldovan origin showed good antibacterial activity against both non-pathogenic Gram-positive/Gram-negative bacteria (*B. subtilis* and *P. fluorescens*) at MBC of 300 μg/mL and good to high antifungal activity against phytopathogenic bacteria (*X. campestris*, *E. amylovora*, *E. carotovora*) and *C. utilis* fungi at MFC of 150–300 μg/mL (Table 5).

The highest antifungal and antibacterial activities were observed for residual water (RW) at 0.08 and 0.125 μg/mL, respectively. Good antifungal and antibacterial activities were ascertained for the SR extract as well (0.50 and 4 μg/mL). The LPM extract showed moderate antifungal and antibacterial activity (0.75 and 6 μg/mL).

The two techniques employed for testing both the disc diffusion and the dilution methods have been developed to yield accurate measurements of antibacterial and antifungal activities and are routinely used in antimicrobial susceptibility testing.

According to the obtained results, the antibacterial activity was similar but the antifungal activity was slightly different, thus suggesting that the activity against different microorganisms could be caused by different components of the oil.

#### *3.5. Statistical Data Analysis*

Univariate as well as multivariate statistical data analysis (SDA) represent one of the most reliable methods that permit extracting useful information and inferring different hypotheses concerning the considered set of data. Given the great diversity of organic compounds which can be found in lavender essential oil, multivariate statistical data analysis was an appropriate method allowing to group samples, in this case, according to the lavender oil producer and based on the concentrations of organic compounds (R mode), or, to classify an experimentally determined organic compound based on the concentration in samples (Q mode) [53,54].

It is worth mentioning that, to avoid any errors induced by missing data, SDA was applied only in the cases of compounds with a non-negligible variation present in all the samples (Table 1), i.e., the compounds which permitted generating the box plots in Figure 1a,b.

**Figure 1.** Box plots representing the distribution of (**a**) 25 components of lavender oil and (**b**) total content of compounds present in all the samples (producers).

Univariate SDA was useful in establishing the extent to which the samples of lavender oil by the seven producers were similar. This information was obtained by analyzing the box plot shown in Figure 1a. It was observed that all the samples were quite similar. To confirm this, we used more univariate tests, such as one-way ANOVA, Tukey's pairwise test, Kruskal-Wallis test of equal medians, as well as Mann-Whitney U tests. All of them confirmed that between the lavender oil samples there are no statistically significant differences. For this reason, we have proceeded with multivariate SDA.

Within multivariate SDA, each sample (case) is characterized by independent parameters (variables), so that the final analysis can be performed in R mode (to study relations between samples based on variables) or Q mode (to study the interrelations between variables based on samples). As both methods were based on the same set of samples and variables, R and Q modes could be considered complementary, which significantly enhanced the analysis.

Depending on the situation, cases/variables can be grouped by a multitude of procedures among which covariance and correlation are frequently utilized.

In the case of lavender samples, the best results were obtained by the principal component analysis (PCA) applied in both R and Q modes. With respect to the other two SDA methods, cluster analysis and K mean clustering, PCA permitted evidencing the association of samples, i.e., seven producers of lavender oil in R mode, as well as 25 lavender oil compounds in Q mode. Moreover, in R mode, a tree diagram corresponding to the cluster analysis (Euclidean distances) is, concerning the number and structure of clusters, similar to PCA based on correlation. For this reason, we restrained our SDA to both R and Q mode PCA.

The results, represented by the principal component (PC) 2 vs. PC 1 bi-plots, are illustrated in Figure 2a,b, respectively. In both cases, the PCA was based on correlations between variables (organic compounds, R mode) or samples (lavender oil producer, Q mode). Moreover, the loadings of each variable or sample were represented by Factor 2 vs. Factor 1 bi-plots in the corresponding insets: Variables in Figure 2a and samples in Figure 2b.

Accordingly, the result of PCA in R mode is illustrated by the bi-plot in Figure 1a. The existence of at least three clusters can be remarked, two of which consist of only one member, i.e., producers P2 and P5, and a third one, grouping the rest of the producers. The bi-plot illustrating the contribution of each compound to the PC1 and PC2 showed a relatively balanced situation, as both Factors 1 and 2 had similar contributions to PC, consisting of 36.46 and 27.25%, respectively. It is worth mentioning that a similar result was obtained by considering the PC3 vs. PC2, which most probably could be explained by their contribution to the total variance, 25.25 and 17.57%, respectively. The corresponding screen plot in Figure 3a illustrated this finding.

Complementary to the R-mode, a Q mode PC2 vs. PC1 bi-plot, shown in Figure 2b, consisted of three clusters, two of which contained a single organic compound, i.e., linalyl acetate and linalool, while the third one included all other 23 compounds. This result was in good agreement with the composition of the investigated samples, according to which, both linalyl acetate and linalool were characterized by the highest concentrations and variances.

On the contrary, Factor 2 vs. Factor 1 (Figure 2b, inset), except for Producers 2 (P2) and 5 (P5), were nearly coincident and negatively oriented along the first axis, which suggested an almost equivalent contribution to the total variance. This finding may explain the fact that PC1 contributed about 96% to the total variance, as shown in the corresponding screen plot (Figure 3b). In this regard, it is of interest to remark, as mentioned before, that P2 and P5 formed two different uni-component clusters (Figure 2b).

**Figure 2.** The results of R (**a**) and Q (**b**) mode PCA. The insets illustrate the contribution of the corresponding principal component (PC) analysis.

**Figure 3.** The screen plots corresponding to R-mode (**a**) and Q-mode (**b**) PCA.

#### **4. Materials and Methods**

#### *4.1. Samples Collection*

The samples of *L. angustifolia* vegetal raw material, by-products, as well as the main product—lavender essential oil (LEO), were provided between 2016 and 2018 by seven producers (P 1-7) from different regions of the Republic of Moldova (Northern, Central, and Southern): P1—Causeni district; P2—Donduseni district; P3 and P6—Rezina district; P4—Falesti district; P5—Dubasari district; and P7—Ungheni district.

For OA and UA characterization, fresh lavender inflorescences were collected directly from the lavender fields near the Pervomaisc village, Causeni district (46◦42 04 N 29◦05 21 E). The inflorescences were dried in shaded places to obtain lavender plant material samples (LPM) (n = 3) which were subjected to HPLC characterization. The byproducts which resulted after hydrodistillation (solid residue—SR (n = 3) and residual water—RW (n = 1)), were collected from the factories, dried, and bottled.

#### *4.2. Chemicals*

All of the used solvents, reagents, and standards were of analytical grade. Anhydrous sodium carbonate, aluminium chloride, sodium acetate, 96% ethanol, methanol, diethyl ether, and petroleum ether were obtained from Merck (Darmstadt, Germany). Deionized water produced by a Milli-Q Millipore system (Bedford, MA, USA) was used for the preparation of aqueous solutions and UHPLC mobile phases.

The standards used for HPLC-PDA analysis (ursolic and oleanolic acids) were HPLC purity and purchased from Sigma-Aldrich (Steinheim, Germany). Stock solutions of all the standards were prepared in methanol. Working standards were made by diluting the stock solutions in the same solvent. Both stock and working standards were stored at 4 ◦C until further use.

#### *4.3. Extracts Preparation*

The selective extraction of triterpenic ursolic and oleanolic acids with ethanol from the LPM and SR was performed using a Soxhlet type extractor after degreasing with light petroleum ether (b.p. 40 ◦C) (Figure 4.). The ethanolic extracts were evaporated to dryness at 35 ◦C under reduced pressure using a rotary evaporator. For HPLC analysis, aliquots of each crude extract were dissolved in methanol using ultrasonication and filtered through a 0.45 μm micro-filter. The extraction and HPLC analysis were performed in duplicate for each plant material and the results were expressed as a mean value. For GC-MS analysis, the industrially produced essential oil samples were dissolved in hexane. The RWs were extracted with diethyl ether and the obtained extracts were subjected to GC chromatographic analysis.

#### *4.4. Analytical GC-MS Analysis*

The GC-MS analysis was performed using an Agilent Technologies 7890A gas chromatograph coupled with a 5975C Mass-Selective Detector (MSD) equipped with a split/splitless injector (1 μL). The analysis was carried out on an HP-5MS fused silica capillary calibrated column (30 m × 0.25 mm i.d.; film thickness 0.25 μm). The injector and detector temperatures were kept at 250 ◦C. Helium was used as carrier gas at a flow rate of 1.1 mL/min; oven temperature program was 70 ◦C/2 min, which was then programmed to 200 ◦C at the rate of 5 ◦C/min, and finally to 300 ◦C at the rate of 20 ◦C/min. The split ratio was 1:50, the MSD ionization energy was 70 eV, scan time was 1 s, the acquisition mass was in the range from 30 to 450 amu, and the solvent delay was 3 min.

#### *4.5. Analytical RP-HPLC Analysis*

The ursolic and oleanolic acids were quantified by an HPLC-PDA method previously reported [55], using a Thermo Finnigan Surveyor Plus HPLC System (Thermo Fisher Scientific Inc., San Jose, CA, USA). The OA and UA from extracts were identified by their retention time and spectral data by comparison with standards. To confirm the peak identity among possible interference peaks, the technique of standard addition to the sample was applied. Moreover, the peak purity for the interest peaks was satisfactory.

**Figure 4.** Flowchart of the UA and OA extract preparation.

Calibration curves of the standards covered the range of 1–400 mg/L for both OA and UA and revealed good linearity, with correlation coefficients higher than 0.995 (0.9989 for OA and 0.9991 for UA) [56]. The accuracy of the method (%) was evaluated for spiked samples at 50 mg/L concentration and the obtained average values were 4.31% for OA and 3.65% for UA.

#### *4.6. Antimicrobial Activity Assessment*

The in vitro antimicrobial activity tests of methanolic extracts from the SR and RW against three species of fungi (*Aspergillus niger*, *Alternaria alternata,* and *Penicillium chrysogenum,* ATCC 53346, 8741, and 20044) and two species of bacteria (*Pseudomonas aeroginosa* and *Bacillus* sp., ATCC 27813 and 15970) were performed using a previously reported method [57].

Antimicrobial activity assessment of the industrially obtained lavender essential oil samples was performed in vitro on the following microorganisms: Non-pathogenic Gram-positive and Gram-negative strains of *Bacillus subtilis* NCNM BB-01 (ATCC 33608) and *Pseudomonas fluorescens* NCNM-PFB-01 (ATCC 25323), phytopathogenic strains of *Xanthomonas campestris* NCNM BX-01 (ATCC 53196), *Erwinia amylovora* NCNM BE-01 (ATCC 29780), *E. carotovora* NCNM BE-03 (ATCC 15713), and fungus strains of *Candida utilis* NCNM Y-22 (ATCC 44638) and *Saccharomyces cerevisiae* NCNM Y-20 (ATCC 4117) following a method described elsewhere [58].

The compounds Caspofungin and Kanamycin, both from Liofilchem (Roseto degli Abruzzi, Italy), were used as standards for antifungal and antibacterial activity tests.

#### *4.7. Statistical Analysis*

All statistical data analyses were performed using the StatSoft Statistica 10 software.

#### **5. Conclusions**

More than 40 main constituents of lavender essential oil from seven Moldavian producers were quantified by means of chromatographic and statistical analyses. The experimental

data for lavender plant material and solid waste residue proved the possibility of their use as sources of biologically active compounds, such as OA and UA triterpene acids. All of the subjects in the present study, essential oil, residual distillation waste water, and extracts from the solid waste residues have shown high antimicrobial activity against 11 strains of bacteria and fungi, including phytopathogenic ones.

**Author Contributions:** Conceptualization, A.A. and I.Z.; microbiological assessments, L.L. and N.V.; GC-MC analysis, I.D.; sample preparation, V.P.; HPLC analysis, E.-I.G.; statistical analysis, O.G.D.; data curation, O.G.D.; writing—original draft preparation, A.C.; writing—review and editing, R.E.I., G.H. and I.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Agency for Research and Development (ANCD), project PLANTERAS 20.80009.8007.03.

**Acknowledgments:** O.G.D. and I.Z. wish to acknowledge that their contribution was done within cooperation protocols no. 4920-4-20/22 between the University of Bucharest and the Joint Institute for Nuclear Research, Dubna, Russian Federation, represented by the Frank Laboratory of Neutron Physics. L.L. is grateful to the National Collection of Non-Pathogenic Microorganisms at the Institute of Microbiology and Biotechnology as well as the Laboratory of the Phytopathology and Biotechnology at the Institute of Genetics, Physiology, and Plant Protection for kindly providing the bacterial strains.

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

#### **Abbreviations**


#### **References**

