α-pinene has been shown to stimulate apoptosis proved by initial disruption of mitochondrial function, ROS formation, improved caspase-3 properties, heterochromatin aggregation, DNA disintegration, and exposure of phosphatidyl serine on the cell surface [33]. Additionally, the environment is supposed to significantly minimize cancer growth, and this was evidenced by Kusuhara et al., who stated that rats placed in fragrant environment with α-pinene exhibit a decrease in melanoma growth [34]. However, direct α-pinene did not display any in vitro effect on melanoma cell production. Chen et al. evaluated the inhibitory action of α-pinene on hepatocellular carcinoma BEL-7402 cells in vitro and in vivo using MTT assay ^[35][36][35,36]. α-pinene prevented BEL-7402 cells through cell cycle arrest at G2/M phase, down-regulated Cdc25C mRNA and protein expression, and decreased the action of cycle dependence on kinase 1 (CDK 1). Other studies have also suggested the use of α-pinene as a potent antitumor drug. Yang et al. measured the inhibitory properties of α-pinene on human hepatocellular carcinoma (HepG2) cells propagation, and found cell cycle arrest at G₂/M phase, which seemed to be associated with down-regulation of miR-221 expression and up-regulation of CDKN1B/P27 and CDKN1C/P57 expression [37]. The effect of α-pinene on cell cycle regulation in HepG2 cells was also investigated and developed as a promising chemotherapeutic drug for administration in hepatocellular carcinoma [38]. Zhao et al. tested the inhibitory potential of α-pinene on human prostate cancer in a mouse xenograft model. β-pinene-based thiazole derivatives were used as anticancer agents via mitochondrial-facilitated apoptosis. Three human cancer cell lines, namely, cervical carcinoma HeLa cells, colon cancer CT-26, and hepatocarcinoma SMMC-7721 cells were used in the study [39]. Out of all derivatives, the compound 5g was found to inhibit cell proliferation by stimulating cell cycle arrest in HeLa cells by ROS-mediated mitochondrial dysfunction signaling pathways [40].

2.4. Genomic Instability

Catanzaro et al. studied the action of α-pinene on genomic instability in Chinese hamster cell line (V79-Cl3). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with fetal calf serum, penicillin, and streptomycin. Different doses of α-pinene (0, 25, 30, 35, 40, and 50 µM) were used to expose cells (3 × 10⁵ per dish) for 1 h. Cytotoxicity was assessed using the standard approach. Morphological study demonstrated considerable growth in micro and multinucleated cell frequencies. Apoptotic cells were seen at 40 and 50 µM. α-pinene stimulated genetic instability, inhibiting mitotic process and leading to irregularity in 50% cells. It was established by flow cytometry that α-pinene stimulated oxidative stress and DNA destruction [41].

2.5. Cytogenetic and Oxidative Effects

Coniferous plants-derived EO are mostly composed of α-pinene. Türkez and Aydin (2013) investigated the cytogenetic and oxidative activities of α-pinene on human blood cells. Cultured human blood cells were supplemented with varying doses (0, 10, 25, 50, 75, 100, 150, and 200 mg/L) of α-pinene for 1–2 days. Lactate dehydrogenase (LDH) and MTT assays demonstrated that α-pinene at 200 mg/L reduced cell viability. Further, no changes were recorded significantly in the rates of genotoxicity endpoints. However, total antioxidant capacity (TAC) and total oxidative stress (TOS) levels revealed dose-dependent changes. TAC levels enhanced post-administration of 25 and 50 mg/L of α-pinene, while TOS levels were reduced only at 200 mg/L α-pinene on human lymphocytes [42].

2.6. Gastroprotective Effect

Gastrointestinal transit is the time it takes food to leave the stomach and travel through the intestines. It is a crucial procedure affected by many parameters that takes a long time. For healing, various medicinal plants and their compounds, such as monoterpenes (α- and β-pinene), have been used as an important source of therapeutic agents for gastrointestinal disorders ^[43][44][43,44]. In a study, Eucalyptus tereticornis-derived EO and its components, like α- and β-pinene, were used as gastroprotective agents [45]. Gastric emptying was measured after supplementation of a liquid test meal containing phenol red at varying time intervals. Gut was divided into consecutive segments. Small intestine transit was analyzed, and liquid test meal was supplemented. Dye retention was determined using spectrophotometric methods. Isometric contractions were observed by isometric transducers and data acquisition system. E. tereticornis EO and its components reduced gastric retention in mice, and α- and β-pinene enhanced gastric tonus in anesthetized mice. α- and β-pinene have been found to contract gastric strips in vitro, to relax the duodenum. In contrast, E. tereticornis soothes the gastric and duodenal strips. E. tereticornis speeds up the gastric emptying of liquid, and part of its effect is related to the contrast action stimulated by α- and β-pinene on gut.

The gastroprotective and antiulcerogenic properties of α-pinene were also studied using Swiss mice. Different doses (10, 30, and 100 mg/kg) of absolute ethanol and indomethacin were used to stimulate the gastric ulcer. Gastric lesions were analyzed by determining the area of lesions using the Scion Image programme. Stomach samples were crushed and sandwiched for further testing. Acute gastric lesions were introduced into the Swiss mice, and these mice fasted for 12 h. Rats were administered with 0.5 mL of vehicle (0.1% tween-80 aq. solution), ranitidine (40 mg/kg), and α-pinene (at 10–100 mg/kg), dissolved in vehicles separately. Rats were sacrificed by cervical dislocation followed by stomachs removal and scanning, with the area of lesions being determined. Gastrointestinal transit ratio was also analyzed. Findings from the study displayed that pretreatment with α-pinene cased a decline in ethanol-induced gastric mucosa lesions. α-pinene provides similar gastroprotective effects for absolute ethanol-stimulated ulcers to ranitidine (40 mg/kg) [46]. Oral pretreatment with α-pinene did not exhibit any considerable influence on indomethacin-sensitized ulcer lesions. Moreover, no considerable variation was recorded between the lesions area of α-pinene and vehicle pretreated rats. However, ranitidine decreases the area of lesions more than alternatives.

Likewise, the antiulcer properties of oleoresin (Pistacia atlantica) and α-pinene were studied on Wistar strain male albino mice [47]. Compounds were isolated from oleoresin using GC-MS. Various doses (250–2000 mg/kg b.w.) of EO were given to eight selected mice. Toxicity (restlessness, dullness, and agitation) was recorded in mice after 72 h of administration. Then, 80% ethanol was supplemented. Mice were sacrificed after 2 h, and stomachs were detached. Gastric ulcers were determined using microscope. The antibacterial properties of cultured Helicobacter pylori strains were screened using disc diffusion assay. Up to 2000 mg/kg EO was found safe. EO exhibited activity against H. pylori strains, with the inhibition zone being found maximum for the clinical strain No. 5. The EO from selected plants exhibited MIC values between 275 and 1100 μg/mL. Ethanol was used to stimulate gastric ulcers, and screening was performed against varying doses of EO and Tween 80 in water (control). Results indicated that varying doses of EO (25, 50, and 100 mg/kg) considerably reduced ethanol-induced peptic ulcers. Histopathological analysis revealed that EO reduced ethanol-induced gastric tissue damage and necrosis.

2.7. Anxiolytic-Like Effects

Yang et al. evaluated the non-rapid eye movement sleep/anxiolytic/hypnotic behavior in a rat model. Electroencephalogram (EEG) and electromyogram (EMG) analysis were used to investigate both behavior and hypnotic effect during sleep. α-pinene and zolpidem were orally pre-supplemented, followed by pentobarbital (45 mg/kg) injection. Ex vivo electrophysiological measurements from brain slices and in silico molecular modeling or molecular docking were performed. α-pinene displayed sleep-increasing behavior by direct binding to GABA_A-Benzodiazepine receptors (GABA_A-BZD) and forcing as a partial modulator at BZD binding site. Varying doses (12.5, 25, 50, and 100 mg/kg, orally) of α-pinene considerably reduced sleep latency and enhanced NREMS duration without any consequences on REMS and delta action. Results from the study indicated that the hypnotic action of α-pinene on rats could be due to its modulation of GABA_A-BZD receptors, similar to zolpidem [48].

Similarly, the effect of α-pinene on emotional behavior, deposition, and expression of related mRNA was investigated in rat brain [49]. Rats were exposed to α-pinene (10 μL/L air), and water was used as negative control for 60/90 min. After inhalation, quantitative measurement of α-pinene in brain and gene expression by RT-PCR was performed. EPM analysis was conducted to determine the anxiolytic-like action on rats after α-pinene inhalation for 60 or 90 min. Distance was considerably enhanced when rats inhaled α-pinene for 60 min longer than the control (water). No notably variation was recorded in the total distance traveled for 90 min. α-pinene inhalation for 60 min in brain was considerably enhanced compared to 90 min. BDNF mRNA expression in olfactory bulb and hippocampus was similar at 60- and 90-min inhalation. TH mRNA expression in middle brain at 60 min inhalation was remarkably enhanced in comparison with the control.

2.8. Neuroprotective Activities

Several neurodegenerative diseases, like Alzheimer’s and Parkinson’s, are triggered by oxidative imbalance [50]. EO of various plant species are able to reduce ROS formation. Excessive ROS production led to injuries in brain functioning; thus, defense against ROS-stimulated injury is vital for appropriate brain functioning, and it diminishes the likelihood of the development of neurodegenerative disorders. With this background, Porres-Martínez et al. investigated the action of α-pinene on H₂O₂-sensitized oxidative stress, using mice pheochromocytoma cells (PC12) as model. Antioxidant and protective effects of α-pinene were assessed for H₂O₂-sensitized oxidative stress in PC12 cells. Cell viability was loosed, and cell morphology was altered after pre-treatment with α-pinene. Intracellular ROS production was prevented by α-pinene; however, it increased catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and hemeoxygenase 1, expression. α-pinene also played an important role in reducing apoptosis; thus, it was concluded that these monoterpenes have ability to defend the nervous system [50].

2.9. Cytoprotective Activity against H₂O₂-Stimulated Oxidative Stress

Excessive ROS (e.g., hydroxyl radical, superoxide anion, and hydrogen peroxide) formation unbalances cellular redox, leading to oxidative stress [51] and randomly oxidizing biological molecules (i.e., peroxidation, mitochondrial dysfunction, protein carbonylation, and DNA strand breaks and base damage) [52]. ROS overproduction protection and cellular redox equilibrium may be promoted by using several natural antioxidants and EO. Salvia lavandulifolia is a species used in traditional medicine to improve memory and cure several diseases. Porres-Martínez et al. studied the constituents of S. lavandulifolia in human astrocytoma 373-MG cell line as a cytoprotective agent against H₂O₂-stimulated oxidative stress. Doses ranged from 10 to 250 mM α-pinene. H₂O₂ significantly reduces cell viability by over 60%. However, pre-supplementation with α-pinene (10, 25, 50, and 100 mM) considerably enhanced cell viability in a dose-dependent fashion (IC₅₀ = 79.70 mM). Pre-administration with α-pinene protected U373-MG cells from H₂O₂-stimulated oxidative damage, through blocking the loss of cell viability (IC₅₀: 79.70 mM to α-pinene) and cell morphology, preventing ROS formation and lipid peroxidation and enhancing the endogenous antioxidant status by enhancing glutathione, CAT, SOD, GR, and GPx activities, and HO-1 properties and protein expression [51].

2.10. Inhibitory Effect on the Growth of Endocarditis Disease

Endocarditis is a disease triggered by cardiac wall or endocardium infection, promoted by different microbes, like bacteria, fungi, and chlamydia. Microorganisms belonging to Streptococcus and Staphylococcus genus, Haemophillus parainfluenzae, H. aphrophilus, H. paraphrophilus, H. influenzae, Actinobacillus actinomycetemcomitans, Cardiobaccterium hominis, Eikenella corrodens, Kingela kingae, and K. denitrificans are the major microbial agents causing endocarditis. In modern times, microbes have shown susceptibility to drugs used in various clinical therapies. Therefore, one study was attempted to slow down the growth of endocarditis, using monoterpenes like α- and β-pinene [53]. The MIC value was determined by solid medium diffusion method, whereas viable cells count was used to investigate the interference of MIC values on bacterial cell viability. Staphylococcus aureus, S. epidermidis, S. pneumoniae, and S. pyogenes strains were utilized for the proposed screening.

Phytochemicals, like α- and β-pinene, showed inhibitory activity against all bacterial strains studied. Some phytochemicals exhibited MIC values between 5 (α-pinene x S. epidermidis SSI 1, S. pyogenes and S. pneumoniae) and 40 μL/mL (β-pinene x S. epidermidis). In addition, some bacterial strains showed resistance to antibiotics, mainly gentamicin. S. aureus exhibited resistance to α- and β-pinene.

3. Bioavailability of α-Pinene and β-Pinene

According to the Codes of Federal Regulations (FDR), Title 21; Part 314 A, bioavailability is defined as the “rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of drug action” ^[54][86]. Various studies have described that α-pinene and β-pinene display antimicrobial ^[55][87], anticancer ^[33][36][33,36], anti-inflammatory ^[56][64], and antiallergic ^[57][69] properties. Nevertheless, most of these studies lack information concerning bioavailability and pharmacokinetics of the active terpenes. However, to take advantage of the bioactivity of a particular natural product, in this case α-pinene and β-pinene, and to further use it as either a supplement or drug in future, it is necessary to study the absorption, distribution, and metabolism Monoterpenes are essentially metabolized by cytochrome P₄₅₀ monooxygenases, epoxide hydrolases, and dehydrogenases to mono- and dihydroxylated substances, as well as higher oxidized metabolites that are conjugated basically to glucuronic acids ^[58][88]. According to the authors, after absorbing α-pinene, the proposed in vivo human metabolites are trans-verbenol (tVER), cis-verbenol (cVER), myrtenol (MYR), myrtenic acid (MYRA), αPNM3, and αPN-M1 ^[59][89]. In contrast, β-pinene metabolites from brushtail possum (Trichosurus vulpecula) were found to be myrtenic acid, present in their urine ^[60][90].

3.1. Dermal Application

Kohlert, et al. ^[61][91] reported the preparation of eucalyptus or pine oils enriched with α- and β-pinene, camphor, 3-carene, and limonene, which have been applied in most works investigating the dermal absorption of EO-based substances, as these terpenes increase percutaneous absorption of drugs and other compounds due to their lipophilic characteristics. According to Cal and Sopala ^[62][92], the maximum concentration of terpenes in the stratum corneum (SC) and epidermis (ED) was obtained within 15 min of application. It was also demonstrated that the extent of absorption depends on the size of treated skin area, skin properties, concentration of administrated compound, and time of exposure ^[61][62][91,92].

Cal and Sopala ^[62][92] investigated the ex vivo skin absorption of Vicks VapoRub^® (α-pinene 4.8%, β-pinene 1.1%) using human cadaver skin of a 40- to 50-year-old Caucasian women. An infinite dose of 100 mg/cm² was applied on a 0.65 cm² skin area; placed in a flow-through diffusion chamber; and left for 15, 30, and 60 min, respectively. For α-pinene, the maximum concentration C_max 40 µg/cm² in the SC was within 15 min of application of Vicks VapoRub. β-pinene reported a C_max 290 µg/cm² 60 min after application. Although their physicochemical properties were similar, their penetration profiles into SC differed. Though the β-pinene content in the product was 4.5 times lower than that of α-pinene, the total accumulation of β-pinene in the SC was seven times greater due to its strong lipophilic nature ^[62][92].

β-Myrcene, limonene, α-pinene, β-pinene, linalool, geraniol, citronellol, and isomenthone are typical monoterpene constituents of rose oil ^[63][93]. In a study investigating the in vitro percutaneous permeation of rose oil using human tissues, Schmitt, Schaefer, Sporer and Reichling ^[63][93] stated that α- and β-pinene had different permeability behaviors when they were applied as a neat solution compared to as a component in rose oil (rose oil constituent). When α- and β-pinene were applied as a neat, single substance, P_app-values (apparent permeability coefficients in cm/s) were reported to be high (α-pinene 6.49 × 10⁻⁵ cm/s and β-pinene 4.48 × 10⁻⁵ cm/s) compared to other monoterpenes in rose oil applied as a neat substance. However, when pinene was available as a component of rose oil, the permeability of α-pinene dropped while the permeability of β-pinene was enhanced. The P_app-value of β-pinene was approximately four times higher (5.76 × 10⁻⁵ cm/s) than that of β-myrcene, limonene, and α-pinene (1.43 × 10⁻⁵ cm/s). It is apparent that the skin permeability was positively or negatively affected depending on the other constituents available in the mixture ^[63][93].

3.2. Inhalation

α- and β-pinene are insoluble in water yet soluble in blood and adipose tissues. Terpenes indicate high respiratory uptake and accumulation in adipose tissues ^[64][94]. In a study by Filipsson ^[65][95], the relative net uptake of α- and β-pinene was reported to be 62% and 66%, respectively (see Table 18).

Table 18. Bioavailability of α-pinene and β-pinene.

	Exposure	Uptake	Distribution	Elimination
				Exhale Air	Blood	Urine
Inhalation (8 volunteers, with light exercise-50W) [66][65][67][81,95,96]
α-pinene (+)	2 h 450, 225, or 10 mg/m3	Relative net uptake 59–62% *	tmax 120 min cmax 20 µMol/L(for 450 mg/m3) cmax 10 µMol/L(for 225 mg/m3) (exposure concentration depended) * cmax 10 µMol/L	7.7%	cl21h 1.9 lkg−1h−1	0.001% In 30 min 4% of total uptake as cis and trans verbenol
					-		Broth microdilution and ethidium bromide deposition	t1/2 (3 phases α, β, γ) α-4.8 min β-38 min γ-695 min
α-pinene (−)	450 mg/m3			7.5%	cl21h 1.16 lkg−1h−1
					Modulation of antibiotic resistance, by reducing MIC value of ciprofloxacin, erythromycin, and triclosan, up to 512 times. α-pinene also affected antimicrobial efflux systems		[	21	]
2	-	Nocardia sp. Strain (P18.3), Pseudomonas putida PX1 (NCIB 10684), Pseudomonas sp. strain PIN18 (NCIB 10687), and P. fluorescens NCIB 11671	-	Strains were cultured into agar slants with α-pinene (3 g/L in media), and strains growth was recorded	Nocardia sp. growth (P18.3) was not remarkable; Pseudomonas strains (NCIB 10684, 10687, and 11,671 and PL) increased promptly when α-pinene (0.3%, v/v) was added	[22]
24	Citrus species	Propionibacterium acnes, Staphylococcus epidermidis	Peel EO	EO was isolated by hydrodistillation	EO demonstrated outstanding antibacterial properties against P. acnes and S. epidermidis	[68]	t1/2 α-5.6 min β-40 min γ-555 h
26	Sigma-Aldrich	Escherichia coli, Micrococcus luteus, Staphylococcus aureus, and Candida albicans	-	Bioautographic method MIC was measured	(+)-α-pinene exhibited modest activity. (−)-α-pinene was unable to display any activity. α-pinene and β-lactams revealed the highest effects. Although (−)-α-pinene revealed no positive activity, the derivatives like β-lactam, amino ester, and amino alcohol exhibited antimicrobial effects	[69]	β-pinene	450 mg/m3 *	Relative net uptake 66% *	cmax 3 µMol/L *	* 5.7%
28	Bursera morelensis	Candida albicans strains (ATCC 14065, ATCC 32354, donated strain, and CDBB-L-1003)	Stems (EO)	EO was extracted by hydrodistillation, and GC-MS was used to isolate compounds Disc diffusion and survival curve assay were used	* cl21h 0.5 lkg−1h−1 * t	Maximum antifungal activity was attributed to the EO and its constituent, namely, α-pinene. Minimum fungicidal concentration of EO was found to be 2 mg/mL. A slight reduction in C. albicans	1/2 α-5.3 min β-41 min γ-25 h	population was recorded after 12 h	Not available
[	70	]	Dermal application in vitro [63][93], Ex vivo [62][92]
α-pinene
	30	-	Staphylococcus aureus and Escherichia coli	-	Disc diffusion test, broth microdilution, and bacterial death kinetics	Inhibition halos of 11 and 12 mm for gram-positive and -negative strains were obtained at 160 µL/mL, respectively. At 1.25 and 2.5 µL/mL, (+)-α-pinene was able to eliminate bacterial colonies formation at one time of exposure of 2 h for E. coli strain	[71]	1000 µL
31	(concentration is not provided) for 27 h	Papp 6.49 × 10−5 cm/s
Syzygium cumini	Swiss mice	Leaves (EO)	MTT assay Cytotoxic effect on macrophages was determined; cells were exposed to α-pinene and tested against Leishmania	Cytotoxic effect of α-pinene against promastigotes of Leishmania amazonensis was observed with different cell death percentages (93.7, 83.2, and 58.4%) at different concentrations (100, 50, and 25 mg/mL respectively)	[72]	100 mg/cm2 applied on 0.65 cm2 at 37 °C ¥		c
40	max 40 µg/cm2 tmax 15 min in SC		-		House fly (Musca domestica)	-
Y-tube and house flies were selected for this test	Solution with lowest concentration did not show significant differences in Y-tube arm choice. (1S)-(-)-α-pinene had maximum repellent efficiency for house flies when compared to (1R)-(+)-α-pinene	[	73]	β-pinene	1000 µL (concentration is not provided) for 27 h	Papp 4.48 × 10−5 cm/s
100 mg/cm2 applied on 0.65 cm2 at 37 °C ¥		cmax 290 µg/cm2 tmax 60 min in SC
Oral administration (four volunteers) [59][89]
α-pinene	9 mg (66 µmol)		Unmetabolized state—not detected (<4 µg/L)		t1/2 MYR-1.7 h tVER-1.0 h cVER-0.8 h	tmax 1.6 h (metabolites)
			tmax 1–3 h Metabolites			t1/2 MYR-1.5 h cVER and tVER-1.6 h MYRA-1.4 h
			cmax MYR-11 µM tVER-26 µM cVER-9.3 µM			cl24h MYR-1.5%, cVER-5.6%, tVER-4.1% MYRA-6.7%.
				78% unknown elimination, which could be exhalation or first-pass metabolism

* Chamber vapour proportions α-pinene-54%, β-pinene-11%, 3-carene-35%; ¥ α-pinene 4.8%, β-pinene 1.1%, eucalyptol 3.3%, camphor 5.7%, and menthol 3.8%; trans-verbenol (tVER), cis-verbenol (cVER), myrtenol (MYR), myrtenic acid (MYRA), αPNM3, and αPN-M1, which are metabolites of α-pinene.

The mean concentration of α-pinene indicated linear kinetics with increasing exposure concentration in blood, as well as in urine ^[65][95]. Blood concentration mainly depends on exposure time, dose, or exposure concentration in the chamber, and the presence of other monoterpenes in the treatment chamber. Terpenes are soluble in oil; so, they are expected to accumulate in adipose tissue and thus exhibit greater t_l/2 in blood. A higher fat: blood partition coefficient for α-pinene resulted in an increase in distribution rate of α-pinene from blood to fat and a higher metabolic rate for α-pinene ^[65][95]. During the early stages of exposure, arterial blood concentration increased rapidly and then levelled off at the end of exposure. After the end of exposure, the decaying of pinene in blood was rapid and the author distinguished three log linear phases of the pinene concentration in arterial blood, namely, initial, middle, and last. The mean half time (t_1/2) of the three phases identified—initial, middle, and last—were 4.8, 38, and 695–555 min, respectively. Likewise, the respective β-pinene t_1/2s were 5.3 min, 41 min, and 25 h. The long half-time of α-pinene implies that it would take more than 2 days for the body to be almost totally cleared of α-pinene (estimated as 5 × t_1/2) ^[65][95]. Therefore, β-pinene may take more than 2 days to completely clear the body. Blood clearance values measured up to 21 h were cl_21h 1.5 lkg⁻¹h⁻¹ for α-pinene and 0.5 lkg⁻¹h⁻¹ for β-pinene. Less than 0.001% of the total α-pinene dose was excreted unchanged in the urine within 0.5 h after exposure, according to Filipsson.

3.3. Oral Administration

Schmidt and Göen ^[59][89] recruited 4 volunteers (receiving orally α-pinene, 9 mg) and studied the metabolic products, including α-pinene, namely, myrtenol (MYR), and cis- and trans-verbenol (cVER; tVER) levels in blood and urine. The maximum blood concentration c_max for MYR was 11 µM. tVER-26µM and cVER-9.3 were recorded and metabolites concentration reached their maxima (t_max) 1.6 h after exposure. The maximum concentration of the urinary α-pinene metabolites was reached 1.6 h after exposure and then within 24 h; after it declined to pre-exposure levels with elimination half-lives of 1.5 h (MYR) and 1.6 h (cVER and tVER). The total eliminated amounts were 1.5% (MYR), 5.6% (cVER), and 4.1% (tVER) of the orally applied dose. The metabolic product, myrtenic acid (MYRA), showed renal elimination of 6.7% share of 9 mg oral dose at t_max 1.6 h after ingestion, and an elimination half-time of 1.4% ^[59][89]. Thus, the study showed that the blood concentration of orally administrated, unmetabolized α-pinene lasts for a short duration at low concentrations (<4 µg L⁻¹) in blood and could be rapidly eliminated from the body unchanged via lungs. Only 22% (MYRA–7%, cVER–6%, tVER–4%, MYR–2%, αPN-M3–2%, and αPN-M1–1%) of the oral dose administrated was detected as metabolites in urine. The amount of α-pinene metabolites found in urine was notably greater compared to blood, with this being indicative of a fast transfer of metabolites from blood to urine with a rapid renal elimination. Detectable metabolite levels and non-detectable α-pinene in blood after low oral doses indicate a fast and approximately entire pre-systemic metabolism, such as hepatic or intestinal first-pass metabolism. However, it could be worthwhile to measure the other pathways of elimination, such as the composition of the exhaled air after oral administration, in order to reveal the unknown 78% (See Table 21) of the oral dose (only 22% is known as mentioned above), according to the authors ^[59][89].

Table 2. Antimicrobial activity of α-pinene and β-pinene.

S. No.	Source/Species	Model	Plant Portion	Method	Result	Ref
α-pinene
1	Sigma Aldrich	Campylobacter jejuni
45
Plectranthus barbatus	Malaria (Anophel es subpictus), dengue (Aedes albopictus), and Japanese encephalitis (Culex tritaeniorhynchus) mosquito vectors	EO (leaves)	GC and GC--MS were performed; larvicidal activity of EO (40, 80, 120, 160, and 200 µg/mL) and its constituents eugenol, α-pinene, and β-caryophyllene (12–100 µg/mL each) were determined by WHO methods. Mortality of larvae was measured at 24 h after exposure	EO showed substantial larvicidal effects with LC50 values of 84.20, 87.25, and 94.34 µg/mL for the selected mosquito species. For Anapheles subpictus, eugenol, α-pinene, and β-caryophyllene revealed larvicidal effects (LC50 = 25.45, 32.09, and 41.66 μg/mL), followed by Aedes albopictus (LC50 = 28.14, 34.09, and 44.77 μg/mL) and Culex tritaenior hynchus (LC50 = 30.80, 36.75, and 48.17 μg/mL, respectively)	[74]
β-pinene derivatives
27	-	Klebsiella pneumoniae, Enterobacter aerogenes, S. aureus, S. epidermidis, and Candida albicans	-	25 3-cyanopyridine compounds of β-pinene were prepared; MIC value was recorded using serial two-fold dilution method	MICs values of all derivatives ranged from 15.6 to 125 mg/l	[75]
29	-	Candida spp.	-	MIC and MFC values and microbial death curve after treatment with (+)-β-pinene enantiomers	MIC values ranged from <56.25–1800 µmol/L (+)-β-pinene. After ergosterol addition, MIC value of (+)-β-pinene was not altered, but was altered with sorbitol addition. (+)-β-pinene displayed anti-biofilm activity against multiple Candida species	[76]
α- and β-pinene
22	Dep. Pharmaceutical Sciences, Ponta Grossa, Brazil	Gram-positive bacteria (Staphylococcus aureus, S. epidermidis, S. pneumoniae, and S. pyogenes)	-	MIC value, viable cells count	All studied bacterial strains were sensitive to α- and β-pinene. MIC values ranged from 5 (α-pinene x S. epidermidis SSI 1; ATCC 12228; S. pyogenes ATCC 19,615; and S. pneumoniae) to 40 μL/mL (β-pinene x S. epidermidis ATCC 12228). Few bacterial strains were resistant antibiotics, mainly gentamicin. S. aureus was resistant to α- and β-pinene	[53]
23	Sigma-Aldrich	Antimicrobial: Escherichia coli (ATCC 11775, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778), and Candida albicans (ATCC 10231). Antimalarial: Plasmodium falciparum (FCR-3)	-	Disc diffusion method. MIC was investigated. Antimalarial properties were analyzed using the tritiated hypoxanthine incorporation assay	(+)-β-pinene was approximately two to 12 times more effective as compared to (+)-α-pinene against both gram-positive and negative bacteria, as well as C. albicans. (+)-α-pinene shows 250-fold more antimalarial activity than (+)-β-pinene	[77]
25	Sigma-Aldrich	Candida albicans, Cryptococcus neoformans, Rhizopus oryzae, and methicillin-resistant Staphylococcus aureus (MRSA)	-	Two-fold serial dilution method was used to evaluate MIC for all the strains	MIC values of α- and β-pinene enantiomers were found to be from 117 to 6250 µg/mL. C. albicans exhibited higher sensitivity to α- and β-pinene enantiomers than MRSA. Positive enantiomers possess capability to kill 100% of C. albicans in 60 min., and 6 h was required for total killing of MRSA	[5]