Chemical composition and biological activities of essential oils from six lamiaceae folk medicinal plants

Essential oils have attracted wide attention in recent years due to their extensive applications in natural functional ingredients, pharmaceutical preparations, biomedical products, and the cosmetics industry. In this study, the chemical compositions and biological activities of essential oils extracted from six Lamiaceae herbs, including Pogostemon cablin (Blanco) Benth. (PCEO), Perilla frutescens (L.) Britton (PFEO), Salvia japonica Thunb. (SJEO), Rosmarinus officinalis L. (ROEO), Lavandula angustifolia Mill. (LAEO), and Agastache rugosa (Fisch. & C. A. Mey.) Kuntze (AREO), were determined and analyzed. A total of 167 components were identified from the six essential oils by GC-MS analysis, with 35, 24, 47, 46, 54, and 37 components in PCEO, PFEO, SJEO, ROEO, LAEO, and AREO, respectively. Hierarchical cluster analysis of chemical compositions showed that the composition of the six essential oils was significantly different in content, and they were clearly divided into six classes. However, all of these six essential oils exhibited promising anti-inflammatory activity by inhibiting the expression of interleukin-1, interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 in rats with adjuvant arthritis, among which PFEO had the best performance. In addition, the six essential oils displayed significant cytotoxicity on B16 (IC50 = 86.91–228.91 μg/mL) and LNCaP cell lines (IC50 = 116.4–189.63 μg/mL). Meanwhile, all of them presented satisfactory antioxidant activity (IC50 = 4.88–13.89 μg/mL) compared with Trolox C (IC50 = 13.83 μg/mL), and SJEO (IC50 = 7.93 μg/mL) served as an optimal candidate natural antioxidant by DPPH assay. Taken together, these results indicate that the six Lamiaceae essential oils manifest excellent and diverse biological activities, enabling them to be used as perfect natural functional ingredients in antioxidant, antitumor, or anti-arthritic drugs. This study provides more references for pharmaphylogeny research and drug discovery from folk medicinal plants.

Essential oils have attracted wide attention in recent years due to their extensive applications in natural functional ingredients, pharmaceutical preparations, biomedical products, and the cosmetics industry. In this study, the chemical compositions and biological activities of essential oils extracted from six Lamiaceae herbs, including Pogostemon cablin , and components in PCEO, PFEO, SJEO, ROEO, LAEO, and AREO, respectively. Hierarchical cluster analysis of chemical compositions showed that the composition of the six essential oils was significantly di erent in content, and they were clearly divided into six classes. However, all of these six essential oils exhibited promising anti-inflammatory activity by inhibiting the expression of interleukin-, interleukin-, tumor necrosis factor-α, and cyclooxygenase-in rats with adjuvant arthritis, among which PFEO had the best performance. In addition, the six essential oils displayed significant cytotoxicity on B (IC = .
-. µg/mL) and LNCaP cell lines (IC = . -. µg/mL). Meanwhile, all of them presented satisfactory antioxidant activity (IC = . -. µg/mL) compared with Trolox C (IC = . µg/mL), and SJEO (IC = . µg/mL) served as an optimal candidate natural antioxidant by DPPH assay. Taken together, these results indicate that the six Lamiaceae essential oils manifest excellent and diverse biological activities, enabling them to be used as perfect natural functional ingredients in antioxidant, antitumor, or anti-arthritic drugs. This study provides more references for pharmaphylogeny research and drug discovery from folk medicinal plants. KEYWORDS Lamiaceae herb, essential oil, chemical composition, folk medicinal plants, biological activities

Introduction
The Lamiaceae family contains 236 genera and about 7,173 species, almost cosmopolitan, except for the coldest regions of high latitude. Seven diversity centers were recognized: (1) Mediterranean and SW Central Asia; (2) Africa south of the Sahel and Madagascar; (3) China; (4) Australia; (5) South America; (6) Northern America and Mexico; (7) Indomalesian region (SE Asia). Based on their distribution, these speciesrich genera fall generally into two groups: those mostly tropical in origin, including Vitex (Viticoideae), Clerodendrum (Ajugoideae), Callicarpa (Incertae sedis), Ocimum, Hyptis (Nepetoideae), and those which probably had a temperate origin, but often extend into the montane tropics, such as Ajuga, (Ajugoideae), Scutellaria (Scutellarioideae), Stachys (Lamioideae), Salvia, Clinopodium, Mentha, (Nepetoideae). In addition, Teucrium (Ajugoideae) has a distinct distribution in the southern hemisphere, but is most species-rich in the north, spreading down to the mountains of tropical Africa. The Lamiaceae plants contain many aromatic and medicinal plants that are widely used in traditional and modern medicine, food, and cosmetics industries (Vukovic et al., 2009;Nieto, 2017;Borges et al., 2019). In traditional and modern medicine, some Lamiaceae species, such as Perilla frutescens (L.) Britton, Pogostemon cablin (Blanco) Benth., Rosmarinus officinalis L., Lavandula angustifolia Mill., and Agastache rugosa (Fisch. & C. A. Mey.) Kuntze, are pervasively applied to dispel fever, expel superficial evils, eliminate stasis, induce diuresis, promote blood circulation, and reduce edema (Guo et al., 2016;Luo et al., 2019). R. officinalis. has been used as an analgesic, antispasmodic, and antidepressant to cure intercostal neuralgia, headaches, migraine, insomnia emotional upset, and depression in folk medicine (Ghasemzadeh Rahbardar and Hosseinzadeh, 2020). For a long time, the biological activities of extracts from these plant species have been studied, such as the antitumor, antioxidant, antimicrobial, and anti-inflammatory activities (Nieto, 2017;Guo et al., 2019;Karpinski, 2020). Essential oil, an important category of plant extracts, has a multidirectional action mode and a variety of biological activities (Wojtunik-Kulesza et al., 2019). Essential oils can disrupt the cell and cell membrane via a permeabilization process. The lipophilic compounds of essential oils can pass through the cell wall; damage polysaccharides, fatty acids, and phospholipids; change the permeability for H + and K + cations to affect cellular pH; and damage organelles and disintegrate mitochondrial membrane (Karpinski, 2020). What is more, essential oils inhibit the biosynthesis of fungal DNA, RNA, proteins, and so on. They are widely applied in cosmetic additives, natural functional food, pharmaceutical preparations, and biomedical products (Nieto, 2017;Zhang et al., 2017bZhang et al., , 2020a. Specifically, essential oils from medicinal plants have attracted increasing attention in recent years for their multifaceted biological activities and diverse chemical compositions (Santos and Rao, 2000;El-Sayed et al., 2014;Xue, 2016). Lamiaceae family plants rich in essential oils have significant values in natural medicine, pharmacology, cosmetology, and aromatherapy. Some Lamiaceae species that are used in traditional medicine have been employed in the characterization of their essential oils, such as bioactivities and phytochemical composition. For example, essential oils of P. cablin (PCEO), P. frutescens (PFEO), R. officinalis (ROEO), and L. angustifolia (LAEO) are mainly composed of patchouli alcohol, linalool, α-terpineol, β-pinene, DL -menthol, and isobornyl acetate, which show strong anti-inflammatory activity by inhibiting the expression of interleukin 6 (IL-6), cyclooxygenase 2 (COX-2), tumor necrosis factor α (TNF-α), and nuclear factor-kappa B (NF-kB) in tetradecanoylphorbol acetate (TPA)-induced inflammation models Zhang et al., 2020b). Moreover, these essential oils also demonstrate high antioxidant, antibacterial, and antifungal activities . The essential oil of A. rugosa (AREO), mainly composed of methyleugenol, estragole, and eugenol, exhibits strong pesticide activity against Meloidogyne incognita, with a LC 50 value of 47.3 µg/mL (Li et al., 2013). ROEO also shows suppression of the lipopolysaccharide (LPS)induced inflammation by inhibiting the expression of COX-2 and inducible nitric oxide synthase (iNOS) and blocking the production of TNF-α (Yu et al., 2013). Lavender essential oils have been used cosmetically and therapeutically for centuries, and their biological activities have been extensively studied (Cavanagh and Wilkinson, 2002). Borges et al. (2019) indicated that ROEO possesses strong anti-inflammatory activity and can be used as a remedy for inflammatory diseases (Borges et al., 2019). Though the essential oil extracted from Salvia japonica Thunb. (SJEO) has been widely used, little is known about its biological activity. Herb essential oils exert their diverse biological activities by acting on various pathways using different chemical components. The composition and bioactivity of essential oils extracted from Lamiaceae plants have been analyzed in many studies; however, their anti-inflammatory, antioxidant, antitumor, and anti-arthritis activities need to be systematically explored from multiple aspects (Nikolić et al., 2014;Vyry Wouatsa et al., 2014;Park et al., 2016;Mamadalieva et al., 2017Mamadalieva et al., , 2019Mouahid et al., 2017;Borges et al., 2019;Karpinski, 2020).
The chemical composition of plant essential oil is influenced by numerous factors, such as the growing environment, harvest time, and plant organ used for essential oil extraction. Therefore, it is necessary to determine the phytochemical composition of the essential oil before carrying out further studies on their .
bioactivities. The current study aimed to elucidate the biological activity through the determination of the chemical composition of the essential oils extracted from six Lamiaceae plants with gas chromatography-mass spectrometry (GC-MS). The diversity of chemical components was analyzed by hierarchical cluster analysis in six essential oils from six Lamiaceae folk medicinal plants. Diverse biological activities, including anti-inflammatory, antitumor, and antioxidant activities, were evaluated through corresponding models. The model of complete Freund's adjuvant (CFA)-induced rheumatoid arthritis was used to evaluate the anti-inflammatory activity and related mechanisms of the six essential oils, and the LNCaP and B16 cell lines were used to estimate the antitumor activity. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical method was carried out to determine free radical scavenging capacity of these essential oils. Our study provides additional data to support the use of essential oils from Lamiaceae plants as a drug treatment.

Plant materials and chemicals
The local name, storage locations, and collection dates of A. rugosa, L. angustifolia, P. frutescens, P. cablin, R. officinalis, and S. japonica samples are shown in Table 1. The leaves of A. rugosa, P. cablin, P. frutescens, R. officinalis were used for essential oil extraction, while the aerial parts of L. angustifolia and S. japonica were used for essential oils extractions. All plant samples were confirmed by Professor Nian Liu of Zhongkai University of Agriculture and Engineering (Guangzhou, China). All chemicals used in this study were purchased from Aladdin Reagent Inc. (Shanghai, China).

Essential oil extraction
The steam distillation method was used to extract essential oils from the six plant samples as previously described (Zhang et al., 2020b). The plants were cleaned, ground, passed through a 0.45-mm sieve, and distilled with a steam distillation device for 3.5 h (Zhang et al., 2017b). The isolated essential oils were dried as previously described in a former research and stored in individual brown glass bottles at 4 • C until use (Xiang et al., 2017).

GC-MS analysis
The phytochemical compositions of PCEO, PFEO, SJEO, ROEO, LAEO, and AREO were identified according to previous methods (Xiang et al., 2017;Zhang et al., 2017b) using a GC-MS system with a DB-5MS capillary column (0.25 mm × 30 m, i.d. 0.25 µm) (Agilent, Santa Clara, CA, USA). The carrier gas was Helium (He) at a flow rate of 1 mL/min. The initial temperature was set as 40 • C for 1 min, then increased to 280 • C by 3 • C/min, and held at 280 • C for 5 min. The split ratio was set as 100:1. For MS conditions, the ionization conditions were as follows: pressure of 50 kPa, electron energy of 70 eV, and ion source temperature of 200 • C.
Each component of essential oils was determined based on the retention index (RI), which was calculated using a series of n-alkanes (C 6 -C 40 ) . Additionally, the mass spectrum of each compound was searched against the NIST Standard Reference Database (NIST Chemistry WebBook, 2014, over 40,000 compounds in this database) and databases published elsewhere (Zhang et al., 2017a). The total ionization chromatography (TIC) was obtained and used for determining the contents of each compound (Supplementary Figure S1).

Animals
The animal experiments were carried out following ethical guidelines of the Laboratory Animal Center of Sun Yat-sen University. Male rats (6-8 weeks old, 210 ± 30 g body weight) were purchased from Sun Yat-sen University and raised under normal conditions (25 ± 2 • C, 12/12 h light/dark cycle). Food and water were fed as required during the experiment.
Rats in the Con. group were not given any treatments. In the model group, CFA (0.1 mL) was subcutaneously injected into each rat after routine sterilization from Day 8 to Day 21 to induce arthritis. Then these rats were raised under normal conditions until use. Tween 80 was given to the rats in the NC group, while ibuprofen (100 mg/kg, dissolved in Tween 80) was given to those in the PC group from Day 8 to Day 20 (Khayyal et al., 2005). Rats in PCEO, PFEO, ROEO, SJEO, LAEO, and AREO groups were treated with corresponding essential oils (100 mg/kg, dissolved in Tween 80) from Day 8 till the end of the experiment. The arthritis score was recorded from Day 8 to Day 20 for all groups. The 5-point method was used to assess and grade the severity of the swelling, erythema, or stiffness in the paw: 0 = no signs of illness; 1 = mild swelling and erythema in the ankle/wrist; 2 = swelling and erythema in the . /fpls. . ankle/wrist; 3 = severe swelling and erythema in the ankle/wrist; and 4 = severe illness in the paw or front leg. Both the hind feet were graded, and the total score was not allowed to exceed 8 (Funk et al., 2010). The rats were then sacrificed and their ankle joints were sampled and stored in 4% (v/v) paraformaldehyde for subsequent histological analysis.

Histological analysis and immunohistochemical staining
As described previously (Zhang et al., 2020a), the ankle joints were paraffin-embedded and the sections were observed using light microscopy (Olympus IX71, OLYMPUS, Japan). The IL-1, IL-6, COX-2, and TNF-α antibodies, all diluted at a 1:200 ratio, were used for immunohistochemical staining. The number of positive cells was counted with ImageJ using photos taken with a fluorescence microscope (NIH, Bethesda, MD, USA).

Determination of antitumor activity
Anittumor activity of essential oil was investigated via human prostate cancer cell model LNCap and mouse B16 melanoma cell lines in vitro. In PCEO, PFEO, ROEO, SJEO, LAEO, and AREO groups, the cytotoxicity of essential oils on LNCaP and B16 cells treated with essential oils was assessed using MTT [3 -(4,5 -dimethyl -2 -thiazolyl) -2,5diphenyltetrazolium bromide] values described in Zhang et al. (2020a). The B16 and LNCaP cells were cultured in DMEM and RPMI1640, respectively, supplemented with 10% FBS, 2 mM glutamine, 100 mg/mL streptomycin, and 100 U/mL penicillin. The cells were maintained in a humidified 5% CO 2 incubator at 37 • C and were subcultured every 3-4 days to maintain logarithmic growth and allowed to grow for 24 h before the treatments were applied. The cells were then treated with different concentrations of the essential oil (Table 3), and then the absorbance at 570 nm was read on a microplate reader. The IC 50 value of MTT assays is defined as essential oils concentration resulting in a 50% reduction of absorbance (Kanipandian and Thirumurugan, 2014). Hydroquinone and Paclitaxel were used as positive controls for B16 and LNCaP cells, respectively (Zhang et al., 2017c;Rodboon et al., 2020).

Determination of antioxidant activity
The antioxidant activity of the six essential oils was evaluated by DPPH free radical scavenging capacity (Xiang et al., 2017;Zhang et al., 2020a). DPPH solution (67 µg/mL) was mixed with each of the essential oils at various concentrations and incubated at 25 • C for 30 min in the dark. The absorbance was then read at 517 nm. The scavenging percentage was calculated as follows:

Statistical analysis
One-way analysis of variance (ANOVA) was used to test statistical significance, and the result was considered significant at P ≤ 0.05. Data were presented as mean ± standard . /fpls. .

Results and discussion
Phytochemical compositions of the six essential oils A total of 167 components were identified from six essential oils (Table 2) using GC-MS. Based on the species and quantity of compositions in each essential oil, the relationship of the six Lamiaceae folk medicinal plants was analyzed via hierarchical cluster. The results showed that the six essential oils were clearly divided into six classes, and the six essential oils have their unique principal components (Figure 1). The proportions of corymbolone, pogostone, patchouli alcohol, pogostole, rotundone, menthol, α-patchoulene, seychellene, αguaiaene, α-gurgujene, and δ-guaijene showed similarity in PCEO and AREO. The proportions of L-α-terpineol, osthole, and safranal showed similarity in LAEO and ROEO. The proportions of nerolidol and linalool showed similarity in LAEO and PFEO. The proportion of terpinen-4-ol showed similarity in LAEO and SJEO. The proportions of (+)-2-bornanone and eucalyptol showed similarity in SJEO and ROEO. The proportion of humulene showed similarity in SJEO and PFEO. The proportion of α-selinene showed similarity in SJEO and PCEO. The proportion of epicurzerenone showed similarity trend in PCEO and ROEO (Figure 1). β-Caryophyllene was present in all six essential oils, while patchouli alcohol and caryophyllene oxide were found in all essential oils except PFEO. In summary, PCEO has the latest relationship with AREO, followed by PFEO, SJEO, ROEO, and LAEO. This relationship was also partly proved by the biological activities of six essential oils in the following results.

FIGURE
Hierarchical cluster analysis for the chemical compositions in six essential oils.

FIGURE
The chemical structure of components in six essential oils. The relative content of each component was more than %.

Anti-arthritis activity of the six essential oils
As shown in Figure 3, all the six Lamiaceae essential oils displayed inhibitory effects on adjuvant arthritis in rats. Compared to the model group, PCEO, PFEO, ROEO, SJEO, LAEO, and AREO at a concentration of 100 mg/kg exhibited inhibitory effects on arthritis, among which PFEO manifested the highest anti-inflammatory activity, while PCEO showed the lowest. This result was consistent with that obtained from the PC group (ibuprofen treatment), which is effective for alleviating joint swelling in the rat models of arthritis.
. /fpls. . The ankle joints of rats in the model group were significantly swollen compared with those of the control (Figure 3). In addition, the arthritis score of the positive control group (ibuprofen treatment) was significantly lower than that of the model group (Figure 4), which implied the success of CFA-induced arthritis. The arthritis scores of six essential oil treatment groups were lower than that of the model group, with PL displaying the lowest score, which indicated that PFEO might possess the strongest anti-arthritis capacity (Figure 4).
To obtain further insight into the anti-arthritis effect of six essential oils, histological and immunohistochemical characterizations were conducted using articular tissues. Severe cartilage damage, capillary hyperplasia, synovial proliferation, and lymphocyte infiltration were observed in the model group, while lymphocyte infiltration and cartilage damage were significantly inhibited in the PC group (ibuprofen treatment) ( Figure 5). PCEO, PFEO, ROEO, SJEO, LAEO, and AREO exhibited similar effects relative to ibuprofen, which may largely decrease the damage.
Complete Freund's adjuvant can induce numerous inflammatory responses of cytokines, including COX-2, iNOS, IL-1, and IL-6 (Zhang et al., 2020c). To further understand the anti-inflammatory mechanism of these Lamiaceae essential oils, the spatial-temporal expression profiles of inflammatory cytokines in rat articular tissues were investigated ( Figure 6). In the model group, CFA treatment greatly induced the expression of COX-2, TNF-α, IL-1, and IL-6 in rat articular tissues, while the essential oil treatments notably reduced that of TNF-α, IL-1, and IL-6 compared to the Con group. Nevertheless, COX-2 expression was slightly decreased after ibuprofen treatment. Caryophyllene was a common component shared by the six essential oils, which was reported to inhibit the expression of TNF, IL-1β, and COX-2 in APP/PS1 mice (Alzheimer-like phenotype) through CB2 receptor activation and the PPARγ pathway (Cheng et al., 2014). In our study, PFEO exhibited the greatest anti-inflammatory capacity inhibiting adjuvant arthritis and largely reduced the expression of inflammatory cytokines including TNF-α, IL-1, and IL-6. Linalool, a dominant component in PFEO, exhibited notably anti-inflammatory potential by reducing the expression of IL-1β and TNF-α in BV2 microglia cell lines (Li et al., 2015). Our results are consistent with those of previous studies, indicating that linalool may be a potential anti-inflammatory compound in those essential oils. In all, our results demonstrated that essential oils of Lamiaceae  herbs play a key role in alleviating inflammation by inhibiting inflammatory cytokine expression.

Antitumor activity of the six essential oils
The antitumor activity was evaluated by examining in vitro inhibitory effects of these essential oils on LNCaP and B16 cells, and the results are shown in Table 3 Previous studies have shown that patchouli alcohol, linalool, caryophyllene, borneol, and camphor achieve anticancer effects by inhibiting the expression of inflammatory factors (Santos and Rao, 2000;de Lima et al., 2014;Fidyt et al., 2016;Lian et al., 2018;Oner et al., 2019). Our results showed that the . /fpls. .

FIGURE
Immunohistochemical staining of rat articular tissues for COX-(A) IL-(B), IL-(C), and TNF-α (D) ( × magnification). (a) normal control (Con) group, (b) model (CFA) group, (c) negative control (NC) group, (d) positive control (PC) group, (e) PCEO group, (f) PFEO group, (g) ROEO group, (h) SJEO group, (i) LAEO group, and (j) AREO group. Statistical significance relative to the model (CFA) group is indicated, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, significantly di erent from the model (CFA) group. Arrows indicate the lesion sites. Statistical significance relative to the positive control (PC) group is indicated, # p ≤ . . dominant components of six essential oils had different degrees of anticancer effects on B16 and LNCaP cells, consistent with previous studies. PCEO and AREO had better performance in inhibiting B16 and LNCaP cells. Patchouli alcohol, a dominant component of PCEO (43.04%) and AREO (45.70%), maybe a key anticancer ingredient in these two essential oils. Linalool was a dominant component in LAEO (29.84%) and PFEO (66.65%) and may be a key component that plays an important role in their anticancer activity. Borneol (14.59%) and camphor (15.10%) were the major components of ROEO, which were likely the key antitumor component in our in vitro cell experiment. Similarly, linalool (8.89%) and camphor (9.54%) may be principal components participating in the anticancer activity of SJEO.

Antioxidant activity of the six essential oils
The DPPH method, which is stable, simple, and fast, was employed to assess free radical-scavenging activity of the six essential oils Zhang et al., 2020a,b). In this study, our results showed that the IC 50 values of the six essential oils were between 7.93 and .

FIGURE
Measurement of DPPH free radical scavenging capacity of the six Lamiaceae essential oils using Trolox C (TC) as the positive control. Statistical significance relative to the Trolox C (TC) group is indicated, ****p ≤ 0.0001, significantly di erent from the positive control group.

Conclusion
The demand for essential oils is growing due to their potential applications in pharmacology and industry. The phytochemical composition of essential oils takes charge of their bio-activities and their pharmaceutical effects. However, the chemical composition of plant essential oils is affected by many factors, such as a growing environment and identification methods. In this study, we used modified methods to identify different chemical component profiles of essential oils extracted from six folk medicinal plants. A total of 167 components were identified and analyzed by GC-MS, among which the dominant components were patchouli alcohol, pogostone, linalool, chavibetol, β-caryophyllene, terpenyl acetate, camphor, chavibetol, verbenone, borneol, and γ-terpineol ( Table 2). HCA of chemical compositions was first analyzed in six essential oils, which provides more reference for pharmaphylogeny research (Figure 1). Meanwhile, the chemical structure of 15 major compounds was exhibited to provide more reference for later research. Our results were remarkably different from the former reported and provide a new insight into the phytochemical composition of the six essential oils.
The six Lamiaceae essential oils exhibited diverse antiinflammatory activities on CFA-induced adjuvant arthritis in rats. PFEO, with a high linalool concentration (67.65%), showed higher anti-inflammatory activity relative to the rest of the essential oils and ibuprofen. Anti-inflammation was achieved by inhibiting the expression of COX-2, IL-1, IL-6, and TNF-α. All six essential oils also demonstrated different DPPH radical scavenging capacities except PCEO. SJEO, with high concentrations of terpenyl acetate (10.52%) and camphor (9.54%), showed the highest antioxidant capacity. The six essential oils also exhibited significantly different antitumor activities on LNCaP and B16 cells. AREO, with a high proportion of patchouli alcohol (45.70%), showed the highest antitumor capacity by inhibiting B16 cells with the lowest concentration of 86.91 µg/mL. LAEO, high in linalool (29.84%), showed promising antitumor capacity by inhibiting LNCaP cells at the lowest dosage of 116.5 µg/mL. Collectively, these six Lamiaceae essential oils, possessing varied chemical compositions and biological activities, exhibit potential for serving as bio-functional additives in biomedical products, such as anti-inflammatory and antitumor drugs.
Five of the six medicinal plants belong to the subfamily Nepetoideae (Dumort.) Burnett, except for P. cablin, which belongs to the subfamily Lamioideae Harley, indicating that species with similar bio-activities and pharmaceutical .
/fpls. . effects are clustered in their phylogenetic relationships. More specifically, based on our study, PCEO showed the lowest effects on anti-inflammatory and antioxidant activities, which is consistent with the phylogeny (Supplementary Figure S2). In the views of pharmaphylogeny, species that are closely related are not only similar in physiological characteristics, but at the same time, it is also reflected in the similarity of phytochemical components. Our results showed that the principal components of the six essential oils were greatly distinct. Perhaps because the genes expressing these chemical compositions have suffered different selection pressures during evolution, or they have been of independent origin and have undergone different evolutionary pathways. However, we can still find chemical compositions that match the phylogenetic tree. For example, L. angustifolia and P. frutescens are sister groups, and the proportion of nerolidol and linalool showed similarity in LAEO and PFEO; R. officinalis and S. japonica are closely related, and the content of (+)-2-bornanone and eucalyptol showed similarity in SJEO and ROEO. Although these six Lamiaceae plants are widely used and cultivated in China, only A. rugosa, P. frutescens and S. japonica are native to China. L. angustifolia and R. officinalis are native to Mediterranean, and P. cablin is distributed around the equator in Southeast Asia. This research provided more references for pharmaphylogeny and drug discovery from folk medicinal plants, and more studies need to be done for further exploring the drugs' function.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by the animal experiments were carried out following Ethical Guidelines of the Laboratory Animal Center of Sun Yat-sen University.