Introduction

Iran is a prominent and historically significant country in Southwest Asia, celebrated for its rich cultural heritage and literary contributions1. Its unique phytogeographical location in the Middle East, coupled with diverse climatic conditions, results in a remarkable variety of flora, housing numerous plant species2,3. The presence of various local communities throughout Iran has led to distinct local names for plants, creating both similarities and differences in plant nomenclature. This variation can result in confusion, as different names may refer to the same species or the same name may apply to different species across cultures and languages4,5,6. Such challenges are particularly pronounced in the trade of medicinal plants with therapeutic properties, often arising from misidentification. Traditional methods for identifying medicinal plants often relied on morphological characteristics, which may not be sufficient to differentiate closely related species or detect adulteration among closely similar species4,5,7,8. Therefore, it is crucial to ensure accurate identification and authentication of these plants and to align their vernacular names with established scientific nomenclature.

“Ostokhudus” an ancient Persian name is a significant medicinal and aromatic plant traditionally used to address a range of health issues, including anorexia, irritability, insomnia, and indigestion9. It is also employed in treating migraines, muscle cramps, and bronchial asthma4. Farsam et al.10 reviewed the historical references of plant species labeled as Ostokhudus, revealing that various specimens with similar morphological traits in their dried forms were identified and utilized under this name in certain traditional Iranian manuscripts. In the eleventh century, Lavandula stoechas L. was recognized as Ostokhudus for treating conditions such as neuralgia, epilepsy, and melancholia, as well as serving as a tonic for urinary organs11. Additionally, in the eighteenth and early twentieth centuries, L. dentata L. was utilized under the same name12,13. Since the mid-twentieth century, Nepeta menthoides Boiss. & Buhse has also been marketed as Ostokhudus in some Iranian herbal markets4,5,7,14. In the Iranian Herbal Pharmacopeia (IHP)5, certain plants have been identified under the name Ostokhudus, which have been mistakenly used instead of Lavandula L., including Nepeta glomerulosa Boiss., Salvia rosmarinus (L.) Schleid. (Syn.: Rosmarinus officinalis L.), Stachys lavandulifolia Vahl, and Stachys inflata Benth. Various ethnobotanical studies have also shown that the Ostokhudus belonged specimens commonly found in the Iranian herbal markets are primarily Lavandula angustifolia Mill.15,16,17,18, to a lesser extent, L. dentata, N. menthoides4,19, N. binaloudensis Jamzad20,21,22,23, N. bracteata Benth. and N. ispahanica Boiss.22. The morphological similarities between certain Nepeta specimens and Lavandula species may have contributed to their misidentification as Ostokhudus7,14.

In Iran, there are three native species of Lavandula: L. pubescens Decne., L. coronopifolia Poir., and L. sublepidota Rech.f. These species have limited distributions, primarily found in the southern provinces of Bushehr, Fars (L. sublepidota), and Hormozgan (L. sublepidota and L. coronopifolia)24,25,26. Despite the presence of these native species, most Lavandula samples available in Iranian herbal markets are imported. In contrast, the genus Nepeta is highly diverse in Iran, with 43 endemic species, representing 54% of its total global species. This makes Iran a significant center of diversity and speciation for Nepeta. The endemic species of this genus often exhibit restricted distributions, typically confined to a single province or, in some cases, to a specific habitat27.

Many herbal market samples are sold in powdered form or lack distinct morphological characteristics, rendering traditional taxonomic and morphometric methods insufficient for accurate identification. This challenge is particularly relevant for herbal-based industrial products, such as soft gel capsules and oral drops, where the original plant material is often processed or altered. In such cases, phytochemical analysis has proven to be a more reliable and effective approach for the precise identification of plant species6,28,29. Numerous studies have shown that anatomical, morphological, micromorphological, and phytochemical characteristics provide valuable taxonomic markers for the identification and differentiation of various genera and species within the Lamiaceae family30,31,32,33,34. These markers have been particularly useful for distinguishing species within the Lavandula35,36,37,38 and Nepeta genera39,40.

The primary goal of this study is to address the challenges associated with the accurate identification and authentication of medicinal plants in Iran, particularly those marketed under the name “Ostokhudus”. Given the historical and cultural significance of Ostokhudus in traditional medicine, coupled with the widespread misidentification and adulteration of plant species in herbal markets, this study aims to: (1) Investigate the taxonomic identity of plant species commonly sold as Ostokhudus in Iranian herbal markets, focusing on the genera Lavandula and Nepeta, which are frequently misidentified due to their morphological similarities, (2) Evaluate and apply phytochemical, morphological, and micromorphological markers to distinguish between Lavandula and Nepeta species, ensuring accurate identification even in processed or powdered forms and (3) Provide a scientific basis for the accurate identification of plant materials used in herbal samples and herbal-based industrial products, to prevent adulteration and ensure product quality. By achieving these objectives, this study aims to contribute to the preservation of Iran’s rich botanical heritage, improve the reliability of medicinal plant trade, and support the sustainable use of these valuable resources in traditional and modern medicine.

Results and discussion

Morphological analysis

Our morphological investigation revealed that the herbal samples labeled as Ostokhudus showed some morphological variation (Fig. 1). The leaf shape of samples HS1, HS2, HS3, HP6, and HP7 was linear-lanceolate, with varying lengths due to their dried form. The leaf color was greyish in HS1 and HS2, and green in HS3, HP6, and HP7. The dried flowers made accurate morphological identification difficult; however, HS1 and HS2 had shades of purple lilac, while HS3, HP6, and HP7 had shades of violet and purple-blue. The bract shape was a key feature for identification, being diamond-shaped in HS1, HS3, HP6, and HP7, and lanceolate in HS2. The size of the bracts varied, with HS1 measuring 1 mm in width and 3–4 mm in length, and HS3, HP6, and HP7 ranging from 3–4 mm in width and 1–3 mm in length. All five samples shared common features such as an appendage on the upper lobe of the calyx and a five- to eight-toothed calyx with 13 nerves, similar to Lavandula species belonging to L. sect. Lavandula25,41. The L. sect. Lavandula is characterized by simple and entire leaves and a calyx appendage that has the same width as the calyx41. The bract forms are crucial for identifying species within the L. sect. Lavandula. In L. angustifolia, L. angustifolia × L. lanata Boiss., and L. × intermedia Emeric ex Loisel., the bracts are rhombic in shape; while in L. lanata and L. latifolia Medik., they are linear to linear-lanceolate. L. angustifolia has bracts where the width is at least twice the length, with small and barely visible bracteoles. On the other hand, L. angustifolia × L. lanata and L. × intermedia have bracts where the length is about three times the width, with visible bracteoles ranging from 1–4 mm. The flowers of L. angustifolia × L. lanata are dark purple-mauve, while L. × intermedia flowers are lilac. The distinction between L. lanata and L. latifolia lies in the characteristics of their calyx teeth; L. lanata has an eight-toothed calyx, while L. latifolia has a five-toothed calyx41. The comparative analysis of the morphological data suggested that the HS1 sample, with its rhombic bract form and different size compared to HS3, HP6, and HP7 samples, may belong to the L. × intermedia species. The similarities between HS3, HP6, and HP7 samples and L. angustifolia indicated that they likely belonged to that species. The HS2 sample, with its lilac flower color, lanceolate bract form, and five-toothed calyx, was likely from the L. latifolia species. The HS4 sample stood out for its dentate, rounded, and shallowly lobed leaf margins, distinguishing it from the other samples. The tuft of enlarged violet-blue floral bracts on the spike’s apex, the 13-nerved calyx, and the presence of a calyx appendage suggested that the HS4 sample could belong to L. sect. Dentatae Suárez-Cerv. & Seoane-Camba41. This section, containing a single species, L. dentata, is characterized by linear-lanceolate leaves with regular shallow-rounded lobes, and a compact flower spike topped by a coma41, further supporting the possibility that the HS4 sample belonged to this species.

Fig. 1
figure 1

Herbal samples labeled as Ostokhudus in Iran. (A,B) HS3 sample; (A) Sample from market, (B) Calyx; (C) Lavandula angustifolia; (D,E) HS1 sample; (D) Sample from market, (E) Leaf; (F) L. × intermedia; (G,H) HS4 sample; (G) Sample from market, (H) Calyx; (I) L. dentata (Photo by Robert Perry (2025); https://waterwisegardenplanner.org/plants/lavandula-dentata/); (J,K) HS6 sample; (J) Sample from market, (K) Leaf; (L) Nepeta glomerulosa (Photo by Shahram Bahadori); (M,N) HS9 sample; (M) Sample from market, (N) Calyx; (O) Stachys lavandulifolia; (P,Q) HS4 sample; (P) Sample from market, (Q) Leaf; (R) Nepeta racemosa Lam. (Photo by Shahram Bahadori).

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The morphological characteristics of the HS5, HS6, and HS7 samples, particularly their leaf forms, were notably different from the rest of the Lavandula specimens. The leaf shape in the HS5, HS6, and HS7 samples was ovate, with HS5 and HS7 exhibiting a dentate-serrate margin, while HS6 displayed a dentate-denticulate margin and an obtuse apex. The reticulate venation, which was more pronounced on the adaxial surface of the leaf in the HS6 sample, provided a distinctive morphological feature for its identification. In terms of indumentum, the HS5 sample featured non-glandular branched and peltate glandular trichomes. In contrast, the HS6 sample was abundant in simple unbranched, vermiform, and peltate trichomes on its leaves and calyces. The indumentum of the HS7 sample was characterized solely by simple unbranched and peltate trichomes. All three samples had calyces with 15 nerves; however, their lengths varied: the calyx of the HS5 sample measured 5–6 mm, that of HS6 was 7–8 mm, and HS7 measured 5–7 mm. Although the distinct structure of the flowers within the HS5, HS6, and HS7 samples was not readily discernible, they exhibited variations in corolla color -from purple-blue and violet in HS5 and HS7 to lilac pink in HS6-suggesting a morphological similarity to Nepeta species. The genus Nepeta exhibits considerable variability in morphological traits, making species identification challenging. Characteristics such as indumentum, leaf shape and size, and calyx morphology can vary significantly even among closely related species. Notably, the presence of branched (dendritic) trichomes is a key feature that distinguishes Groups 1–3 of Nepeta species from others, while species in Groups 4–6 are characterized by simple indumentum42. Additionally, ethnobotanical studies conducted in the northeast and northwest regions of Iran—specifically in Razavi Khorasan and Ardabil provinces, where the HS5, HS6, and HS7 samples were collected—have identified traditional medicinal plants referred to as Ostokhudus, which are used for treating various ailments and are classified as Nepeta spp.20,21,43,44,45. This ethnobotanical context supported our morphological identification with greater certainty. Considering the morphological characteristics observed in the HS5 sample—particularly the presence of branched trichomes on its leaves and calyx, along with its collection region from Razavi Khorasan province in northeastern Iran—it was suggested that this sample may belong to Group 1 of Nepeta species and exhibit greater similarity to N. binaloudensis. The reticulate venation, vermiform trichome type, and lilac pink color of the HS6 sample were key diagnostic features within Group 2 of Nepeta species, suggesting it may belong to N. glomerulosa species. The presence of only simple unbranched trichomes in the HS7 sample differentiated it from other investigated cf. Nepeta samples, indicating it may belong to Group 4 of Nepeta species. However, for accurate identification, more detailed information was needed.

Based on the distinct morphological features of the HS8 and HS9 samples compared to other herbal samples, they were clearly separated. Morphologically, the elliptic-oblong leaf shape and verticillasters with four to six pinkish-purple flowers of the HS8 herbal sample resembled Stachys lavandulifolia, an important medicinal plant distributed in different regions of Iran25. The aerial parts of this plant have traditionally been used in Iranian folk medicine to alleviate various ailments, including infections, asthma, inflammation, and rheumatism46. The odor and morphological features of the HS9 sample, particularly its linear-lanceolate cauline leaves and purplish spiked inflorescence with a corolla tube slightly longer than the calyx tube, resembled Ziziphora tenuior. This aromatic herb is extensively distributed throughout Iran25 and is used in Iranian folk medicine for the treatment of fever, dysentery, and uterine infections47,48 .

Micromorphological analysis

Different types of non-glandular and glandular trichomes could be distinguished on the leaf and calyx of the investigated samples (HS1-HS7, HP6, and HP7) (Fig. 2). Based on the variation observed, the non-glandular trichomes were subdivided into subtypes: simple unbranched and branched (dendroid) trichomes. Peltate (sessile) and capitate (short-stalked trichomes) constitute two subtypes of glandular trichomes.

Fig. 2
figure 2

SEM micrographs of trichome types in certain herbal samples labeled as Ostokhudus. (A,D) HS1 sample; (B,E) HS2 sample; (C,F) HS3 sample; (G–I) HS4 sample; (J) HS6 sample; (K,L) HS5 sample; (M–O) HS7 sample.

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HS1 had pubescent, densely dendroid, and peltate glandular indumentum (Fig. 2A,D). In HS2, HS3, HP6, and HP7 samples, the presence of densely papillate dendroid trichomes was prominent (Fig. 2B,C,E,F). Dendroid trichomes, as a type of glandless multi-branched hairs, have different numbers of arms at each node. The peltate glandular trichome was also observed in these samples (Fig. 2C). HS4 had dendroid trichomes with smooth surfaces and both peltate and capitate (short-stalked) glandular trichomes (Fig. 2G–I). In HS5, branched non-glandular trichomes were frequent and no types of glandular trichomes were observed (Fig. 2K,L). HS6 possessed pubescent and vermiform unbranched indumentum, as well as peltate glandular trichomes (Fig. 2J). HS7 had densely-pubescent and peltate trichomes (Fig. 2M–O). The presence of papillae on the surface of its simple trichomes was considerable.

Simple unbranched non-glandular and capitate glandular trichomes were the common types of trichomes among the investigated species. Considering the morphological investigations and previous studies on the trichome types of different Lavandula species and cultivars37,49, our micromorphological data further confirmed that HS1, HS2, HS3, HP6, and HP7 samples could have belonged to L. sect. Lavandula. Despite the presence of different types of capitate trichomes (short- medium- and long-stalked) in various cultivars of L. angustifolia49, no types of capitate glandular trichomes were observed in HS3, HP6, and HP7 samples morphologically identified as cf. L. angustifolia. It seems that this dissimilarity may be related to the origin of different cultivars and species investigated in different studies. Yet, analyzing more samples may validate the finding results. Among common trichome types observed within the L. sect. Lavandula, the presence of pubescent indumentum differentiated HS1 among others, which could be a key diagnostic feature for its identification as L. × intermedia species. The contribution of different studies conducted on L. dentata indumentum35,50 confirmed our morphological findings on the HS4 and its presumable identification as L. dentata species. Following the reported morphological characteristics of the HS5, the presence of branched trichomes on its leaves and calyx was confirmed by SEM micrographs. It characterized this sample as a member of Group 1 of the Nepeta species25. In complement to prior morphological investigations, it was confirmed that the HS5 sample may be attributed to N. binaloudensis species. The presence of vermiform unbranched trichomes in the HS6 could serve as a diagnostic key for its identification and validated by SEM micrographs. Densely-papillate pubescent indumentum in the HS7, confirmed by its micromorphological analysis, characterized it among other investigated samples. Generally, our micromorphological investigations revealed that the type of trichomes exhibited significant variation among different genera, yet remained consistent within the species of the same section or genus, thereby providing valuable and complementary diagnostic characteristics for the identification of different species and genera. The taxonomic significance of indumentum and its implication in systematics and phylogenetic studies have also been well-documented within Lamiaceae and allied families, including Verbenaceae and Scrophulariaceae51,52,53. Therefore, trichome micromorphology could be considered as a complementary method for different species identification.

Phytochemical analysis

The importance of phytochemical studies in complementing morphological research and authenticating true species has recently been highlighted6,28,29. This study investigates the essential oil composition of commercially available herbal samples labeled as “Ostokhudus” in the Iranian herbal markets as well as seven commercial herbal products were evaluated using GC-FID and GC–MS analyses. The results of the analysis of the chemical constituents of the essential oils are represented in Table 1 and Fig. 3, indicating altogether 34 compounds, including monoterpene hydrocarbons (1.4–17.1%), oxygenated monoterpenes (70.1–95.7%), sesquiterpene hydrocarbons (0.1–3.9%), oxygenated sesquiterpenes (0.1–5%) and the other compounds (0.1–1.2%). The essential oil composition of the samples varied significantly. In HS1, the major components were linalool (37.3%), camphor (14.4%), and linalyl acetate (12.3%). The HS2 sample was primarily composed of 1,8-cineole (29.3%), linalool (22.3%), and camphor (10.7%). Linalool and linalyl acetate were the dominant compounds in HS3 (33.5%, 19.9%), HP1 (66.2%, 16%), HP2 (linalool alone at 68.8%), HP3 (51.5%, 33.1%), HP4 (44%, 41.2%), HP5 (40.9%, 27.1%), HP6 (38.1%, 21.2%), and HP7 (36.8%, 20.1%). In contrast, HS4 was characterized by a high concentration of camphor (64.3%), HS5 by 1,8-cineole (75.8%), and HS7 by two nepetalactone isomers: 4aα,7β,7aα-nepetalactone (63.3%) and 4aα,7α,7aα-nepetalactone (14.9%). Lastly, HS6 was primarily composed of 1,8-cineole (23.2%), α-terpinene (19%), and isobornyl acetate (10.9%). Overall, the presence of 4aα,7α,7aα-Nepetalactone, 4aα,7α,7aβ-Nepetalactone, and 4aα,7β,7aα-Nepetalactone in the essential oils of HS5, HS6, and HS7 samples chemotaxonomically distinguished these three samples from the others, whose chemical profiles were more consistent with those of the genus Nepeta. Nepetalactone, a notable bioactive compound, is primarily found in the essential oils of various Nepeta species39,54,55,56. Baser et al.57 further demonstrated that Nepeta species can be categorized into two distinct groups based on the presence or absence of nepetalactone isomers, resulting in nepetalactone-containing and nepetalactone-less species. This underscores the significance of nepetalactone as a key chemotaxonomic marker for identifying Nepeta species and differentiating them from other genera. Within Lavandula, the primary components of the essential oils are typically linalool and linalyl acetate58,59,60. However, the concentrations of these constituents vary significantly across different species61,62. Phytochemical analyses of the essential oils derived from various Lavandula species, hybrids, and cultivars reveal considerable inter- and intraspecific differences. These variations can be attributed to factors such as climatic and geographical conditions, seasonal changes, agricultural practices (e.g., irrigation and fertilization), genotypic differences, and the methods used for essential oil extraction60. Notably, commercial Lavandula hybrids often exhibit varying concentrations of 1,8-cineole and camphor, while these compounds are present only in trace amounts in L. angustifolia60. The high percentages of linalool in HS1, HS2, HS3 (37.3%, 22.3%, and 33.5%, respectively) and HP1-HP7 (66.2%, 68.8%, 51.5%, 44%, 40.9%, 38.1%, and 36.8%, respectively), when compared to the major component of L. angustifolia essential oil (44.4%)63, suggested that these samples likely belong to different Lavandula species. Furthermore, the chemical profiles of HS3, HP6, and HP7 were compared with those of L. angustifolia63 and published data58,60,62,64, revealed a strong correlation. Overall, based on both the morphological characteristics of HS3, HP6, and HP7 samples and the phytochemical findings, these samples were identified as L. angustifolia. In addition to the high concentration of linalool in HS2, elevated levels of 1,8-cineole, camphor, and α-terpineol, along with low percentages of linalyl acetate, distinguished this sample from other examined Lavandula samples, such as HS1, HS3, HP6, and HP7. The phytochemical analysis, when compared to previous data58,64,65,66, indicated that HS2 could be identified as L. latifolia. The high similarity in linalool and linalyl acetate concentrations, coupled with the low content of 1,8-cineole in HS1 compared to HS3, HP6, and HP7, along with its relatively high camphor content (second only to HS2), suggested that HS1 might represent an intermediate or hybrid between L. angustifolia and L. latifolia. This conclusion was further supported by the chemical profile and morphological analysis of HS1, which aligned with findings from Bajalan et al.67, Wells et al.66, and Batiha et al.58, confirming its identification as L. × intermedia. HS4, on the other hand, exhibited distinct differences from the other Lavandula samples, particularly in its low concentrations of linalool and linalyl acetate. The high percentages of camphor in HS4 served as a key chemotaxonomic marker, further distinguishing it from other Lavandula species, as corroborated by Wells et al.66 and Batiha et al.58. Morphological analysis, including flower structure, along with the phytochemical profile of HS4, supported its identification as L. dentata. However, El Abdali et al.68 reported that the essential oil of L. dentata is primarily composed of linalool (45.06%), camphor (15.62%), and borneol (8.28%). The phytochemical data of HP1-HP5, when compared to the chemical profile of L. angustifolia63, confirmed the presence of L. angustifolia essential oil in these herbal-based products. The high percentage of 1,8-cineole (75.8%) in the HS5 sample, as the predominant constituent of its essential oil, distinguished it from other investigated Nepeta specimens. A study by Nadjafi et al.23 revealed that the essential oil of N. binaloudensis grown in two regions of Razavi Khorasan (Dowlat Abad and Freizi) was primarily composed of 1,8-cineole (77.8% and 73.2%, respectively), with no traces of nepetalactone isomers detected. Similarly, Talebi et al.39 reported that the essential oil of N. binaloudensis contained 1,8-cineole (43.5%) and 4aα,7α,7aβ-nepetalactone (23.5%) as its main components. Mohammadpour et al.69 identified 1,8-cineole (68.3%) and α-terpineol (5.2%) as the predominant constituents of N. binaloudensis essential oil, while Rustaiyan and Nadji70 found 1,8-cineole (42.3%) and 4aα,7α,7aα-nepetalactone (25.2%) to be the major components. These findings highlight the variability in the chemical composition of N. binaloudensis essential oils across different studies and regions. Although 1,8-cineole was identified as the main component in all reported species, the varying percentages of other constituents can likely be attributed to environmental factors. Numerous studies have highlighted that phytogeographical origin, as well as physical and chemical conditions such as temperature, humidity, and harvesting time, can significantly influence the yield and composition of essential oils derived from different samples71,72,73,74. Tounekti et al.75 demonstrated that environmental factors, including salinity stress, can affect specific secondary metabolites such as 1,8-cineole. Similarly, Hashemi-Moghaddam et al.76 found a significant positive correlation between oil yield and precipitation, while a negative correlation was observed with temperature. Additionally, they reported a positive correlation between 1,8-cineole content and altitude, whereas nepetalactone content showed a negative correlation with altitude. These findings underscore the variability in essential oil composition among populations of the same species, which is not uncommon. Regarding the HS5 sample, its morphological and micromorphological characteristics, combined with ethnobotanical data from northeast Iran20,21,22,23, suggested its identification as N. binaloudensis. Ethnobotanical records indicated the use of certain Nepeta species, including N. binaloudensis as “Ostokhudus” in Iranian traditional remedies. Furthermore, N. binaloudensis is an endemic species native to the Binalud Mountains in northeast Iran, supporting the identification of HS5 as N. binaloudensis. The chemical profile of the HS6 sample, along with its major components, differed significantly from the phytochemical constituents of other Nepeta species previously reported56,77,78,79. Notably, the very low percentage of nepetalactone isomers in the essential oil of HS6 set it apart from other Nepeta specimens in this study. In a previous study by Talebi et al.39, no nepetalactone isomers were detected in the essential oil of Nepeta glomerulosa subsp. carmanica (Bornm.) Rech.f. (Syn.: Nepeta glomerulosa var. carmanica Bornm.), which was instead dominated by 1,8-cineole (23.3%), isobornyl acetate (6%), geraniol (5.4%), terpinen-4-ol (5.3%), and borneol (4.3%). In contrast, α-terpinene (19%) emerged as the second major component in the HS6 sample, a compound not reported as a primary constituent in the study by Talebi et al.39. Based on the morphological and micromorphological characteristics of the HS6 sample, as well as the findings of the phytochemical analysis, HS6 could be identified as belonging to the N. glomerulosa species. The high percentages of nepetalactone isomers, specifically 4aα,7α,7aα-Nepetalactone (14.9%) and 4aα,7β,7aα-Nepetalactone (63.3%), in the HS7 sample distinguished it from other Nepeta specimens examined in this study. In species where nepetalactone isomers are the predominant constituents, 1,8-cineole, and caryophyllene oxide are typically minor components of the essential oil80. This pattern has been further confirmed by studies on N. racemosa54,82,83, N. racemosa subsp. crassifolia (Boiss. & Buhse) A.L.Budantzev (syn.: N. crassifolia Boiss. & Buhse)81, and N. persica Boiss.84. In the HS7 sample, low concentrations of 1,8-cineole (2.3%) and caryophyllene oxide (0.6%) were also observed, consistent with these findings. Combining the phytochemical data with the morphological and micromorphological features of the HS7 sample, it was suggested that this sample likely belongs to N. racemosa species. Overall, the chemical profiles of HS5, HS6, and HS7 samples, along with their morphological and micromorphological analyses, confirmed the historical mistaken substitution of different Nepeta specimens for Lavandula species over time4,5,7,14. The resemblance in flower shape and color of these samples in their dried forms to those of Lavandula species has contributed to this substitution. Additionally, the similar therapeutic effects of these specimens have further perpetuated this practice10.

Table 1 Percentage chemical composition of the essential oils obtained from herbal samples and products labeled and sold as Ostokhudus in the Iranian herbal market.
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Fig. 3
figure 3

GC-FID chromatograms of the studied samples.

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Statistical analyses

To construct the dendrogram of studied samples based on their chemical profile, cluster analysis using the Ward method was performed (Fig. 4). There were two major clusters: 1st cluster included two subclusters 1a and 1b, differentiating by the presence of high percentages of linalool and linalyl acetate in the essential oils of the samples. HS3, HP6, and HP7 samples identified as L. angustifolia, along with HS1 as L. × intermedia were placed in subcluster 1a based on the similar percentages of linalool and linalyl acetate in their chemical profile. Subcluster 1b consisted of HP1-HP5 commercial herbal products in which the presence of L. angustifolia essential oil in their composition was confirmed. The extremely high percentages of linalool in the chemical profile of HP1-HP5 compared to HS1, HS3, HP6, and HP7 separated them in subcluster 1b. 2nd cluster was subdivided into three subclusters: HS2, HS4, and HS5 in 2a, HS7 in 2b, and HS6 in 2c. HS2 and HS4 were set apart from other Lavandula species in the 2a′ subcluster due to their high percentages of 1,8-cineole and camphor, respectively. HS5, HS7, and HS6 samples were separated from others in 2a″, 2b, and 2c subclusters, respectively, based on the presence of nepetalactone isomers in their essential oils. However, the HS5 sample was put together with HS2 and HS4 samples in 2a due to its high percentages of 1,8-cineole and low percentages of linalool which were among its similarities with HS2 and HS4 samples, respectively. Moreover, the high percentages of 1,8-cineole and isobornyl acetate in HS6, compared to other Nepeta specimens in the 2nd cluster, separated it into the 2c subcluster. To validate the results of cluster analysis and demonstrate the predicted position of each sample in a two-dimensional space, the scoring plot of the studied samples based on the first and second components derived from principal component analysis (PCA) was constructed (Fig. 5). Overall, five components accounted for ca. 69% of the variation among the samples. The first three components with  48% and 21% variances respectively, had the most significant effect on differentiating the samples from one another. Based on the first component (PCA1), the first and second main groups of the samples were obviously set apart. In the first group, subgroup 1a (including HS1, HS3, HP6, and HP7) and 1b (HP1-HP5), as well as three subgroups of the second group (2a, 2b, and 2c), were differentiated and separated based on the second component (PCA2).

Fig. 4
figure 4

Dendrogram obtained from 34 phytochemical constituents of the studied samples using Ward’s method.

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Fig. 5
figure 5

PCA plot performed based on the principal components of essential oils determined by GC-FID data.

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Conclusion

In this study, the authentication and identification of nine herbal samples labeled as Ostokhudus in the Iranian herbal markets, and seven commercial herbal-based products were investigated and analyzed using morphometric methods, and GC-FID/GC–MS analyses. Considering the crashed form of certain herbal samples in the markets, as well as herbal-based industrial products lacking distinctive characteristics for identification, our findings revealed that using complementary approaches including morphological, micromorphological, and phytochemical techniques is recommended to achieve accurate identification and authentication of plant species. Based on morphological, micromorphological, and phytochemical analyses, HS1-HS4, HP6, and HP7 samples exhibited calyces characterized by five- to eight teeth and 13 nerves, as well as dendroid trichomes covering both leaf and calyx surfaces. Furthermore, GC–MS analysis revealed that linalool and linalyl acetate constituted the dominant chemical components in HS1, HS2, HS3, and HP1-HP7 samples, were more consistent with those of Lavandula species. HS5, HS6, and HS7 samples, however, displayed various leaf forms and trichome types. The presence of 15-nerved calyces, along with the identification of nepetalactone isomers within their chemical profile, characterized these samples from the others which were suggested to be those of the genus Nepeta. The HS8 sample, characterized by its elliptic-oblong leaf form and its distinct morphological features was identified accurately as Stachys lavandulifolia. The HS9 sample displayed characteristic features diagnostic of Ziziphora tenuior, specifically its linear-lanceolate cauline leaves and a purplish spiked inflorescence. Since there was no comprehensive research has been conducted on the status of plant species named Ostokhudus in the herbal markets and industries of Iran, this was the first study on the authentication of these samples and their probable substitutions. Utilizing multidisciplinary collaborative approaches combining molecular and phytochemical techniques will help and enhance the accurate identification of medicinal plants and also would be a promising perspective for their standardization.

Materials and methods

Plant material and market samples

Among the diverse herbal samples labeled as Ostokhudus in herbal markets across various provinces of Iran (locally known as attari in Persian), nine herbal samples and seven commercial herbal products were selected for further examination (Table 2). The commercial products included three soft gel capsules and two oral drops claimed to contain L. angustifolia essential oil, as well as one herbal tea and a bulk product labeled as Ostokhudus. Our study is compiled with relevant national guidelines and legislation. Herbal samples with distinguishable morphological features were examined using a stereomicroscope at ×80 magnification and identified by the first and last authors; A. Babaei determined the herbal samples: MPH-3266, MPH-3267, MPH-3269, and MPH-3270, and A. Sonboli identified herbal specimens: MPH-3264, MPH-3265, MPH-3268, MPH-3271, and MPH-3272, using key references, including Flora Iranica (Vol. 150, Labiatae)26, Flora of Iran (Vol. 76, Lamiaceae)25, and Lavender: The Genus Lavandula41. Voucher specimens of the collected herbal samples (MPH-3264 to MPH-3272) were deposited in the Medicinal Plants and Drugs Research Institute Herbarium (MPH) of Shahid Beheshti University, Tehran, Iran.

Table 2 Information on herbal samples and products labeled and sold as Ostokhudus in Iranian herbal markets.
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Micromorphological study

For scanning electron microscopy (SEM) analysis, small sections of leaves and calyces from the nine herbal samples (HS1-HS7, HP6, and HP7) were mounted on aluminum stubs using double-sided adhesive tape and coated with a thin layer (approximately 25 nm) of gold–palladium. The SEM images were obtained using an SU3500 microscope, operating at an accelerating voltage of 15 kV.

Essential oil isolation procedure

The essential oil isolation from the air-dried aerial parts and flowers of herbal samples, as well as commercial herbal products, was performed using hydro-distillation, employing a Clevenger-type apparatus. 30 g of the powdered and pulverized dried herbal samples, and for the commercial products, 10 mL of oral drops and five soft gels, were macerated in 500 mL of distilled water within a round-bottom flask. Boiling stones were also added to promote the procedure stable. Upon heating, the volatile oil components co-distilled with water, and the resulting two-phase distillate was collected and separated85. After a three-hour isolation period, the essential oil was collected, dried using anhydrous sodium sulfate, and analyzed by Gas Chromatography (GC-FID) and Gas Chromatography–Mass Spectroscopy (GC–MS) techniques.

Gas chromatography (GC)

The GC-FID analysis of the oil was performed using a ThermoQuest-Finnigan instrument equipped with a semi-polar HP-5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 μm). The carrier gas used was Nitrogen, which was maintained at a constant flow rate of 1.1 mL/min. The oven temperature was initially set at 60 °C and then programmed to increase to 250 °C at a rate of 5 °C/min. The temperature of the injector and flame ionization detector (FID) temperatures were maintained at 280 °C throughout the analysis.

Gas chromatography–mass spectrometry (GC–MS)

The GC–MS analysis was performed using an Agilent Technologies TRACE MS instrument equipped with a quadrupole analyzer on an HP-5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 μm). The oven temperature was programmed to increase from 60 to 250 °C at a rate of 5 °C/min. The transfer line temperature was maintained at 250 °C. Helium was employed as the carrier gas at a flow rate of 1.1 mL/min.

Identification of essential oil constituents

The composition of the essential oil was determined through the calculation of retention indices under temperature-programmed conditions, utilizing n-alkanes (C6–C24) on an HP-5 column, with identical chromatographic conditions. Finally, the constituents were confirmed by comparing their retention indices and mass spectra with published literature data86, thereby ensuring the accuracy of the identification process.

Statistical analysis

Cluster analysis is a statistical method employed for the processing of GC-FID or GC–MS data, wherein the percentage chemical composition of the essential oils (> 1%) from GC-FID data of all the samples was analyzed and categorized into groups and clusters based on their associations. The Ward method was employed to construct the dendrogram, utilizing squared Euclidean distance as the distance metric. The Principal Component Analysis (PCA) illustrates the predicted positions of the principal components (PCs) of each sample component, thereby facilitating the statistical determination of similarities between samples. Minitab software version 22.1 was used for cluster analyses and PCA.