Introduction

The use of medicinal and psychoactive plants by humans is a tradition with profoundly ancient origins1,2,3,4. Selecting, processing, and harnessing their bioactive properties marked significant breakthroughs in human history, which laid important foundations for modern pharmacology and traditional medicine5. Across cultures, these plants were highly valued for their ability to provide therapeutic benefits and induce altered states of consciousness, playing integral roles in medicinal, sanitary, ritualistic and recreational practices across various cultures4,6,7,8,9. Through careful observation and experimentation, ancient communities cultivated a deep understanding of their local flora10,11. This rich body of knowledge, which included the identification, harvesting, and application of medicinal and psychoactive plants, was refined over generations and passed down through centuries if not longer11,12,13,14. However, much of the ancient expertise has been lost over time, primarily because these knowledge systems were often transmitted only orally. In contrast to other regions, such as ancient Mesopotamia, Greece, Egypt, or China11,15,16, where botanical and medicinal knowledge has been extensively documented also in texts, we lack such ancient written sources from Arabia before the Classical Greek and Islamic periods. Translations of ancient Greek medical texts, such as Galen’s medical works, into Syriac and Arabic occurred only in Late Antiquity17.

A promising approach for gaining more information about the use of medicinal and psychoactive plants in earlier periods of history is the analysis of preserved plant residues found in archeological artifacts7,8,18. Using techniques such as metabolic profiling, these residues can reveal direct insights into the ancient use of botanical resources, including the identification of the original plant materials, their bioactive properties, and methods of application. This approach offers a unique window into the past, shedding light on the types of plants used for purposes that are often challenging to investigate in archeological contexts, such as medicinal, therapeutic, sensorial, olfactory, sanitary, and recreational practices7,9,19. These challenges arise primarily from the limited preservation of these substances, which were often burned during fumigation, directly consumed or processed. In this context, preserved organic residues in archeological artifacts and contexts serve as important archives18, offering critical information for plant identification.

The present study investigates the use of plant-based substances in Iron Age Arabia, focusing on the archeological site Qurayyah, an ancient oasis settlement in Saudi Arabia (28° 47’ 00” N and 36° 00’ 27” E), which thrived as a significant ‘urban’-like center during the Bronze and Iron Ages (Fig. 1A)20,21. This site has yielded numerous censers and fumigation devices from these periods, notable for the preserved organic residues inside them. Although evidence exists for the ancient consumption of drug plants dating back to prehistory in the Americas8,22,23,24,25,26, Europe6,27, North Africa9, and Central Asia7,28,29, such practices remain unexplored in pre-classical Arabia, despite the region’s rich diversity of drug plants. Today, among the ca. 2,250 identified plant species in Arabia, nearly 25% are documented for their medicinal uses13,30, suggesting that past societies in the region may already have harnessed these plants for their therapeutic and psychoactive properties.

Fig. 1: Residential structures and censers from the oasis of Qurayyah.
Fig. 1: Residential structures and censers from the oasis of Qurayyah.
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A Drone view of Qurayyah with the localization of the excavated Areas D, N, and R, indicated by circles (Photo A. M. Abualhassan). B Iron Age residence of Area D with censer QU.D.1167.F.6 and painted vessel QU.D.1167.F.1 in situ (Photo S. McGlone), and Iron Age elite dwelling of Area N (Photo A. M. Abualhassan). C Photos of the censer from Area D: QU.D.1167.F.6 and of the two censers from Area N: QU.N.2340.F.3 and QU.N.1253.F.1 (Photos: H. Sell [Area D] and C. Jäger [Area N]). Graphics: Michelle O’Reilly, MPI-GEA.

The practice of burning substances for ritual purposes in the oasis of Qurayyah can be traced back to the 3rd millennium BCE, evidenced by a censer found in a final Early Bronze Age grave (2135-1952 cal BCE (IntCal20, 95.4% probability))31. Recent excavations have revealed additional fumigation devices from residential areas dating to the Middle Iron Age (first half of 1st millennium BCE; see Supplementary Table 1 for radiocarbon dates). Notably, one such burner (QU.D.1167.F.6) was discovered in Area D, in the courtyard of an Iron Age residence (Fig. 1B). From this object’s inner surface (Fig. 1C), two residue samples (DA-QU_D-1 and DA-QU_D-2) were collected for analysis. Additional devices were discovered in Area N, an elite dwelling of the same age as the Area D residence (Fig. 1B). While one burner (QU.N.1253.F.1) was found in the food production area, another (QU.N.2340.F.3) was discovered in the cellar of the home (see Supplementary Information for additional contextual information). These devices also contained traces of burning and residues on the surface. Sample DA-QU_N-1 was taken from the object in the cooking area, a cuboid burner, which belongs to the building’s latest phase, and sample DA-QU_N-2 was retrieved from the burner in the cellar corresponding to the building’s earliest phase. All censers are made of fired clay.

By employing high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) in multiple reaction monitoring (MRM) mode, we aim to identify the origins of these organic residues and explore their significance in their contexts. MRM is a targeted mass spectrometry technique that facilitates the monitoring of specific precursor and product ion pairs, significantly enhancing the specificity and sensitivity of the analysis. This proves particularly advantageous for detecting compounds present in low concentrations or contained within complex sample matrices, as often encountered in archeological contexts. Here, we present the first material evidence that the plant Peganum harmala (commonly known as Syrian Rue, Harmal, or Esfand) was used in fumigation devices. P. harmala is known for its antibacterial32, psychoactive33,34, and multiple therapeutic properties35,36, and is widely used in traditional medicine37,38. Beyond its well-documented medicinal and psychoactive applications, this discovery invites further exploration of its broader potential uses in ancient Arabian society, including its role in daily life for sanitary purposes, cleansing rituals, and other practical functions.

Results

HPLC–MS/MS

The HPLC–MS/MS results show the presence of two tricyclic beta-carboline alkaloids, notably harmine and harmane (Fig. 2; Supplementary Data 1–4), in samples DA-QU_D-1, DA-QU_D-2, and DA-QU_N-1, confirmed through comparison with analytical standards. Harmine and harmane were detected in the archeological sample through specific optimal transitions of precursor and product ions under MRM mode. For harmine, the precursor ion with m/z 213.0 fragmented under specific collision energies (CE), producing three product ions: 170.1 at CE: 30 eV, 198.1 at CE: 23 eV, and 169.2 at CE: 42 eV. Similarly, harmane was identified with the precursor ion m/z 183.2, which fragmented to yield the product ions 115.1 at CE: 34 eV, 119.1 at CE: 52 eV, and 168.1 at CE: 28 eV (Fig. 2). These transitions, as well as the observed chromatographic retention times, aligned with those obtained from analytical standards, confirming the presence of harmine and harmane in the archeological sample with high specificity. Sample DA-QU_N-2 differed from the other samples as it did not contain these compounds.

Fig. 2: Alkaloids in archeological residues linked to Peganum harmala and its psychoactive and therapeutic properties.
Fig. 2: Alkaloids in archeological residues linked to Peganum harmala and its psychoactive and therapeutic properties.
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Archaeological samples (with DA-QU_D2 shown here as an example) were analyzed using targeted metabolomics to detect alkaloids via multiple reaction monitoring (MRM) mode. MRM chromatograms show transitions corresponding to the β-carboline alkaloids harmine and harmane, detected in the archeological sample and compared to analytical standards. For harmine, three transitions are shown: yellow trace (m/z 213.00 → 170.10, collision energy −30 eV), red trace (m/z 213.00 → 198.10, −23 eV), and purple trace (m/z 213.00 → 160.15, −42 eV). For harmane, the following transitions were recorded: yellow trace (m/z 183.08 → 115.10, −34 eV), purple trace (m/z 183.08 → 89.10, −52 eV), and red trace (m/z 183.08 → 168.10, −28 eV). The retention times and transition patterns matched those of reference compounds, confirming the presence of harmine and harmane in the archeological residue with high specificity. The icons below summarize known psychoactive and therapeutic properties of P. harmala, including antidepressant, psychoactive, antibacterial, anti-inflammatory, antiparasitic, and pain-relieving effects32,36,37,39,46,69,75,76. Graphics: Michelle O’Reilly, MPI-GEA.

These alkaloids occur naturally in species of the Peganum genus (Zygophyllaceae family)35. These perennial herbaceous shrubs bloom with whitish-yellow flowers and produce globular capsules as fruits, housing blackish seeds35. This genus includes four species with a wide distribution, extending from the Mediterranean to East Asia, and even reaching certain areas in America34. Specifically, P. harmala is widespread across the Mediterranean, Central Asia, North Africa, and Western Asia, including Saudi Arabia, thriving in arid and semi-arid regions39. In contrast, P. nigellastrum and P. multisectum are primarily found in northwestern China, Mongolia, and Russia, while P. mexicanum is native to North America34. Given that only P. harmala is endemic to Saudi Arabia and the other species grow at a considerable geographical distance, P. harmala is identified as the most likely and locally available source based on current vegetation. Beyond P. harmala, these alkaloids also appear in plants, such as Passiflora incarnata, Banisteriopsis caapi (also known as the ayahuasca vine), and other members of the Malpighiaceae family40. However, these species are endemic to the Neotropical Region and the Americas, making them unlikely candidates as sources used in Iron Age Arabia. Furthermore, the compound harmane has been identified in tobacco leaves (Nicotiana sp.), which are also indigenous to the Americas, as well as in coffee beans, which originated in Africa41,42. While the coffee plant (Coffea sp.) is currently cultivated in Yemen, its introduction to South Arabia from Ethiopia occurred only in the mid-fifteenth century CE43. There is no evidence of coffee cultivation, either in Arabia or Ethiopia, as early as the Iron Age. The chronological gap of more than two millennia between the Iron Age occupation at Qurayyah and the start of coffee cultivation further supports the exclusion of Coffea sp. as a potential source. Thus, due to the significant geographical distance and habitat differences of some plant sources and the chronological distance of others, we propose P. harmala as the most likely source.

Harmala alkaloids are reversible inhibitors of monoamine oxidase A (MAO-A)44. This means that they temporarily inhibit the MAO-A enzyme, which is responsible for breaking down certain neurotransmitters in the brain, such as serotonin and norepinephrine45. As a result, these neurotransmitters accumulate to higher levels, stimulating the central nervous system and contributing to the health-benefiting effects of P. harmala. Unlike irreversible inhibitors, which permanently deactivate enzymes, harmala alkaloids allow enzyme activity to recover once the compounds are metabolized. Additionally, the broad receptor affinity of harmala alkaloids contributes to their diverse psychopharmacological effects, ranging from sedation to stimulation46, as well as exhibiting antibacterial32, antiparasitic36, and anti-inflammatory activities39,47 (Fig. 2). Additionally, they possess immunomodulatory and potential cardiovascular effects, demonstrating a broad spectrum of actions on the human body48. However, the effects vary with the dose: small amounts can act as mild and therapeutic stimulants, while larger doses may induce hallucinations, and excessive consumption can even lead to poisoning49,50. This dose-dependent response exemplifies the concept of hormesis, where low doses of a substance may trigger beneficial or stimulatory effects, whereas higher doses can lead to adverse or inhibitory outcomes51.

In addition to the alkaloid content, samples DA-QU_D-1 and DA-QU_D-2 are characterized by a high abundance of sterol molecules, such as campesterol, β-sitosterol, stigmasterol, and sitostanol, alongside cholesterol (Supplementary Table 2 for further information about lipids). Samples DA-QU_N-1 and DA-QU_N-2 only contained low amounts of β-sitosterol and cholesterol. Phytosterols are widespread among a diverse array of plants52, and therefore, undiagnostic for specific plant identification. Common sources are plant oils, seeds, nuts, whole grains, legumes and fruits53. Given the high oil content of P. harmala seeds, it is plausible that these phytosterols originate from the harmala seeds. Research into P. harmala seed oil has revealed anti-inflammatory properties, underscoring the bioactive potential of the oil itself54. Nevertheless, the possibility of another plant oil’s contribution cannot be discounted without further evidence.

An additional substance was found in the two burners from Area N. This material was the main substance preserved in sample DA-QU_N-2, which lacked harmala alkaloids. We identified the presence of pentacyclic triterpenoids, specifically α-amyrin and β-amyrin, along with their derivatives (Supplementary Data 3 and 4). These compounds are prevalent across the plant kingdom, notably in plant resins and extracts from the Burseraceae family, which includes species like Canarium, Protium, Bursera, and Commiphora55. Although these compounds alone do not allow for precise species identification, resins from the Burseraceae family are recognized for their beneficial biological activities, including anti-inflammatory, antidepressant, and gastroprotective effects55,56,57. In sample DA-QU_N-1, these compounds co-occurred with beta-carboline alkaloids, suggesting a possible mixture of two substances. Alternatively, the two substances might have been used sequentially within the same fumigation device. The presence of mixtures of substances is known from the oasis of Tayma, another Northwest Arabian oasis settlement 280 km south-east of Qurayyah, where censers from Iron Age graves also revealed residues containing amyrin compounds mixed with conifer and Pistacia resins19,58. Additionally, at Madaʾin Salih, a settlement part of the oasis of al-ʿUla, 300 km south of Qurayyah, amyrin compounds were recovered from textiles used in Nabataean burial practices59. They were interpreted as constituents of elemi resins (Canarium sp.). However, there is no evidence of P. harmala or other drug plant use from settlement contexts in either Tayma or in Madaʾin Salih, thus corroborating a pattern of use of P. harmala in settlement contexts and not in funerary ones. The several censers from funerary contexts in Qurayyah (Area R) have not been analyzed yet.

Discussion

The metabolic analysis of the alkaloid content in the samples provides the most ancient, radiometrically dated, material evidence of P. harmala in fumigation devices. P. harmala was traditionally consumed in two main forms: by ingestion, often prepared as a tea or concoction, and through the inhalation of smoke from burning its seeds and roots1,9,60. Occasionally, it has been reported for external application, e.g., to treat skin conditions61,62. Our case study specifically highlights the practice of burning, as the residues were recovered from fumigation devices at the oasis of Qurayyah, setting it apart from ingestion-based practices and topical treatments. To our knowledge, this represents the earliest material evidence for P. harmala’ use as a substance to be burnt in censers in the past. In contrast, earlier documented cases, such as seeds of P. harmala found at the Predynastic site of Maadi, Egypt, only confirm the plant’s presence but provide no evidence of its specific applications63. Additionally, the archeological context at Qurayyah reveals that the fumigation devices containing harmala alkaloids were recovered exclusively from residential areas. P. harmala was used within dwellings, indicating its role at the oasis was most likely for domestic purposes connected to the household rather than for public ceremonies or funerary rituals.

While we have identified the original plant material, its bioactive properties, the method of application, and the context of use, the specific purposes for burning P. harmala at Qurayyah may have been multiple. Drawing on its historical and traditional use in other regions, one plausible hypothesis is its use for medicinal and therapeutic purposes, given its well-documented health benefits34. Today, the plant is still part of ethnomedicine in Saudi Arabia, where reliance on medicinal plants continues to be a common and valued practice13. In traditional medicine, particularly in West Asian and North African systems, P. harmala seeds have been recognized for a variety of properties, including analgesic, antithrombotic, carminative, anthelmintic, anti-inflammatory, galactagogue, and emmenagogue effects35,38. The administration of P. harmala is used to manage conditions such as joint pain, chronic headache, toothache, and rheumatoid arthritis64. Other applications described in traditional medicine include its use as a sedative for alleviating nervousness, as well as its potential roles as an antidepressant and mood stabilizer44,65. Concerning women’s health, it has been reported as an abortifacient, as well as for regulating menstrual flow and promoting or increasing breast milk production66,67. Also in ancient Greek texts, the plant is recognized for its medicinal value, being utilized as a vermifuge to expel tapeworms and as a treatment for fevers68.

It is plausible that, beyond human health applications, these plants may have also been used for veterinary purposes. P. harmala’s known sedative effects when consumed by farm animals69,70 could suggest a dual use in managing human and animal health at the oasis settlement. However, the method of application—fumigation—argues against this, as the plant was typically ingested by animals rather than inhaled as smoke. Similarly, for some human health benefits, the substance is more commonly consumed as a beverage or applied topically. Nevertheless, there are applications in traditional medicine where the burning and inhalation of P. harmala seeds was used, for instance, to relieve toothaches and headaches, for anti-arthritic and anti-inflammatory purposes, as a mood stabilizer, and generally to relieve pain38,64.

Beyond its therapeutic applications, P. harmala was documented in current traditional fumigation practices, particularly in ritualistic and spiritual contexts where its psychoactive properties were harnessed to induce altered states of consciousness60. At high doses, harmala alkaloids can produce intense hallucinations, euphoria, and other central nervous system effects46. The use of P. harmala at the oasis settlement could, therefore, suggest intentional exposure to its psychoactive effects. However, in order to have a hallucinogenic-like effect71, it must have been consumed in high concentrations, but the excessive consumption of P. harmala as a recreational psychoactive agent can be toxic, with several cases of harmala-related poisoning through overdosing already reported50. Beyond dosage, the modality of administration is crucial as well, where fumigation differs from oral intake, as inhalation delivers the active compounds through the respiratory system, potentially altering their absorption, distribution, and impact on the body. Another ritualistic and purifying purpose attested in modern-day Iran is the burning of P. harmala seeds to ward off evil.

In connection with purifying and cleansing rituals and routines, the practical applications of P. harmala must also be considered, particularly its potential use for sanitary and hygienic purposes. Due to its antibacterial and antifungal properties, the smoke produced from burning P. harmala seeds was traditionally used as a disinfectant agent to cleanse spaces and reduce the spread of illnesses72. At an oasis settlement like Qurayyah, maintaining hygienic habits in daily life would have been essential for minimizing health risks. In this context, P. harmala smoke may have played a role in air purification and disinfecting living spaces. Its practical properties further extend to its use as an insect repellent, offering protection against pests in domestic contexts—a significant concern in warm, oasis environments. In the context of air purification, the use of P. harmala primarily for its fragrance is unlikely because P. harmala smoke has a rather pungent scent, making it less suitable for olfactory purposes. Parallels from other northwest Arabian oases in the Iron Age indicate that other aromatic substances, such as Commiphora and coniferous resins, as well as Pistacia were commonly used for incense burning19,58.

Given its repeated association with households and its absence from tombs and temples in other oases (Tayma, al-ʿUla58,59), the most likely purpose of fumigating P. harmala at the oasis settlement of Qurayyah appears to be primarily medicinal or practical in nature. The plant’s documented properties suggest its use for sanitary and hygienic purposes, such as air purification, disinfection, and pest control, which would have been particularly relevant in a domestic oasis environment. Additionally, traditional medicinal practices, both historical and modern, attribute P. harmala a range of therapeutic benefits, making it plausible that its smoke was used to alleviate ailments and all sorts of pains. While the plant’s psychoactive potential cannot be entirely dismissed, achieving hallucinogenic effects would have required significantly higher doses, which seems unlikely in this context. Therefore, the use of P. harmala at Qurayyah may reflect a combination of practical hygienic applications and medicinal fumigation for treating health conditions within a domestic setting.

The evidence for the burning of P. harmala at Qurayyah as early as the Middle Iron Age—approximately 2700 years ago—underscores the deep historical roots of this traditional use of native plants. This discovery not only revives knowledge of ancient practices and highlights a longstanding legacy of medicinal plant use but also contributes to safeguarding the region’s intangible cultural heritage. At the same time, it is equally important to protect and preserve the traditional knowledge that still exists today. In Arabia, traditional plant-based remedies remain deeply valued within their communities13. However, such practices are increasingly disappearing. This underscores the urgent need to document and preserve this rich ethnobotanical knowledge before it is lost entirely, along with its historical context. Furthermore, utilizing the information stored in ancient organic remains could enable the recovery of bioactive compounds that have been forgotten over time73, potentially leading to the development of innovative plant-based therapies.

Methods

Sampling

The organic residue samples come from the archeological site Qurayyah in Saudi Arabia. The excavation permit for research in Qurayyah, as well as for analysis, was issued by the former Saudi Commission for Tourism and Antiquities (now Heritage Commission of the Ministry of Culture). Sampling of organic residues from the incense burners located in Area N was conducted at the archeological site post-excavation, while the samples from Area D were taken within the laboratories of the Max Planck Institute of Geoantropology in Jena, Germany, following established protocols19,52. Samples were exported in full accordance with relevant permits and local laws. Prior to sampling, the uppermost inner layer of each burner was meticulously abraded to eliminate potential surface contaminants. The prepared sampling spots then underwent targeted sampling, where visible incrustations were excised using a scalpel, and deeper matrix penetration was achieved by employing a Dremel 200 drill outfitted with a tungsten carbide abrasive bit to extract approximately 2 g of powder from the residual compounds absorbed into the clay matrix. Drill bits were rigorously cleansed with methanol between samplings to preclude any cross-contamination. Typically, a 1 × 1 cm section was drilled to a depth of 2–3 mm. The resultant powder was collected on sterile aluminum foil before being transferred into pre-cleaned glass vials for subsequent analysis.

Materials

HPLC grade methanol (MeOH) and Dichloromethane (DCM) were obtained from Sigma-Aldrich (Munich, Germany), acetonitrile (ACN) and ultrapure water from Biosolve (Valkenswaard, Netherlands), and formic acid (FA) from VWR (Leuven, Belgium). The analytical standards α- and β-amyrin, cholesterol, campesterol, β-sitosterol, stigmasterol, harmane, and harmine were purchased from Sigma-Aldrich (Munich, Germany), and sitostanol from Avanti (Darmstadt, Germany).

Extraction and analysis

Plant secondary metabolites and lipids were isolated from ancient organic residues by pressurized solvent extraction (PSE), adhering to previously established protocols74. The extraction was facilitated by a Büchi E-916 Pressurized Speed Extractor, which utilized high temperatures and pressures to optimize the recovery of organic compounds. Prior to extraction, samples were homogenized to a uniform particle size using a mortar and pestle. These homogenized samples were then combined with quartz sand (Büchi, 0.3–0.9 mm) at an approximate ratio of 1:5 (sample to sand) and transferred to stainless steel extraction cells positioned within the PSE device’s heating block. The samples were extracted using DCM and MeOH (2:1, v/v) over three extraction cycles, each comprising a 1-min heat-up phase, a 15-min hold, and a 2-min discard interval. Conditions were set at 50 °C and 100 bar. The resultant extracts were collected in glass vials capped with Teflon septa. These extracts were then concentrated to about 1 mL via rotary evaporation. Aliquots of the concentrated extracts were evaporated and resuspended in HPLC-grade methanol.

HPLC–MS/MS analyses were conducted using a Shimadzu LCMS-8050 triple-quadrupole system with an electrospray ionization (ESI) source. The HPLC setup included LC-30AD binary pumps, a DGU-20A5R solvent degasser, CTO-20AC column oven, and a SIL-30AC auto sampler. Analytes were separated on two different columns: a Shimadzu Shimpack Velox SP-C18 and a Restek Raptor Biphenyl, both 100 mm × 2.1 mm with a 2.7 µm particle size. The samples were run with a gradient mobile phase of A, H2O:0.1% FA, and B, ACN, in duplicates with blanks in between. A consistent column temperature of 25 °C was maintained throughout the gradient program, which started with 0.5% B for the first minute, increased to 80% B at 10 min, reached 100% B at 15 min with a hold until 17.5 min, and then reverted to 0.5% B, holding until 20 min. Flow rates were adjusted to 0.2 mL/min for the C18 column and 0.3 mL/min for the biphenyl column. Injection volumes of 1 or 2 µL based on sample concentration were injected onto the system and analyzed in positive and negative ESI mode.

Data collection and processing were conducted using LabSolutions software (Shimadzu, Kyoto, Japan), which also facilitated the optimization of MRM mode parameters for the targeted compounds. Authentic analytical standards were employed to optimize the MRM parameters, essential for screening specific compounds in archeological samples. These parameters included precursor and product m/z, dwell times, collision energy and Q1 and Q3 pre-bias voltages (refer to Supplementary Data 5 for detailed MRM parameters).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.