Saudi Arabia has one of the oldest and most extensive herbal medicine traditions, and the locals have significant knowledge of medicinal plants. Due to the significance of these plants in Prophetic medicine [1] and the Middle East’s long history of medicinal plant study [2].

The researchers counted more than 400 species belonging to 89 families that were used in traditional medicine in Saudi Arabia [1]. For example, over a hundred medicinal plants were used in Makkah [3], and 85 medicinal plants were used in Jeddah [2], 124 medicinal plants were used in popular medicine in Jazan region [4]. More than 60 herbs were frequently used to treat and prevent respiratory illnesses and more than 20 plants were commonly used to immune system [3].

The ethnobotanical expertise might aid in developing a different strategy or identifying novel pharmacological compounds derived from therapeutic plants. Potential antiviral treatments can be made from the extracts or formulations of these plants. Additionally, they contain a variety of phytochemicals and other metabolites with immunity-boosting characteristics. Enhancing the human body’s immune system to combat viral diseases like the Coronavirus disease (COVID-19) and its variants [5].

Herbal traditional remedies have been employed since the beginning of the COVID-19 outbreak in December 2019, and they have had a good impact on the health of COVID-19 patients [6,7]. For instance, a team of doctors from Wuhan University’s Zhongnan Hospital highlighted the use of traditional remedies in their recommendations for the care and prevention of COVID-19. For the prevention of COVID-19, several strategies utilizing medicinal plants were suggested [6,8].

According to a recent study by Alqethami et al. [2] 85 medicinal herbs have been utilized in Jeddah traditionally. Sixty-one of them are used to treat respiratory diseases. Here and for the first time, the chemical composition of 5 medicinal plants commonly used to treat respiratory diseases in folk medicine in Jeddah was investigated, by the analysis methods of High-performance liquid chromatography (HPLC) with Gas chromatography-mass spectrometry (GC-MS) analysis. The objectives of this research focus on revealing the chemical constituents of these plants which will help in developing new drugs or improve natural formulations to help the immune system in fighting COVID-19 and its mutations.

A. Study area

The city of Jeddah is situated in the center of the Red Sea’s eastern side on the west coast of Saudi Arabia. On the Red Sea, it boasts the biggest seaport. The low heights of the Hejaz region and the Tihama plains are to the east of the city. The arid climate of this area, with its high summer temperatures and humidity, has an immediate impact on Jeddah. The main entry point for pilgrims to Makkah and Medina, two of Islam’s holiest towns and well-liked tourist destinations, is Jeddah. With a population of more than 4,082,184, Jeddah is the second-largest city in Saudi Arabia (2016 estimates). Population growth in Jeddah is 3.8% annually, which is faster than the national average. Immigration from rural areas and abroad is to blame for this increase in population [9].

B. Collection and identification of plants

Based on ethnobotanical fieldwork conducted in Jeddah over a year from August 2018 to September 2019, many plants were collected. Based on the results of phytochemical screening [10] that showed a favorable result with the test of flavonoids, samples of five plant species were chosen (Table 1). Approval was received from King Abdulaziz University (KAU), Unit of Biomedical Ethics Research Committee, Ethics Committee (Reference No 671-19). The ethical guidelines of the International Society of Ethnobiology (ISE) Code of Ethics [11] and the American Anthropological Association [10] were adopted. Ethnomedicinal data was published in [2]. In KAU herbarium (KAUH) specimens were mounted and identified using herbarium specimens, Flora of KSA [12,13]. The authors verified the identity. Families and nomenclature adhere to the Catalogue of Life [14]. Voucher specimens were preserved in KAUH.

C. Plant extraction

Plant extracts were hydrolyzed and subjected to analysis using the method previously described by [15]. In 10 ml of 1.2 mol HCl in 50% aqueous methanol, one gram of dried plant material was dissolved. Before being utilized for high-performance liquid chromatography, the mixture was refluxed at 80 °C for two hours, cooled, made to 10 ml in the same solvent, and filtered (HPLC).

D. Standard preparation

As a standard, quercetin, rutin, caffeic acid, cinnamic acid, and gallic acid were chosen. 25 mL of HPLC-grade methanol was used to dissolve 25 mg of each standard, resulting in a stock concentration of 1 mg/mL. Later, while still using the same mobile phase, they were dissolved in a variety of concentrations to construct the standard curve. For each standard, a calibration curve was created, and the relative standard deviation (RSD) was computed. Then, the amount of any phenolic or flavonoid acids present in each sample was determined as follows:

"Concentration of standard in the sample= " "Area under the peak of detected standard in the sample X standard concentration" /"Area under the peak of standard "

E. HPLC

The HPLC model 1260 Infinity II from Agilent Technology served as the liquid chromatography facility. The Zorbbax (SB)-C18 column, with dimensions of 150 mm, 4.6 mm, and 3.5 m (USA), was used for separation, and the column oven was set to room temperature. The solvent gradient program was as follows, using a mobile phase consisting of (A mobile phase: Methanol, B mobile phase: 0.7% Acetic acid HOAC) (Table 2). Ninety-minute runtime at a flow rate of 0.8 ml/min. The injection has a 10 L volume. At wavelengths of 254nm, 268-280nm, and 320-370nm, quantitation was carried out. At room temperature, a diode array UV detector (10X sensitive, 1260 DAD HS, G7117C) and an auto-sampler (1260 Vialsampler, G7129A) were used. Openlab CDS-Chemstation Rev. C. 01.08 [210] is the HPLC program.

F. GC-MS

Clarus 500 GC-MS (Perkin Elmer, Shelton, CT, USA) was used throughout the investigations. Gas chromatography-mass spectrometry. Version 5.4.2.1617 of TurboMass was the software controller/integrator. A Crossbond® 100% dimethyl polysiloxane (30-meter 0.25 mmID 0.25 m df), Optima® 1 GC capillary column, manufactured by Macherey-Nagel GMBH, Duren, Germany, was utilized. Helium (quality 99.9999%) served as the carrier gas, and the flow rate was 0.90 ml/min. Source (EI+): The source was 180 °C whereas the GC inlet line was 210 °C. The trap emission was 100 v, and the electron energy was 70 eV. 275 °C, injector. The oven was set to heat up from 80 °C (hold for 3 minutes) to 170 °C (rate 10 °C/min, hold for 11.0 min), and then to 280 °C (rate 10 °C/min, hold for 5.0 min). The split ratio was 50:1, and the injection volume was 1 L. By using a complete MS scan from 40 to 500 m/z (500 scan/sec), samples were obtained. The eluted chemicals were characterized using NIST 2008.

A. HPLC

Flavonoids are present in all 5 plant species that were examined using the HPLC technique. Utilizing five distinct standards (Table 3, Figure 1). The findings revealed that five different plant species included the antioxidants quercetin, rutin, caffeic acid, cinnamic acid, and gallic acid (Table 4). Four samples (3,5,14,26) had rutin, two samples (12,26) contained cinnamic acid, two samples (12,14) contained gallic acid, two samples (5,26) contained quercetin, and one sample contained caffeic acid (14).

Rutin concentration in Anethum graveolens L. seeds was 0.105 mg/mL. (Table 4, Figure 2). Rutin and quercetin concentrations in Cinnamomum cassia (L.) Presl bark were 0.121 mg/mL and 0.022 mg/mL, respectively (Table 4; Figure 3). About 0.309 mg/mL of cinnamic acid and 0.304 mg/mL of gallic acid were present in the seeds of Lepidium sativum L. (Table 4; Figure 4). About 0.030 mg/mL of rutin, 0.112 mg/mL of gallic acid, and 0.017 mg/mL of caffeic acid were present in the seeds of Helianthus annuus L. (Table 4; Figure 5). Cuminum cyminum L. seeds had rutin concentrations of 0.272 mg/mL, cinnamic acid concentrations of 0.012 mg/mL, and quercetin concentrations of 0.086 mg/mL. (Table 4; Figure 6>).

B. Gas chromatography-mass spectrometry

The extracts from all 5 plant species were analyzed by GC-MS. Thirteen chemical compounds were observed during GC-MS analysis of Anethum graveolens extract (Table 5; Figure 7). GC-MS analysis of Cinnamomum cassia extract resulted in the identification of 20 compounds (Table 6; Figure 8). Nineteen chemical compounds were detected in Lepidium sativum extract during GC-MS analysis (Table 7; Figure 9). Two chemical compounds were identified in Helianthus annuus extract (Table 8; Figure 10). Twentynine chemical compounds were observed during GC-MS analysis of Cuminum cyminum extract (Table 8; Figure 11).

Here and for the first time, the chemical reasons behind the use of 5 medicinal plants in folk medicine in Jeddah to treat respiratory diseases were highlighted. The HPLC and GCMS analysis revealed the chemical composition of these 5 medicinal plants. Cuminum cyminum was frequently cited in Jeddah’s traditional medical literature (72 citations [2], and the findings presented here indicate that it contains rutin, cinnamic acid, and quercetin [16]. Other unidentified peaks were also revealed in Cuminum cyminum extract, which could be coumarin, apigenin, luteolin, salicylic acid, or gallic acid. Additionally, the outcomes here demonstrated that Cinnamomum cassia included rutin and quercetin (64 citations) [2]. According to Prasad et al. [17], Cinnamomum cassia contains quercetin and kaempferol. Additionally, Lepidium sativum was found to contain gallic acid and cinnamic acid (40 citations) [2], while Agarwal and Verma [18] noted the presence of quercetin and kaempferol in it. Because of this, the chemical ingredients are present to promote the effectiveness of using these medicinal plants in Jeddah’s ethnomedical practice. Although Helianthus annuus and Anethum graveolens were only referenced twice each in Jeddah’s traditional medical literature [2], the findings indicated that both species contain antioxidants. Helianthus annuus included rutin, gallic acid, and caffeic acid in their entirety. Additionally, Helianthus annuus extract had other unidentified peaks that could be heliannone, quercetin, kaempferol, luteolin, or apigenin, as found by Guo et al. [19]. Additionally, the outcomes demonstrated that Anethum graveolens contains rutin. Anethum graveolens extract also showed other unidentified peaks that might contain quercetin or isorhamnetin [20]. The significance of Helianthus annuus and Anethum graveolens in medicine can now be emphasized. Helianthus annuus seeds include phenolic compounds, flavonoids, vitamins, and polyunsaturated fatty acids, which have antioxidant, antihypertensive, anti-inflammatory, antibacterial, and cardiovascular effects [19,21]. In ethnomedicine, they are used to cure a variety of illnesses including heart disease, laryngeal and lung infections, bronchial, whooping cough, coughs, and colds [19,22]. Anethum graveolens is said to have pharmacological properties, including antibacterial and antihypercholesterolemic action. It is typically used to treat colic, indigestion, and gas. On the gastrointestinal smooth muscles, it exerts an antispasmodic action [20].

The HPLC and GCMS analysis revealed the chemical composition of these 5 medicinal plants which were used in folk medicine in Jeddah to treat respiratory diseases. The results revealed that all these 5 medicinal plants contained antioxidants. Therefore, it can be concluded that the chemical composition of these therapeutic plants and their ethnomedicinal significance are consistent. From this point on, the many metabolites can be separated to conduct pharmacological testing and develop into a secure medication for a variety of ailments. Additionally, ethnobotanical investigations must be the primary focus of phytochemical screening to finish traditional medicine research and find novel medications.

The authors appreciate the contributions and support of the staff at King Abdulaziz University’s herbarium. Additionally, we would like to thank the anonymous reviewers whose suggestions helped to enhance the first manuscript.

Ethics approval was received from King Abdulaziz University (KAU), Unit of Biomedical Ethics Research Committee, Ethics Committee (Reference No 671-19).

The authors declare no conflict of interests. All authors read and approved final version of the paper.

All authors contributed equally in this paper.

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