Introduction

Mood disorders are the most prevalent and disabling diseases among neuropathic disorders [1, 2]. One of the most important diseases in this category is depression. Currently, 17% of people in the world are suffering from this disease. Depression is increasing worldwide, which makes global society concerned [3, 4]. Annually, 351 million people suffer from depression [2].

It is reported that females are twice as likely as males to be depressed [5, 6]. The most important intervention factors are genetic and environmental [7, 8]. The main feature is feeling depressed for at least two weeks in different situations. Besides, it is usually accompanied by sleep and eating disorders, fatigue, weakness, pain, and lack of self-confidence [9, 10].

Different pharmacological and non-pharmacological methods are used to treat depression. Pharmacological treatment includes tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), and serotonin-norepinephrine reuptake inhibitors (SNRIs) [11, 12]. Various non-pharmacological therapies have been studied to relieve a patient’s depression, including complementary medicine, massage therapy, exercise, acupuncture and acupressure, music therapy, and yoga [13, 14]. Other non-pharmacological interventions, such as transcranial direct current stimulation (TDCS) and electroshock therapy, have also been reported [15, 16].

Along with common medicines, medicinal plants have been widely considered as alternative or complementary remedies for depression. Complementary documents are a valuable resource for discovering new medicines. Traditional Persian Medicine (TPM), consisting of many medical and pharmaceutical manuscripts, has introduced numerous herbal medicines to manage different diseases [17–19]. As mood-elevating supplements, TPM has reported a class, namely Mufarrah (exhilarators). These medicines regulate mood disorders and simultaneously enhance memory and thinking [20, 21].

Reported in Qarabadin-e-Salehi, one of the most famous formulary textbooks in TPM, Mufarrah-e-Bared-e-Saghir (simple cold exhilarator) is an effective multi-ingredient traditional formulation consisting of Coriandrum sativum L. (C. sativum) (2 parts), Rosa × damascena Herrm (R. damascena) (1 part), and Melissa officinalis L. (M. officinalis) (0.6 part) [22].

R. damascena (Rosaceae) is traditionally used for headaches, exhilaration, and improving the heart function, lungs, stomach, and liver [21]. It is effective in the treatment of depression [21, 23], convulsions [23, 24], dementia and nervous stress [23, 25], and insomnia [26, 27]. It also has anti-nociceptive effects [28] and antioxidant activity [29, 30]. M. officinalis (Lamiaceae) is also used for exhilaration, toothache, enhancement of memory, and brain function [21, 30]. It is revealed to be effective in insomnia, reducing stress, and enhancing memory, convulsion, Alzheimer’s, depression, and anxiety [31–34]. This plant has been observed to have considerable antioxidant, anti-inflammatory, and anti-nociceptive effects [35, 36]. C. sativum (Apiaceae) is traditionally used for exhilarating, insomnia, preventing smallpox and jaundice, relieving dental pain, and suppressing cough [21, 37]. Different pharmacological effects have been reported for C. sativum, including effects on insomnia, anticonvulsive activity, enhancing memory, anti-Alzheimer effects, antidepressant effects, and reducing anxiety [38–41]. Besides, it has analgesic, antioxidant, and anti-inflammatory activities [40, 42].

This study aimed to introduce, formulate, and determine the related phytochemicals in the prepared product via chromatographic and spectrophotometric methods.

Materials and methods

Purchased samples of each plant were authenticated by a botanist from the Department of Phytopharmaceuticals, Shiraz School of Pharmacy, with a voucher number (R. damascena: PM1329; M. officinalis: PM1318; C. sativum: PM1248). Polyvinylpyrrolidone was from Sigma-Aldrich (CAS 9003-39-8), magnesium stearate from Merck (415057), ethanol from Taghtir Khorasan Co. (Iran), methanol (Merck: 39302), linalool (Sigma-Aldrich: C39288), gallic acid (Sigma-Aldrich: G7384), quercetin (Sigma-Aldrich, CAS 117-39-5), aluminum chloride (Merck: 206911), Folin-Ciocalteu (Sigma-Aldrich: F9295), and sodium carbonate (Supelco: 1.06329).

Determination of the total phenolic content of extracts

Phenolic and polyphenolic compounds are the main group of natural antioxidants available in plants [44, 45], and they would be measured using the Folin-Ciocalteu reagent and colorimetric method via a UV spectrophotometer [46]. In this method, metal oxides will be reduced by polyphenolic antioxidants such as gallic acid and catechin, and a blue-colored solution will be created [44, 47].

In this test, gallic acid was used as the standard, and total phenolic content was reported based on gallic acid (mg/g). Firstly, gallic acid stock solutions (6.25, 12.5, 25, 50, 100, and 200 mg/mL) were prepared by dissolving them in methanol, and 60 mg of the product was then dissolved in 20 mL of methanol and passed through a filter paper (Table 2). Afterward, 0.5 mL of various concentrations of gallic acid was diluted by 2.5 mL of Folin-Ciocalteu and mixed with 2 mL sodium carbonate (75 g/L) and kept in the dark at room temperature. The absorbance was measured at 765 nm using a UV spectrophotometer (methanol as the blank). This test was performed three times for every concentration of gallic acid stock solutions. Then the graph of absorbance versus concentration was drawn, and the line equation was calculated (Figure 1) [4].

Table 2. Standard phenolic content calibration

Standard	Conc. mg/mL	Abs.
Gallic acid	6.25	0.037
Gallic acid	12.5	0.059
Gallic acid	25	0.134
Gallic acid	50	0.242
Gallic acid	100	0.468
Gallic acid	200	0.948

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Abs.: absorbance; Conc.: concentration

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Figure 1. A gallic acid standard curve. Abs.: absorbance; Conc.: concentration

Determination of the flavonoid content of the prepared tablet

The Dowd method was applied [48], and 2 mL of the extract was mixed with 2 mL of aluminum chloride (2%); it was kept in the dark for 10 minutes at room temperature. The UV absorption range was measured via a UV spectrophotometer (415 nm). Flavonoid content in the dried plant (mg/g) was calculated based on quercetin, and the calibration curve was drawn. Various concentrations of quercetin (3.125, 6.25, 12.5, 25, and 50 mg/mL) were prepared in methanol (Table 3) and used as the standard to draw the calibration curve so that flavonoid content would be measured (Figure 2). The injection volume was 1 microliter in all cases.

Table 3. Standard flavonoid calibration

Standard	Conc. mg/mL	Abs.
Quercetin	3.125	0.091
Quercetin	6.25	0.172
Quercetin	12.5	0.357
Quercetin	25	0.703
Quercetin	50	1.46

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Abs.: absorbance; Conc.: concentration

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Figure 2. Quercetin standard curve. Abs.: absorbance; Conc.: concentration

Results

Analysis of the essential oil compositions in the final formulation

According to the data from GC/MS (Figure 3), EO ingredients were identified, and linalool was considered the main constituent (Table 4).

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Figure 3. GC/MS spectrum of the final product. GC/MS: gas chromatography/mass spectrometry

Table 4. Essential oil compositions. (KI indices were calculated based on the reference, Adams RP [49]. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy: Allured, Carol Stream, IL 60188, USA.)

Number	Component	Retention time (min)	Area (%)	KI (calculated)	KI (reference)
1	γ-Terpinene	7.406	1.21	1,058.309	1,060
2	n-Octanol	7.639	0.58	1,068.062	1,070
3	Linalool	8.525	54.60	1,104.720	1,101
4	Phenethyl alcohol	8.793	2.06	1,115.004	1,114
5	4-Terpineol	10.454	1.01	1,178.741	1,180
6	β-Citronellol	11.725	2.02	1,227.221	1,228
7	Geraniol	12.424	1.14	1,253.759	1,255
8	Thymol	13.409	3.79	1,291.154	1,292
9	Carvacrol	13.677	5.01	1,301.365	1,300
10	Eugenol	15.170	7.11	1,359.571	1,362
11	Geranyl acetate	15.782	2.11	1,383.431	1,386
12	trans-Caryophyllene	16.802	4.22	1,424.128	1,420
13	α-Humulene	17.623	0.72	1,457.421	1,456
14	Curcumene	18.288	0.70	1,484.388	1,483
15	Zingiberene	18.591	1.15	1,496.675	1,495
16	β-Sesquiphellandrene	19.285	0.99	1,526.065	1,525
17	Caryophyllene oxide	20.748	0.59	1,588.373	1,589
18	Dillapiole	21.634	1.13	1,627.378	1,625
19	Turmerone	22.630	0.69	1,671.862	1,672
20	n-Nonadecane	27.381	1.75	1,898.033	1,899
21	Palmitic acid	28.575	0.68	1,959.454	1,960
22	Oleic acid	31.909	0.59	2,137.075	2,141
-	Identification (%)	93.85

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KI: Kovats index

After injecting the EO and the linalool at a similar temperature and comparing the linalool peak in the spectrum generated by the EO, the similarity of both peaks and their locations in both spectra was ensured (Figure 4), and the calibration curve was drawn (Figure 5). Three injections were performed for each standard, and the standard curve was plotted via the mean value of the three injections (Table 5). Considering the point that the density of the obtained EO was estimated as 1, the linalool sample concentration was calculated at 2,379.65 ± 262.13 μg/mL (LOD: 0.06 mg/mL, LOQ: 0.18 mg/mL) in 1 mL injection of the EO (Figure 6).

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Figure 4. Comparison of the appearance of standard linalool and the same marker in the EO. EO: essential oil

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Figure 5. The standard curve of linalool

Table 5. The intra-day and inter-day values

Days	Area (%)			Mean SD (intra-day)	RSD% (intra-day)	RSD% (inter-day)
	A1	A2	A3
1	1,117.2	1,191.1	1,130.8	1,146.37 ± 39.33	3.43	3.20
2	1,129.9	1,180.5	1,198.5	1,169.63 ± 35.57	3.04
3	1,138.5	1,202.2	1,110.7	1,150.5 ± 46.90	4.08

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RSD: relative standard deviation; SD: standard deviation

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Figure 6. Determination of linalool on the calibration curve of standard linalool

Discussion

Considering the high prevalence of depression among people of different ages, its increasing prevalence can be attributed to economic and social reasons, etc. [50, 51]. Accordingly, it is important to provide a variety of medicines to control this complication. This research aimed to introduce a reformulated natural medicine, Mufarrah-e-Bared-e-Saghir, from TPM to improve social and public welfare and introduce a related product.

Similar effects have been reported in modern medicine for the herbal ingredients used in this formulation. Herein, some of them are referred to. In a study, the aqueous extract of C. sativum seed at 100 mg/kg showed an anxiolytic effect in male albino mice compared to the control group in performing the elevated plus-maze (EPM) test as an animal model of anxiety [52, 53].

Another study evaluated the antidepressant and anti-anxiety effects of the ethanol extract of C. sativum seed in Albino mice. Compared with the standard treatment group, the evaluation of the antidepressant activity of 200 mg/kg ethanol extract of C. sativum seed showed a significant reduction in immobility time, while 200 mg/kg of it would result in a significant reduction of locomotion as a criterion for anxiety [54].

Animal studies have shown that M. officinalis at a dose of 25 to 300 mg/kg has antidepressant effects [55]. In another study, it was observed that the ethanol extract of M. officinalis would increase the neurotransmission of norepinephrine, which in turn produced antidepressant effects in the forced swimming test [56]. An animal study on mice showed that an aqueous extract of M. officinalis can significantly reduce immobility and increase climbing behavior similar to that observed with imipramine. Besides, compared to the control group, the EO obtained from the plant can reduce immobility and increase climbing in mice. However, a considerable increase was observed in the forced swimming test at the highest dose (300 mg/kg) [57].

In respect of R. damascena, a study has been performed on rats using a forced swimming test, and the results indicate that oral rose drops with 10, 20, and 40% concentrations significantly reduce depression acutely and in a short period compared to placebo, which was similar to amphetamine. Considering this, the effect may be due to the release of presynaptic amines [58]. In another study, the aqueous extract of R. damascena (15 mg/kg) used in male rats caused a significant increase in swimming time and reduction of immobility duration compared to the control group, and this shows that it has an antidepressant effect [59]. Besides, R. damascena can lead to a reduction in neurogenic stress [60]. In some studies, the effectiveness of linalool (the main substance of this product) has been reported in the treatment of brain diseases and mood disorders, especially depression and anxiety. A study conducted on Wistar male rats using a forced swimming test showed that linalool has considerable antidepressant effects [61–63].

In another study performed on mice using a forced swimming test and antagonist drugs to receptors related to the depression process, such as 5-HT1A, it can be concluded that linalool has antidepressant-like effects by interacting with the monoaminergic system [64].

Linalool has also been shown to have antidepressant-like activity in the tail suspension test in male mice [65]. Besides, some other pharmacological properties have been reported for linalool, such as antiepileptic effect, sedative activity, mood-stabilizing, anti-nociceptive, and anti-inflammatory activity [38, 66], as well as competitive NMDA receptor inhibition, which has resulted in antidepressant and anti-anxiety properties in pre-clinical studies [61, 67].

In this study, Mufarrah-e-Bared-e-Saghir was prepared based on the citations found in TPM texts. Subsequently, phytochemical research and standardization were conducted. Standardization is defined as the optimal technical application of shared knowledge, which includes processes for selection that facilitate appropriate choices for approval, along with consistent decisions to maintain established standards [19].

Subsequently, GC studies were conducted on the EO and extract of the final product, determining the EO components as well as the total phenols and flavonoid content in the extract. Overall, this formulation could be utilized in clinical and industrial settings to address anxiety and depression. The obtained spectra may also serve as a reference for evaluating market samples.