Improvement of phytochemical and quality characteristics of Dracocephalum kotschyi by drying methods

Abstract This experiment was conducted to evaluate the effects of different drying methods on drying parameters and qualitative characteristics of Dracocephalum kotschyi in a completely randomized design with three replications. Treatments included shade drying as control, sun drying, cabinet drying (CD at 50 and 60°C), refractance window drying (RWD), infrared drying (IRD) at 200 and 300 W, and combination of RWD+ IRD at 200 and 300 W. According to the results, IRD, RWD, and RWD+ IRD effectively maintained valuable secondary metabolites compared to the conventional drying methods. The maximum total phenol content (2.7 and 2.66 mg GAE/g dry weight), total flavonoid content (2.26 and 2.33 mg QE/g dry weight), antioxidant activity (79% and 78.33%), and essential oil content (0.65% and 0.76%) were obtained from plants dried by RWD and IRD. Samples dried by RWD, IRD, and RWD+ IRD had high color quality, acceptable green color, and less browning. Also, RWD and IRD methods effectively reduced microbial contamination of dried plants compared to the control and other methods. The minimum aerobic mesophiles, mold, yeast, and coliforms were observed at 3.11, 0, and 1.47 log CFU/g in IRD 300 W and 3.17, 1, and 1.30 log CFU/g in RWD. D. kotschyi dried at CD 50°C had the maximum microbial contamination. Generally, according to the obtained results, RWD and IRD methods are suggested for drying of D. kotschyi and similar herbs due to shortening the drying time, preserving and improving the quality properties of dried plants.

such as infrared and refractance window drying have been reported that increase drying rates and maintain product quality. Infrared (IR) drying is a combination of radioactive and conductive heating techniques used for heating, drying, and sterilization (Nozad et al., 2016). Once electromagnetic radiation (like infrared) hits the plant or food material, some of the radiation is absorbed and others are either reflected or transmitted. Absorbed radiation by material creates vibration in water molecules, so the material heats up and finally causes moisture evaporation and drying (Delfiya et al., 2021).
Infrared drying (IRD) caused uniform heating, high drying rate, high quality of dried products, more energy efficiency, microbial decontamination, and sterilization. Due to the many advantages of IR drying, it is used for drying medicinal plants and food processing (Sakare et al., 2020). The positive role of IRD on MAPs has been reported by many researchers. Boateng et al. (2021) stated that IRD made ginkgoes more practical for the food industry by reducing their toxic compounds and ginkgolic acid. Moreover, the IR desiccant increased the content of pyridoxine, total phenol content, total flavonoid content and antioxidant activity in dried ginkgo compared to fresh herbs. IRD positively increased the amount of perilla aldehyde, Dgermacrene, and trans-caryophyllene of Dracocephalum kotschyi essential oil and the highest IR wavelength level (0.5 w/cm) leading to the maximum production of these components (Samadi et al., 2018).
In addition to shortening the drying time, IRD increased the EO content and composition of Lippia citriodora samples (Ebadi et al., 2016).
It is reported that IRD reduced the microbial load of peppermint and cumin with no change in color and phytochemical compounds (Eliasson et al., 2014;Erdogdu & Ekiz, 2011).
Refractance window drying (RWD) is an innovative and energyefficient method (Baeghbali et al., 2020) that is appropriate for heat-sensitive products. RWD-dried products had high retention of sensory properties such as aroma, flavor, and color, as well as vitamins and antioxidants than other conventional methods (Baeghbali et al., 2020). Also, as the RWD has significant microbial inactivation capability, the dried product's safety will be increased (Nindo & Tang, 2007). This technology is relatively simple, inexpensive, and easy to use. Owing to the many benefits of this drying technology, it has found many applications in the pharmaceutical, food, and cosmetic industries (Ortiz-Jerez et al., 2015). The positive effect of RWD has been shown in several studies. Drying bananas with RWD at 90°C caused the preservation of TPC, TFC, AOA, and ascorbic acid retention with minimum color change compared to the other conditions studied (Rajoriya et al., 2021). Drying apple slices with RWD and IRD+ RWD methods preserved taste, color, phenol, flavonoids, and antioxidant compounds, as well as increased energy efficiency and shortened drying time (Rajoriya et al., 2020). In other research, RWD increased the picrocrocin, safranal, crocin, and anthocyanin of saffron (Aghaei et al., 2018).
Due to the importance of the drying process in the MAPs industry and the usage of dried D. kotschyi, the present study was conducted for the first time to compare various drying methods (RWD, IRD, and RWD+ IRD in comparison to conventional drying methods) to select the appropriate method with an emphasis on protecting the phytochemical characteristics accompanied by reducing microbial contamination.

| Natural drying
In shade drying (SHD), fresh plants were dried at room temperature (25°C) with natural airflow, and for sun drying (SD), the plants were dried under the sunlight at 36°C. Samples were weighed by digital balance (LutronGM-300p) until they reached the final weight (10% of base moisture).

| Cabinet drying (CD)
In this method, a cabinet dryer (Artaco, Arvin Tabriz, Iran) equipped with a ventilation and air circulation system was used at two temperatures 50 and 60°C.
2.2.3 | Infrared-assisted refractance window drying (RWD+ IRD) and refractance window drying (RWD) To dry plants by these methods, a laboratory-scale infrared-assisted refractance window dryer was used (Department of Food Process Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran-Schematic IRD, Figure 1a). In this dryer, a Mylar film (DuPont) was placed on a hot water source at 90°C, and plant samples were spread on the surface of the Mylar film. The infrared lamp (NIR, Noor Lamp Company) was another main component of the dryer. In the RWD method, the IR lamp of the dryer was turned off, while in the combination method (RWD+ IRD), the IR lamp was turned on. For regulation of IR's power, a variable device (LS-1P-3K-VA, Gold star) was used and RWD+ IRD 200 and 300 W were considered as combined treatments.

| Infrared drying (IRD)
In this experiment, a laboratory-scale infrared dryer with a singlesided radiation system (Department of Food Process Engineering, Gorgan, Iran-Schematic IRD, and Figure 1b) was used. An IR lamp (NIR, Noor Lamp Company) was placed above and at 15 cm from the sample tray. About 30 min before the drying process, the IR lamp was turned on with the required power so that the temperature conditions inside the chamber and the lamp temperature reach a stable state. The IR lamp's power was adjusted by a variable (LS-1P-3K-VA, Gold star) and samples were dried at two powers (200 and 300 W).

| Moisture content
The fresh samples (50 g) were dried in a conventional oven (INB 400, Memmert) at 105°C for 24 h. Moisture content (based on fresh weight) was obtained using Equation (1): M w : moisture weight based on fresh weight (%), W w : fresh weight (g), and W d : dry weight (g).

| Drying time
Drying time in each method was recorded based on the time (min) to reach the final weight (10% of base moisture).

| Drying rate
The drying rate was obtained by measuring the amount of weight loss during drying and reported as grams per hour.

| Total phenol content
TPC was measured by the Folin-Ciocalteu method. First, 70 μL of the methanolic extract was mixed with 130 μL distilled water and 1 mL of 10% Folin, and kept at room temperature for 5-8 min, and then, 800 μL of sodium carbonate (7.5%) was added. The mixture was placed in a water bath at 40°C for 1.5 h in darkness Schematic diagram of IR-assisted refractance window dryer (RWD + IRD) and refractance window dryer (RWD); (b) Schematic diagram of infrared dryer (IRD). and immediately read by spectrophotometer (T80+ UV/VIS Spectrophotometer, PG Instrument Ltd) at 760 nm. TPC was expressed in mg gallic acid equivalent per gram dry matter (mg GAE/g DM; Singleton et al., 1999).

| Total flavonoid content
TFC was estimated by the aluminum chloride method (Du et al., 2009)

| Free radical scavenging activity
Free radicals scavenging activity (AOA) was calculated using the free radical DPPH method. Fifty microliter of the plant extract and 950 μL of DPPH 0.1 mM were placed in darkness for 15 min. Then, the sample was read at 517 nm (Chiou et al., 2007). The antioxidant capacity was calculated as the percentage of DPPH inhibition using Equation (2):

| Glandular trichome analysis by scanning electron microscopy (SEM)
The influence of drying methods on dried plants' microstructure was assessed by scanning electron microscopy. The samples were mounted on double-sided carbon tape and coated with a thin layer of gold and scanned with an SEM (XL 30, Philips) at 25 kV.

| EO content
To prepare EO, 20 g of the powdered plant was hydrodistillated by a Clevenger apparatus for 3 h. EO percentage was calculated by To measure the color parameters, 6 g of dried and powdered plants were poured evenly on a Petri dish and photographed by the camera (HS 20 EXR, Fujifilm) and analyzed by Color Average software to obtain the color parameters L* (lightness or brightness, 100: white and 0: black), a* (red/green value, positive is red and negative is green), and b* (blue/yellow value, positive is yellow and negative is blue).

| Chroma index
This parameter indicated the degree of saturation or color intensity and was calculated by the following equation (4): a: a* and b: b*.

| Hue angel index
It indicated the dominant color which was obtained by Equation (5): a: a* and b: b*.

| Browning index (BI)
It showed the color changes of a plant to brown (Equations 6 and 7): L: L*, a: a*, and b: b*.

| Total color difference (ΔE index)
The ΔE index indicated the color changes during drying. The higher value indicated a greater difference with the control. ΔE index was obtained by Equation (8): L 0 , a 0 , and b 0 : color indices before drying, L: L*, a: a*, and b: b*.

| Microbial load
The microbial contamination was determined by dilution method and culture in specific media. First, 10 g of the plants were sterilized with 90 mL of buffer peptone solution in sterilized conditions. Then, Free radical scavenging percentage = A control −A sample ∕ A control × 100.
a series diluted up to 10 −3 was prepared.

| Drying rate
The highest drying rate (9.01 g/h) observed in IRD 300 W had significant difference with control (0.11 g/h). IRD 200 W showed the highest drying rate. RWD and RWD+ IRD also effectively increased the drying rate compared to the control and other conventional methods ( Figure 2b).
The preservation of the qualitative characteristics of MAPs in the post-harvest stage is one of the main concerns of MAPs production and processing industries (Ebadi et al., 2015). Drying is used to reduce the moisture content of foodstuffs to reduce the microbial spoilage speed and chemical changes, increasing product shelf-life and reducing weight and required space (Sui et al., 2014). The time required to dry plant samples depends on the material's volume and moisture content as well as drying temperature. As shown, the most prolonged time and the lowest drying rate were observed in the control. Similarly, Mokhtarikhah et al. (2020) reported that the longest drying time (28 h) was recorded in shade-dried mint. Drying the Clinacanthus nutans leaves at higher IR power increased the drying rate and reduced drying time (Abdullah et al., 2022). In Artemisia absinthium L., drying duration decreased by increasing IR power (Beigi, 2018). Also, in Stevia Rebaudiana (Bakhshipour et al., 2020) and Mentha spicata L.

| Total phenol content (TPC)
According to  probably related to the ability of IR radiation to break covalent bonds and release polyphenols (Lee et al., 2003). In the current study, a high amount of TPC was observed in samples dried by RWD+ IRD 300 W. Similarly, the TPC of apples dried by RWD+ IRD 50 and 60°C was increased compared to the hot air dryer (Rajoriya et al., 2020).
According to reports, the increase in phenolic compounds in drying conditions may be due to several reasons, and prolonging drying time and high temperature might destroy the cellular structure and further release some bound phenolic compounds (Zhou et al., 2016).
Also, some phenolic compounds were produced due to the structural transformation of polyphenols during high drying temperatures (Lopez et al., 2010). On the other hand, the formation of phenolic compounds could be due to the availability of phenolic precursors through non-enzymatic interconversion (Que et al., 2008;Zhou et al., 2016).

| Total flavonoid content (TFC)
The maximum TFC (2.33 mg catechin/g dry weight) was obtained from the plants dried by IRD 300 W that had no significant difference with RWD (2.26 mg catechin/g dry weight). Also, RWD+ IRD The TFC of B. subtilis-fermented polished adlay dried by IRD was 1.60 times higher than that of freeze-vacuum drying samples (Wen et al., 2020). Also, drying D. kotschyi by RWD and RWD+ IRD increased the TFC. Talukdar and Uppaluri (2021) reported that the turmeric dried by RWD at 95°C had the maximum TFC. Since the high temperature of the RWD reduced the drying time, the samples were exposed to heat for a shorter time, and consequently, retained
Antioxidants inactivate the free radicals and protect the cells from destructive effects (Nora et al., 2014). Many researchers stated that high AOA in fruits and vegetables after drying might be due to the more antioxidant activity of oxidized polyphenols than non-oxidized polyphenols (Nora et al., 2014;Önal et al., 2019). IRD had significant antioxidant activities compared to hot-air dryers (Rajoriya et al., 2020). In saffron stigma, the maximum antioxidant compounds such as crocin, picrocrocin, and anthocyanin were observed in RWD at 70 and 80°C (Aghaei et al., 2018). IRD is an effective technique to increase the products' oxidative capacity. The B.
subtilis-fermented polished adlay dried by hot air and infrared had AOA significantly higher than under vacuum, freeze-vacuum, and microwave-vacuum drying methods (Wen et al., 2020). Similarly, our findings showed that IRD 300 W increased the plant's AOA.

| Glandular trichomes
Trichomes are specialized structures with many morphologies and functions that initiate from young epidermal cells and cover aerial tissues (Feng et al., 2021). The deformation of trichomes in some methods was obvious.
Rupturing of the secretory structure in samples dried by CD 50°C can be due to long-term exposure to the dryer temperature. The minimum changes and damages in the secretory trichomes were observed in SHD. Our findings about the inappropriate effects of drying methods on glandular trichomes were in accordance with some studies. In spearmint, some methods such as vacuum, microwave, and oven drying with temperatures less than 50°C caused trichomes to break or wrinkle (Mokhtarikhah et al., 2020).
In lemon verbena, glandular trichomes were damaged by increasing temperature and time prolongation in the oven and vacuum dryer, respectively (Ebadi et al., 2015). An evident collapse and shrinkage in the chrysanthemum structure dried by IR-HAD was observed which could be due to the different vapor pressure between the outside and inside of samples during drying (Xu et al., 2022). An et al. (2016) reported that the microstructures of ginger IR dried were well preserved and less damaged due to the less heating and shorter duration of IR radiation, which is in accordance with our observations on glandular trichomes of the IR-dried plants compared to CD.

| EO content
Drying methods had a significant effect on the EO content of D.
kotschyi (Table 1). As shown in Figure 5, the maximum EO content (0.76%) was obtained from IRD 300 W followed by IRD 200 W and RWD (0.67% and 0.65%, respectively). The minimum EO content was observed in the SD and CD 50°C method.
EOs are a group of secondary metabolites that are influenced by post-harvest processing. EOs' quantity and quality depend on the drying duration, drying method, and plant species (Yazdani et al., 2006 kotschyi which was consistent with another study on saffron. Aghaei et al. (2018) reported that the safranal content (as saffron aroma factor) was increased in the RWD method at 70 and 80°C. The quality of food products obtained by RWD is better than the other dryers.
Quick and gentle drying in RWD minimizes heat-induced degradation and oxidation and maximizes product fragrance and flavor (Bernaert et al., 2019). An increase in the EO content in some drying methods (e.g., IRD and RWD in the current study) may be due to the gradual stress in plants (Mokhtarikhah et al., 2020). Respiration of plants continues until thoroughly dry, so they try to preserve their intracellular balance during drying through the activation of internal mechanisms. Therefore, the plant may prevent water loss by producing active ingredients such as EO (Castelló et al., 2006;Lewicki et al., 2001;Mokhtarikhah et al., 2020). Generally, the increase or decrease in EO content during drying process may be related to differences in plant species, glandular trichomes structure, and EO chemical components (HamrouniSellami et al. (2011)).

| Color
As shown in Table 2, the drying methods significantly affected the color parameters (p < 0.01). The highest and lowest L* index (56.52 and 46.65) was obtained from RWD and CD 50°C, respectively.
The IRD and RWD+ IRD methods were also at a high level of the L* index and showed a significant difference compared to the control Color is one of the most important appearance attributes and a significant quality index to assess thermal damage that directly affects consumer acceptability and marketability of dried products (Aral & Beşe, 2016;Maskan, 2001). Thus, the color indices were used to evaluate the changes in the color characteristics of the dried products. Alteration of the L*, a*, and b* index occurs due to pigment decomposition during drying, which can increase the value of ΔE and BI and ultimately reduce the product quality (Maskan, 2001). Drying conditions affected color changes and quality. The methods with high drying rates could preserve color by inhibiting the destruction of the pigments. In blackberry, the best color quality was obtained by microwave at higher temperatures, which means the fast-drying process leads to better color preservation (Kaveh et al., 2020). Inshade drying led to rapid degradation of photosynthetic pigments due to the longer drying process, which was consistent with results on savory and thyme (Rahimmalek & Goli, 2013). According to the results on dried bananas, the highest values of L*, b*, chroma, and ΔE along with the lowest values of a*, hue, and browning index were F I G U R E 5 Essential oil content (EO) of Dracocephalum kotschyi by different drying methods. CD, cabinet dryer; IRD, infrared dryer; RWD + IRD, combination method of refractance window and infrared dryers; RWD, refractance window dryer; SD, sun drying; SHD, shade drying.

EO (%)
Drying methods observed in the RWD method. L*, a*, and b* indices increased with increasing RWD temperature (Rajoriya et al., 2021). The L*, a*, and b* indices of cherries, strawberries, and cranberries dried by RWD, and freeze-drying have been maintained and improved which is related to less decomposition of pigments during drying owing to mild drying conditions compared to hot air (Nemzer et al., 2018). kotschyi dried and they had high color purity, acceptable green color, and less browning.

| Microbial load
Based on Table 2, the effect of different drying methods on the microbial load of D. kotschyi was significant (p < .01). The maximum aerobic mesophiles, mold and yeast, and coliforms were observed at 3.65, 1.9, and 2.17 log CFU/g in the plants dried by CD 50°C, respectively. The minimum number of aerobic mesophiles was observed in IRD 300 W (3.11 log CFU/g) and RWD (3.17 log CFU/g) which showed less microbial load than the control. The combined method of RWD+ IRD was effective in reducing the plant's microbial load. No mold and yeast grew in the plants dried by IRD 300 W.
Subsequently, the plants dried at IRD 200 W and SD had the minimum amount of mold and yeast contamination (1 log CFU/g). Slight contamination was observed in the plants dried by RWD and control (1.30 log CFU/g). According to Figure 7, the minimum coliforms (1.30 log CFU/g and 1.47 log CFU/g) were recorded in plants dried by RWD and IRD 300 W, respectively. Generally, RWD and IRD were more effective in reducing the microbial contamination of D. kotschyi than the control and other methods (Figures 7 and 8). due to the presence of contaminants such as soil, insects, and water (Eliasson et al., 2014). Pathogenic microorganisms such as Salmonella, Escherichia coli, Bacillus cereus, Clostridium perfringens, and toxigenic molds and yeasts have been detected in MAPs that could cause disease (Banerjee & Sarkar, 2003;Sagoo et al., 2009). Consequently, the decontamination of MAPs for pathogenic microorganisms' removal and prevention of food spoilage and food-borne diseases is very important (Eliasson et al., 2014;Erdogdu & Ekiz, 2011). In addition, microbial contamination in plant products has limited their usage in the pharmaceutical, food, cosmetic, and health industries.
Therefore, to use MAPs as food or herbal medicines, they should be adequately monitored to minimize the side effects of allergenic reactions and contaminants and to provide safe, efficient, and standard products to the consumer (Mimica-Dukic et al., 2004).
Based on the results, RWD, IRD, and RWD+ IRD were more effective in reducing microbial contamination than other treatments.
Among the electromagnetic radiations used for the combined drying method, IR is chosen due to its fast and uniform heating characteristics (Zeng et al., 2019). IR heating is a thermal technology and therefore it is presumed to inactivate microorganisms by heat.

(f)
However, as the IR spectrum lies between microwaves and UV light, an overlapping effect involving induction heating and damage to DNA has also been reported to be responsible for microbial inactivation (Krishnamurthy, 2006 (Eliasson et al., 2014). According to Erdogdu and Ekiz (2011), IR disinfected and reduced aerobic mesophilic bacteria and eliminated mold and yeast, while no change was observed in the amount of EO and pigments of the cumin seeds. RWD technique, due to heat transfer by both radiation and conduction, in addition to shortening the drying time, is effective on the final quality of dried plants in terms of color, nutritional value, and microbial load (Nindo & Tang, 2007;Tontul & Topuz, 2017). RWD had a strong inactivation effect against aerobic mesophyll, coliforms, Escherichia coli, and Listeria nomoa of pumpkin purees that can be due to damage to the cell membrane and DNA of pathogenic microorganisms during the drying period (Nindo et al., 2003). In the RWD method, the low thickness and high transparency of the Mylar film allow IR rays to pass, so that more radiation is transferred to the product, hence it is effective in reducing the microbial load (Nindo & Tang, 2007). Here, the CD 50°C method did not successfully reduce the microbial load.
Similarly, Mirmostafaee et al. (2014) reported that the most bacterial contamination was observed in the oven at 45°C. Since the oven had a closed environment with limited ventilation, the water vapor increased at the first hours of drying and by condensation of water, favorable conditions for the growth and reproduction of microorganisms were provided. On the other hand, the oven temperature (45°C) was not efficient to prevent bacteria growth.
Cluster analysis was applied to determine the relationship between the different drying methods based on all studied traits. A dendrogram of cluster analysis separated the drying methods into three major groups that were distinguished both in quality and quantity ( Figure 9). Group 1, included the IRD (200, 300 W), RWD, and combination method (RWD+ IRD 200 and 300 W). Conventional drying methods such as CD 50, 60°C, and SD were placed in group 2. The SHD method was placed in a separate group (group 3). According to previous studies, the classification of different drying methods was affected by various factors. In Lippia thymoides and Stachys lavandulifolia, differences in EO components led to the grouping (Hazrati et al., 2021;Nascimento et al., 2021) while in Thymus daenesis, drying temperatures were the main factor of the grouping (Rahimmalek & Goli, 2013).

| CON CLUS ION
IRD, RWD, and IRD+ RWD significantly reduced the drying time compared to all treatments. In these methods, the D. kotschyi plants were dried 16 to 80 times faster than the control. Among the drying methods, IRD, RWD, and IRD+ RWD had a positive effect on maintaining qualitative characteristics. The maximum total phenol content (2.7 and 2.66 mg GAE/g dry weight), total flavonoid content (2.26 and 2.33 mg QE/g dry weight), antioxidant activity (79% and 78.33%), and EO content (0.65% and 0.76%) were reported in RWD and IRD methods, respectively. Evaluation of color indices revealed that plants dried by RWD, IRD, and RWD+ IRD were better than other treatments. In these methods, dried plants had high color purity and quality, acceptable green color, and less browning. The microbial load was also measured as an important indicator in determining the product's health for the consumers. Our results revealed that RWD and IRD were more effective in reducing microbial contamination than the control. IRD 300 W completely inactivated the mold and yeast growth. Also, the combined method of RWD+ IRD had a positive effect in reducing the plant's microbial contamination. Plants dried by CD at 50°C had the maximum microbial load. As shown in obtained results and dendrogram, there was a distinction between modern and conventional drying methods. Generally, to produce dried D. kotschyi with less time, more active substances content, high and acceptable color quality, and less microbial contamination F I G U R E 9 Dendrogram obtained by hierarchical cluster analysis of all traits in Dracocephalum kotschyi dried by different methods. CD, cabinet dryer; RWD + IRD, combination method of refractance window and infrared dryers; IRD, infrared dryer; RWD, refractance window dryer; SD, sun drying; SHD, shade drying.
is preferred to use modern drying methods such as RWD, IRD, and combination RWD+ IRD instead of conventional methods (SHD, SD, and CD). writing -review and editing (equal).

ACK N OWLED G M ENTS
The authors thank the University of Guilan and Gorgan University of Agricultural Sciences and Natural Resources for financial support and laboratory facilities.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
Our research did not contain any animal experiments or human subjects.