Effects of different harvesting times and processing methods on the quality of cultivated Fritillaria cirrhosa D. Don

Abstract Fritillaria cirrhosa D. Don is the major source plants of traditional Chinese medicine Fritillariae Cirrhosae Bulbus (FCB). Domestication, introduction, and cultivation is an important strategy to alleviate the shortage of endangered medicinal plants of F. cirrhosa. However, until now, the yield and quality changes of FCB in different harvest periods and drying treatments after harvest were not well understood. Therefore, in this paper, we investigated the yield and quality of cultivated F. cirrhosa at different harvest periods and postharvest processing methods. The results showed that dry weight per bulb ranged from 0.8913 to 1.4681 g and reached the highest at the wilting stage. The soluble sugar content ranged from 0.075% to 0.127% and reached the highest at the wilting stage. The content of total alkaloids ranged from 0.088% to 0.218% and reached the highest at the late‐flowering stage. The contents of peimisine, sipeimine, peimine, and peiminine were 0.01178%‐0.02615%, 0%–0.01713%, 0%–0.00745%, and 0%–0.00621% and reached the highest at the late‐flowering period, wilting period, young fruit period, and initial flowering period, respectively. For the two different postharvest processing methods, the contents of total alkaloids and the 16 main characteristic peaks did not exhibit significant differences. Still, the alkaloid contents of the oven drying after washing were slightly higher than the sun drying. In conclusion, the best harvest period is the wilting period of F. cirrhosa, and oven drying after washing is more beneficial to ensure the quality of FCB and improve productivity.


| INTRODUC TI ON
Committee., 2020a). As a critical FCB source, F. cirrhosa has always been regarded as a representative of good quality and high production added value (Wang et al., 2016b).
FCB mainly contains steroidal alkaloids, nucleosides, saponins, terpenoids, and many other compounds (Duan et al., 2011;Wang et al., 2017). Several studies have shown that alkaloids in F. cirrhosa extracts are the main active ingredients, which have the effects of antitussive, expectorant, and antiasthmatic Wang et al., 2016a). In recent years, studies have found that they also appeared to have antitumor, anti-inflammatory, and antihypertensive pharmacological effects, which have further broadened its application scope (Guo, Wu, et al., 2020;Li et al., 2020;Zhao et al., 2018). However, the natural reproductive capacity of the wild F. cirrhosa is low, and the population self-renewal is slow. Coupled with uncontrolled harvesting in recent years, there has been a situation of short supply, causing its price to soar, reaching about 3,100 CNY (= 449 US$) per kg in 2020 (Tiandi Network of Traditional Chinese Medicine., 2020).
Nowadays, artificial cultivation has become an effective way to supplement the wild resources of F. cirrhosa to meet clinical medicine needs (Cunningham et al., 2018). However, until now, we still know little about the ingredient content of the cultivated F. cirrhosa.
Moreover, the cultivation of F. cirrhosa could not only effectively alleviate the resource pressure of wild plants but also could improve the yield of medicinal materials and the content of active substances by changing plant growth conditions and standardizing postharvest processing methods. Among them, the appropriate harvest period is regarded as one of the essential factors to affect the yield and quality of medicinal material during production (Alqahtani et al., 2015). Some studies were employed to investigate the content changes of active ingredients during the growth of F. cirrhosa in the wild or the tending condition (Konchar et al., 2011;Liu et al., 2009). However, there was no report about the ingredient changes in different phenological periods under artificial cultivation conditions until now. Besides, postharvest processing also plays an essential role in the production of herbal medicines and may affect the organoleptic and chemical properties, as well as clinical efficacy and safety (Zhu et al., 2018). It has been reported that there are more than five postharvest processing methods of F. cirrhosa bulbs, among which sun drying and low-temperature drying are the two main methods that are easy to operate and laborsaving in practice . Therefore, investigating the effects of different postprocessing methods and drying times on the change of active ingredients in bulbs could remarkably promote the production of F. cirrhosa and alleviate the shortage of F. cirrhosa supplement.
In this study, we have systematically investigated the content changes of active ingredients in different harvest periods and postharvest processing methods, and analyzed the most suitable harvest period and postharvest processing method. Our study will provide detailed information for quality control of artificial cultivated F. cirrhosa.

| Plant material and growth conditions
The research site was located in the Fritillaria planting base of Qinghai
Ten bulbs were harvested randomly in each growth stage, immediately placed in liquid nitrogen, and then stored at −80°C (Table 1).
The dried samples were milled into powder, sieved through 80 mesh before used for content determination.
Materials in wilting period (H6) were used for postharvest processing studies. Briefly, (1) Sun drying: about 300 g of fresh bulbs was put into a container and dried under sunlight for 15 days. Samples of 40 g (P1-P5) were collected every 3 days and stored at −80℃ for further research.
(2) Oven drying after washing: the bulbs were washed with running water to remove the soil, and the materials were placed in an oven at 60℃ after the water droplets drained away. Samples of 40 g (P6-P9) were collected every 6 hr and stored at −80℃ for further research (Table 3). Samples of 20 g treated by different drying treatments were placed in a grinding miller (IKA, Germany) grounded to powder with liquid nitrogen before being used for moisture and alkaloid analysis.

| Determination of moisture content
The moisture content of the samples was detected by reference to the method of Chinese Pharmacopoeia (State Pharmacopoeia Committee., 2020b). Approximately 2.0 g of sample powder was dried in ovens (Langgan, China) at 105℃ for 5 hr and then weighed.
The drying and weighing were continued until the difference between two consecutive weightings was within 0.005 g, and the measurement was repeated three times for each sample.

| Determination of soluble sugar content
Soluble sugar was extracted and quantified according to the method described by literature (Ma et al., 2009). About 50 mg of sample powder was extracted with 10 ml of 80% ethanol in a water bath (70°C) for 30 min. After cooling to room temperature and centrifuged (Cence, China), the operation was repeated once and the supernatants were combined. The reaction mixture contained 2 ml of diluted supernatants, 1 ml of 2% anthrone reagent, and 10 ml concentrated H 2 SO 4 and was heated in a water bath (100°C) for 1 min.
After the reaction, the absorbance of each sample was measured at a wavelength of 620 nm using an ultraviolet spectrophotometer (Aoe, China). The soluble sugar content was measured using a calibration curve, which was constructed by using the glucose solution.
Data were expressed as gram of glucose equivalents for per gram dry weight (DW).

| Determination of total alkaloid content
The method recorded in Chinese Pharmacopoeia was used to determine the total alkaloids of F. cirrhosa bulbs (State Pharmacopoeia Committee., 2020a). Dried sample powder of 2 g was accurately weighed and placed in a 50 ml conical flask with stopper, then added 2 ml of ammonia solution and standing for 1 hr. Subsequently, 40 ml of chloroform-methanol (4:1, V/V) mixture solution was added, and the extract was refluxed at 80°C in a water bath with heat for 2 hr.
The extract was vacuum suction filtered and evaporated, then dissolved and diluted with methanol to a 50 ml volumetric flask.
Finally, the determination of total alkaloids was based on the reaction between alkaloid and bromocresol green (BCG), and after the reaction was completed, the absorbance was determined at 415 nm (Aoe, China). The standard curve was plotted with sipeimine solution, and the total alkaloid content of each sample was calculated. Data were expressed as gram of sipeimine equivalents for per gram DW.

| Preparation of standard solution
The 5.0 mg of each alkaloid reference substance, peimisine, sipeimine, peimine, and peiminine, was accurately weighed into a 5 ml volumetric flask and made up to the volume with methanol. Different volumes (1, 2, 5, 10, 15, and 20 μl) of mixed standards solution were taken into the HPLC system to measure the peak areas. The linear regression equation of each reference substance was obtained by taking the logarithm of the peak area of mixed standards solution and the injection volume. The mixed solutions were filtered through a 0.45 μm microporous membrane before injection into the HPLC system.

| Preparation of sample solution
The samples were prepared for alkaloid analysis as described by Wang (2013) with minor modifications. An accurately weighed sample of 2.0 g dried powder and 4 ml of ammonia solution were added in a 100 ml round-bottomed flask for 2 hr. Then, the extraction and concentration operations were the same as 2.5. Finally, the extract was diluted to a 2 ml volumetric flask with methanol. The samples were filtered through a 0.45 μm Millipore filter membrane before injection.

| Chromatography conditions
The HPLC-ELSD conditions were used for the determination of the alkaloids, and an LC-20AT Shimadzu high-performance liquid chromatography system (Kyoto, Japan) equipped with an Alltech 2000 evaporative light-scattering detector (Grace,) was used. The chromatographic separations were performed over a ZORBAX Extend-C18 (250 mm × 4.6 mm, 5 µm; Agilent) column at a column temperature of 25℃. The column was eluted with a mixture of acetonitrile (mobile phase A) and water-ammonium bicarbonate (PH = 10.10, mobile TA B L E 1 Changes in individual dry weight, water content, soluble sugar content, and total alkaloid content in bulbs of cultivated F. cirrhosa at different phenological stages

| Statistical analysis
All experiments were performed at least three times, and data were analyzed using IBM SPSS 26.0 (IBM Corp) and Microsoft Excel 2010 (Microsoft Corp). The variance was analyzed by one-way ANOVA, followed by Duncan's test for multiple comparisons. p <.05 was considered significant.

| Biomass
At each harvesting period, ten bulbs were used to determine the fresh weight and dry weight, and the water content was calculated ( As we all know, sugars are largely produced in functional green leaves via photosynthesis, and most harvested organs, including seeds, fruits, tubers, corm, and bulbs, depending on an external supply of sugars for growth and development (Liu et al., 2020).
We speculated that fewer carbohydrates were produced by pho-  bulbs gradually rises to a level close to the germination period, making final preparation for the subsequent long-term overwintering.

| Total alkaloid contents
Total alkaloid is an essential index in the quality control of BFC. In this study, the total alkaloid contents in different harvest periods were analyzed. The results showed that the total alkaloid contents in the bulbs of F. cirrhosa increased rapidly from the germination stage (0.088%), reached the highest level around the late flowering period (0.218%), and then decreased rapidly from the late flowering stage to the young fruit stage (0.125%). However, the total alkaloid contents increased again from the fruit maturity stage (0.135%) to the wilting stage (0.140%). It was evident that total alkaloid contents were significantly higher in flowering periods than in other growth periods. The same results were also found in the study of two Turkish Hypericum species (Cirak et al., 2013), Origanum vulgare L. (Towler & Weathers, 2015), and

| Content of four monomeric alkaloids
Since each medicinal product has its content requirement for different bioactive compounds, the best harvest time might be identified according to the accumulation dynamics of target compounds (He et al., 2010). Isosteroidal alkaloids as target compounds are usually regarded as chemical markers in FCB or related medicinal products for quality control (State Pharmacopoeia Committee., 2020a;Wu et al., 2018). To investigate the dynamic changes of different alkaloid monomers in the various harvest periods, we have detected the contents of them, respectively. The results showed that the changing trend of the four alkaloid monomers was basically the same as the total alkaloid content except for sipeimine, whose peak value was in the wilting period. The other three alkaloid monomer contents had their maximum values around the flowering period of the plants

F I G U R E 1
The HPLC fingerprints of drying treatment. Mixed standards (S1), sun drying (S2-S6), and oven drying (S7-S10); peimisine (1), sipeimine (2), peimine (3), and peiminine (4) ( Table 2). This result was further evidence that the production or translocation of defensive compounds (alkaloids in this study) was not only inducible by stressful situations but also was related to specific plant reproduction stages, such as the beginning of flowering or fruit development.
Based on our data, we suggested that the most appropriate period to harvest the cultivated F. cirrhosa was the wilting period. At this time, the amount of dry matter can reach the maximum, the active substance content can be maintained at a high level, and the seeds can be harvested to expand the production scale.

| Water loss
The changes in sample water loss under different postharvest treatments were investigated (Table 3). For the sun drying method, the results showed that the moisture content could meet the requirements of Chinese Pharmacopeia through 15 days of treatment (12.58% < 15%). By contrast, after 18 hr, the moisture content of oven-dried samples could satisfy the pharmacopeia standard (13.96% < 15%).

| Total alkaloid contents
Due to the fact that certain active components of traditional Chinese medicine will be changed because of the influence of temperature, enzymes, residual moisture contents, or other factors during the drying or other processing processes (Tetteh et al., 2019;Zhu et al., 2018). Shi et al., (2017) reported the active and nutritional ingredients content in harvested medicinal chrysanthemum (Chrysanthemum morifolium Ramat) was not only related to drying methods but also significantly influenced by the specific conditions of each processing method. To investigate whether the total alkaloids of F. cirrhosa were changed under the different postharvest processing, we have compared the contents of total alkaloids in the two processing methods. Our results showed that the total alkaloids did not demonstrate a significant difference (p > .05) in the two postharvest processing methods as well as different processing times (Table 3).
It is widely known that drying will cause fluctuations in the content of active ingredients in plants. A lot of research has shown that secondary metabolites such as phenolics, flavonoids, tannin, terpenoids, and saponin were remarkably influenced by sun drying or oven drying processed (Ademiluyi et al., 2018;Ni et al., 2013).
Meanwhile, the effect of drying on alkaloids also has been proved. Chen et al., (2016)  (peimisine, peimine, and peiminine) content in the oven drying than other drying methods. These findings demonstrate the unique advantages of the oven-drying method for bulb processing of genus Fritillaria. However, in our present study, whether it is sun-dried or oven-dried, or different drying time under the same drying method, it has little effect on the content of total alkaloids. The reason may be that alkaloids being stable compounds were unlikely to be degraded by ambient light and temperature under the relatively mild conditions of the current two processing methods (Hossain et al., 2015).

| HPLC fingerprints of drying treatment
Chinese materia medica (CMM) fingerprint technology is an effective method to evaluate the pros and cons, identify the authenticity, distinguish species, and ensure consistency and stability of CMM (Liu et al., 2018). To illustrate the changes of the main active ingredients in the two different postharvest processing methods, we have imported the HPLC-ELSD chromatogram of the mixed standards (S1), sun drying (S2-S6), and oven drying (S7-S10) samples into the Similarity Evaluation System for Chromatographic Fingerprint of TCM (2004 Editon A) for analysis. The characteristic peaks 1, peak 2, peak 3, and peak 4 were peimisine, sipeimine, peimine, and peiminine, respectively (Figure 1). Other characteristic peaks include D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, and D12, and the contents were calculated based on the peak area of peimisine.
Subsequently, the total content of 16 main characteristic peaks was used to evaluate the differences between the two postharvest treatments. We found no significant difference (p > .05) in the total content of 16 main characteristic peaks between sun drying and oven drying (Table 4). However, some characteristic compounds such as peimine and peiminine were not detected in the experimental group of H6, P1, and P2. Thus, we inferred that some derivatization and transformation reactions might occur between few compounds during the drying process, but most compounds are relatively stable.
In summary, this study compared the two postharvest processing methods on F. cirrhosa bulbs, and the dynamic changes of alkaloids during the drying process were well revealed. Suggested that as a defensive secondary metabolite, the alkaloids in F. cirrhosa were related to the plant's reproductive activities and not be easily affected by different processing methods and processing times. Compared with sun drying, oven drying has the advantages of timesaving, continuous operation, and pleasing appearance and color after processing. Therefore, drying in an oven at 60°C for 24 hr can be used as a postharvest processing method for cultivated F. cirrhosa.

| CON CLUS IONS
In this study, we dynamically monitored the physicochemical characteristics of F. cirrhosa bulbs under different growth stages and different postharvest processing methods. Our results suggested that the best harvest time of the cultivated F. cirrhosa was the withering period, and the different processing methods did not show a significant difference in the contents of the active ingredients. Through our research, necessary experimental data were provided to growers and related enterprises for daily production activities and the full utilization and rational development of F. cirrhosa resources.

ACK N OWLED G M ENTS
This research was supported by the National Key R&D Program of China (No. 2018YFC1706101). We also thank Qinghai Lvkang Biotechnology Development Co., Ltd. for providing us with the test site.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

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.