**1. Introduction**

Ginseng (*Panax ginseng*) has long been used as a traditional medicine for the prevention and treatment of various diseases, including cancer, diabetes, inflammation, allergy, and cardiovascular

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diseases, in the East Asia, particularly in Korea and China [1,2]. Ginseng is one of the best known and most recognized medicinal herbs. Its pharmacological e ffects have been successfully demonstrated by numerous studies worldwide [3]. However, most studies have focused mainly on the root of ginseng, while studies on other parts of ginseng such as its berry and leaf are relatively limited [4].

More than sixty di fferent ginsenosides have been identified from various parts of ginseng [2]. In particular, ginseng berry is known to have a distinct phytochemical profile. It contains significantly higher ginsenoside content than ginseng root [5,6]. Oral bioavailability of ginsenosides is generally very low. It is only 0.64% for Rb1 and 3.29% for Rg1 in rats [7,8]. However, oral absorption of ginsenoside Re is significantly higher (by 1.18–3.95 fold) after oral ingestion of a ginseng berry extract than pure ginsenoside Re [9]. To date, a few in vitro and in vivo studies have reported a variety of biological activities of ginseng berry on cancer, diabetes, sexual dysfunction, skin whitening, immunity, and liver injury. These studies are summarized in Table 1 [3,4,10–16]. A randomized and placebo-controlled clinical trial has also been performed to evaluate the safety and e fficacy of ginseng berry extract on glycemic control [17].


**Table 1.** Chemical constituents and pharmacological activities of ginseng berry extracts reported in previous literature.

A recent study reported the alterations of metabolomes during five di fferent ginseng berry maturation stages and their e ffects on the functional bioactive compounds in ginseng [18]. Thus, information regarding the optimal harvest time of ginseng berry is needed to standardize the collection and pretreatment process of the plant material for its further development as functional foods or medicinal preparations. However, only a few studies have reported the influence of harvest time on the chemical and biological properties of ginseng berry. In a previous study, five di fferent flower and berry development stages (flower bud, flowering, early berry, green berry, and red berry) were tested with respect to ginsenoside biosynthetic gene expression and ginsenoside contents in biochemical and molecular aspects [19]. However, we focused on the e ffect of harvest time on biological activities and chemical profiles of green-to-red ginseng berries, which is more relevant to the agricultural and industrial aspects. Here, the objective of the present study is to determine the e ffects of harvest time on yields, phenolic contents, ginsenoside contents, antioxidant activity, and the elastase inhibitory activity of ethanol extracts of three and four years old ginseng berry.

#### **2. Results and Discussion**

#### *2.1. Drying and Extraction Yields of Ginseng Berry Extracts*

To date, there have been no reports on yields related to the preparation of ginseng berry extracts. As shown in Table 2, after drying the harvested ginseng berry with hot air, the yield of this process ranged from 29.9% to 34.8% (31.8% on average). After extracting the dried ginseng berry with 70% ethanol, the yield of this process ranged from 8.8% to 12.6% (mean: 10.8%). The overall production yield of the extract was calculated to be 3.4%.


**Table 2.** Drying and extraction yields of ginseng berry extracts.

#### *2.2. Antioxidant Properties of Ginseng Berry Extracts*

Antioxidant properties of ginseng berry extracts were assessed by measuring DPPH radical scavenging activity, reducing power, and total phenolic contents. DPPH antioxidant assay is a fast and easy method to evaluate free radical scavenging capacity of a given sample [20]. As shown in Figure 1, DPPH radical scavenging activities of three-year-old ginseng berry extracts tended to increase from 26.8% to 62.5% when the harvest time was delayed. Those of four-year-old ginseng berry extracts also showed similar tendency of increase from 11.0% to 72.7%. Extracts of four-year old ginseng berry harvested in the 3rd and 4th weeks of July exhibited DPPH radical scavenging activity comparable to the positive control (vitamin C), which tended to be higher than other groups (Figure 1). As shown in Figure 2, the reducing power tended to increase as the harvest time was delayed from the 3rd year 1st week (3Y1W) to 4th year 5th week (4Y5W). Extracts of four-year-old ginseng berry harvested in the 3rd week of July exhibited significantly higher reducing power than other groups (Figure 2). As shown in Figure 3, total phenol contents of three-year-old ginseng berry extracts tended to increase from 13.6% to 29.7% as the harvest time was delayed from 1st week to 5th week of July. Those of four-year-old ginseng berry extracts showed a similar tendency, increasing from 3.2% to 13.6%. Extracts of three-year-old ginseng berry harvested in the 4th week of July exhibited significantly higher reducing power than other groups (Figure 2). Although the temporal changes of mean DPPH activity tended to be roughly similar to those of mean total phenols, the harvest time to exhibit the highest DPPH activity (4Y3W and 4Y4W) was different from that for total phenols (3Y4W). This discrepancy could be attributed to other antioxidant phytochemicals besides phenols in the ginseng berry extract, which warrants further investigation.

**Figure 1.** DPPH radical scavenging activity of ginseng berry extracts harvested at various time points. Rectangular bars and their error bars represent means and standard deviations, respectively (*n* = 3). The '*m*Y*n*W' on the x-axis means *m*-year-old ginseng berry harvested in the *n*th week of July. \*, significantly lower than the 'Vitamin C' group (positive control).

**Figure 2.** Reducing power of ginseng berry extracts harvested at various time points. Rectangular bars and their error bars represent means and standard deviations, respectively (*n* = 3). The '*m*Y*n*W' on the x-axis means *m*-year-old ginseng berry harvested in the *n*th week of July. \*, significantly different from other groups.

**Figure 3.** Total phenolic contents of ginseng berry extracts harvested at various time points. Rectangular bars and their error bars represent means and standard deviations, respectively (*n* = 3). The '*m*Y*n*W' on the x-axis means *m*-year-old ginseng berry harvested in the *n*th week of July. \*, significantly different from other groups.

#### *2.3. Elastase Inhibitory Activity of Ginseng Berry Extracts*

Figure 4 shows inhibitory effects of ginseng berry extracts on elastase activity. Elastase inhibitory activities of three-year-old ginseng berry extracts tended to increase from 32.5% to 70.0% as the harvest time was delayed from 1st week to 5th week of July. Those of four-year-old ginseng berry extracts

showed a similar tendency, increasing from 43.2% to 84.6%. Extracts of three-year-old and four-year-old ginseng berry harvested in the 3rd, 4th, and 5th weeks of July exhibited significantly higher inhibitory activities than the phosphoramidon group (as positive control).

**Figure 4.** Elastase inhibitory activities of ginseng berry extracts harvested at various time points. Rectangular bars and their error bars represent means and standard deviations, respectively (*n* = 3). The '*m*Y*n*W' on the x-axis means *m*-year-old ginseng berry harvested in the *n*th week of July. \*, significantly higher than the 'phosphoramidon' group as positive control.

#### *2.4. Contents of Ginsenosides in Ginseng Berry Extracts*

Contents of ginsenosides Rb3, Rc, Rd, Re, and Rg1 in ginseng berry extracts were determined by LC-MS/MS analysis. Typical mass chromatograms are shown in Figure 5. Contents of five ginsenosides in extracts of ginseng berry harvested at various times are shown in Figure 6. As shown in Figure 6B,C, Rc and Rd contents were significantly higher in extracts of four-year-old ginseng berry harvested in the 1st week of July than those in other groups. Similarly, Rb3 and Re contents tended to be the highest in extracts of four-year-old ginseng berry harvested in early July (Figure 6A,D). However, Rg1 content exhibited a slightly different tendency from other ginsenosides. It tended to be the highest in extracts of three-year-old ginseng berry harvested in the 4th week of July and four-year-old ginseng berry harvested in the 2nd week of July (Figure 6E). Contents of all ginsenosides studied were the lowest in extracts of four-year-old ginseng berry harvested in the last week of July.

**Figure 5.** *Cont*.

**Figure 5.** Representative chromatograms of ginsenosides Rb3, Rc, Rd, Re, and Rg1 in calibration standard (**A**), three-year-old ginseng berry extract sample (**B**), and four-year-old ginseng berry extract sample (**C**).

**Figure 6.** Contents of ginsenosides Rb3 (**A**), Rc (**B**), Rd (**C**), Re (**D**), and Rg1 (**E**) in ginseng berry extracts harvested at various time points. Rectangular bars and their error bars represent means and standard deviations, respectively (*n* = 3). The '*m*Y*n*W' on the x-axis means *m*-year-old ginseng berry harvested in the *n*th week of July. \*, significantly different from other groups.

#### *2.5. E*ff*ects of Harvest Time on Chemical Constituents and Biological Activities of Ginseng Berry Extracts*

In both 3- and four-year-old ginseng berry extracts, antioxidant (DPPH radical scavenging activity and reducing power) and anti-elastase activities tended to increase as berries ripened from the first week to the last week of July. However, contents of ginsenosides except Rg1 tended to be higher in four-year-old ginseng berries harvested in early July than those in other groups. These results indicate that biological activities and ginsenoside profiles of ginseng berry extracts depend on their age and harvest time in July, suggesting a need to optimize harvest time for the development of functional foods and medicinal products containing ginseng berry extracts. To the best of our knowledge, this is the first study to report the impact of harvest time on antioxidant and anti-elastase activities as well as ginsenoside contents of ginseng berry extracts.

#### **3. Materials and Methods**
