D. kaki has various medicinally bioactive compounds such as carotenoids, tannins, flavonoids, sugars, hydrocarbons, lipids, hydrocarbons, aromatics, terpenoids and steroids. The astringent
D. kaki is a wild species and have an astringent taste until fully ripened, at which stage the tannin content is completely transformed into an insoluble structure. Astringent
D. kaki containing water-soluble tannin components have a bitter taste and the tannic acid has been shown to have anti-aging, anticancer and herbivory defense effects [
13,
20,
25]. We obtained extracts from the stalks of astringent
D. kaki and analyzed the contents of tannic acid according to their level of maturity. The tannic acid contents of three kinds of astringent
D. kaki at different stages of maturity indicated that all the immature stalks had higher tannic acid content than the mature and dried stalks. The results of this study are consistent with those of a study by Jeong [
18,
34], showing that the concentration of tannic acid in
D. kaki decreases as the astringency is removed during the drying process.
D. kaki show a three-stage S-shaped growth curve and their sugar content has been reported to be significantly affected by sunshine, especially after October [
36]. In this study, the useful component content was significantly different depending on the growth stage of the
D. kaki. Del Bubba et al. [
37] found that the contents of soluble tannic acid in cultivated Rojo Brillante and Kaki Topo, increased from July to November but decreased rapidly in December, while the content of sugar increased from September. Additionally, various weather factors including temperature, monthly precipitation, duration of sunshine, and diurnal range, have been observed to influence
D. kaki fruit weight and quality [
17]. Of the nine samples analyzed, the stalks of immature astringent
D. kaki from Hamyang Gojongsi had the highest content of tannic acid (2.597 mg/g). The contents of tannic acid from Myrang Bansi samples decreased considerably from 2.217 mg/g to 0.615 mg/g in the immature and mature stages, respectively. Lee [
38] also reported that the phenol contents of Cheongdo Bansi fruits rapidly decreased during July and August and during September and October. In this study, the tannic acid contents of astringent
D. kaki stalks from the three different regions showed substantial differences in tannic acid content at the mature stage. Previous analysis of the tannic acid contents of astringent
D. kaki from three different regions (Sancheong Gojongsi, Sancheong Danseongsi, and Miryang Bansi) showed that tannic acid content was 30% higher in undried
D. kaki than in dried ones [
39]. The change in tannic acid contents of three cultivars from the Gyeongsangnam-do province in 2015 and 2016 showed similar tendencies. To increase the utilization of astringent
D. kaki, we investigated tannic acid content according to maturity and established it’s important to harvest astringent
D. kaki when they are fully matured; at a time when the tannic acid is at its lowest. However, further analysis needs to be performed across various seasons for comprehensive results. In addition to the effects due to the variation in environmental conditions, the astringency of
D. kaki may also be attributed to its genetic factors [
40].
Diospyros kaki is known to accumulate a large amount of proanthocyanidins in its fruit, resulting in its astringent taste [
41]. The transcriptional regulation of proanthocyanidin biosynthesis pathways may be one of the many factors attributed to the varying level of astringency in the different cultivars of persimmon fruits [
40,
42]. The Myb transcription factor (DkMYB4) and bHLH transcription factor (DkMYC1) which forms a protein complex with each other for proanthocyanidins regulation may have a central role in the differential expression of proanthocyanidins in the different
D. kaki cultivars [
41] and hence the variation in the level of astringency in them. Furthermore, a WD40-repeat protein from persimmon has been observed to interact with the regulators of proanthocyanidin biosynthesis DkMYB2 and DkMYB4 to form a MYB-bHLH-WD40 complexes in persimmon to regulate proanthocyanidin accumulation in fruits [
42].
The stalks of astringent
D. kaki have been used as traditional medicine in Korea (known as Kaki calyx) and are used for the treatment of bed-wetting, vomiting and hiccups. Studies have also shown that
D. kaki has anti-inflammatory effects. Macrophages are critical for the development of inflammatory reactions, as they produce various pre-inflammatory mediators. NO plays an important role in biological defense mechanisms [
4] but excessive production of NO results in the development of inflammatory diseases such as rheumatoid arthritis and autoimmune disorders [
43]. Therefore, to inhibit NO production is a major target for anti-inflammatory treatments. We therefore investigated whether extracts from the stalks of astringent
D. kaki blocked NO production and iNOS protein expression in a LPS-stimulated inflammatory condition. Immature astringent
D. kaki was found to be the most effective and acted dose-dependently (Fig.
4). As shown in Fig.
5, extracts obtained from the stalks of immature astringent
D. kaki decreased the protein expression of iNOS in a dose-dependent manner. In most types of cells NF-κB is an important transcriptional factor related to inflammation. NF-κB is a family of dimeric molecules with pro- and anti-inflammatory properties. In mammals, the NF-κB family comprises five proteins: NF-κB1 (p50/105), NF-κB2 (p52/100), Rel A (p65), Rel B and c-Rel [
34,
44]. Of these, the p65/p50 heterodimer is the most predominant pro-inflammatory complex and p65 nuclear translocation and DNA binding is typically defined as the activation of the NF-κB pathway [
45]. Upon activation by external stimuli, such as LPS, the IκB protein is phosphorylated, degraded, and translocated into the nucleus. Therefore, degradation of IκB makes NF-κB free to translocate to the nucleus, where it regulates gene transcription. NF-κB activation leads to the transcription of pro-inflammatory mediators and cytokines such as iNOS, COX-2 and NO [
46]. We found that stalk extracts suppressed NO production and decreased the expression of iNOS at transcriptional and post-transcriptional levels, as well as reducing p65 nuclear translocation in LPS-stimulated RAW 264.7 cells. These results indicate that stalk extracts inhibit the expression of iNOS through inactivation of NF-κB by reducing p65 phosphorylation. The inhibition of the NF-κB pathway in RAW 264.7 macrophages down-regulated the pro-inflammatory mediators hence exhibiting the anti-inflammatory effects of the stalks of immature astringent
D. kaki. Apart from the stalks, previous study also showed that the fruits of
D. kaki has the potential to ameliorate
inflammatory responses owing to its ability to scavenge free radicals [
15,
34]. Consequently, the anti-inflammatory potential of the stalks of immature astringent
D. kaki can be exploited for use in treatment of disease conditions.