Seasonal variations of triterpene acid contents in Viscum album L. on typical host trees of Hyrcanian forests

Viscum album L. (mistletoe) is a semiparasitic plant of the Santalaceae family. A valuable group of bioactive compounds in mistletoe are triterpene acids (TTAs), which possess anti-inflammatory and anticancer properties. Parrotia persica and Carpinus betulus are the most common hosts of mistletoe in the Hyrcanian forests of Iran. This study was performed to compare the content of oleanolic acid (OA), betulinic acid (BA), and ursolic acid (UA) in the mistletoe foliage (stems and leaves) from P. persica and C. betulus in various seasons for the first time. The results showed that OA was the prevailing TTA in all samples, while UA was found in none of them. The maximum amount of OA (12.38 mg/g dry weight [DW]) and BA (1.68 mg/g DW) was detected in V. album from P. persica in summer. The minimum amount of OA (5.58 mg/g DW) and BA (0.72 mg/g DW) was observed in that growing on C. betulus in winter. However, the mistletoe from C. betulus showed the greatest level of OA in spring (9.06 mg/g DW) and BA in summer and autumn (0.92 and 0.97 mg/g DW, respectively). The data collected in this study complement existing research on this subject from around the world.

According to various studies, the active ingredients of mistletoe and its therapeutic effects can significantly change relating to the host species, harvest season, and growing conditions such as temperature and climate 14,[18][19][20][21][22][23] .
Regarding the effect of harvest time, V. album plants from various deciduous host trees harvested at different seasons in Poland indicated the highest concentrations of TTAs in summer. Also, OA was the most abundant TTA in their extracts 18 . Likewise, Turker et al. 23 reported that water and ethanol extracts of V. album plants inhabiting different host trees revealed various antitumor activities according to the host species. In their study, the water extract of V. album from cherry (Prunus divaricate) and wild pear (Pyrus elaeagnifolia), as well as the ethanol extract of mistletoe samples on pine (Pinus sylvestris) showed the highest antitumor effect, respectively 23 .
To the best of our literature review, there is no report on the TTAs content of Iranian mistletoe under the effects of host species and harvesting season. Because the content of bioactive compounds in mistletoe can vary depending on the host species and harvesting time, this study was conducted for the first time to evaluate the variations of TTAs content in Iranian V. album from P. persica and C. betulus in the Hyrcanian forests of Iran during different seasons.

Results
As mentioned, to study the effect of two host species, P. persica and C. betulus, on the OA, BA, and UA content of mistletoe, the samples were harvested in summer (early September), autumn (early December), winter (early March), and spring (early June). The extract analysis indicated that OA and BA existed in all samples, but no UA was found.
Effect of host species, P. persica and C. betulus, on the OA and BA content of mistletoe. According to Fig. 1, the mistletoe OA content was significantly affected by host species in summer and winter, so that the mistletoe growing on P. persica indicated the maximum concentration of OA in both seasons, summer (12.38 mg/g DW) and winter (7.48 mg/g DW) (Fig. 1B,D). It is noticeable that the level of OA in mistletoe from C. betulus was somewhat higher than that in mistletoe from P. persica only in spring, when they were 9.06 mg/g DW and 7.71 mg/g DW, respectively. www.nature.com/scientificreports/ Betulinic acid was significantly affected by host species in all seasons except autumn. Mistletoe growing on P. persica produced the highest content of BA in three seasons, including spring, summer, and winter, with concentrations of 1.00 mg/g DW, 1.68 mg/g DW, and 1.02 mg/g DW, respectively (Fig. 1A,B,D).
The seasonal variations of OA and BA content in mistletoes from different host species. The analysis of variance (ANOVA) indicated that the type of host species, season, and their interaction had a significant effect on the OA and BA content of mistletoe at the 1% probability level ( Table 1).
As shown in Fig. 2, OA was the prevailing TTA in mistletoe samples from both host species in all seasons. The quantity of OA ranged from 5.58 to 12.38 mg/g DW, which was approximately 7.5-fold higher than the amount of BA, which varied from 0.72 to 1.68 mg/g DW.
The maximum concentration of OA (12.38 mg/g DW) and BA (1.68 mg/g DW) was found in the mistletoe from P. persica species in summer, while the lowest content of OA (5.58 mg/g DW) and BA (0.72 mg/g DW) was detected in the mistletoe growing on C. betulus in winter (Fig. 2).
In the mistletoe from P. persica, there was no significant difference in OA quantities between spring, autumn, and winter samples, although the lowest amount of that was detected in winter (7.48 mg/g DW). Also, the minimum concentration of BA in these samples was observed in spring and winter (1.00 and 1.02 mg/g DW, respectively) (Fig. 2).
The TTAs analysis of the mistletoe growing on C. betulus during four seasons showed that there was no significant difference in the OA values in the spring, summer, and autumn samples; nevertheless, OA concentration was at the highest level in spring (9.06 mg/g DW) (Fig. 2). As well, the highest content of BA in the mistletoe from C. betulus was detected in summer and autumn (0.92 and 0.97 mg/g DW, respectively) ( Fig. 2).
Overall, as shown in Table 2, the mistletoe growing on P. persica had the maximum content of total OA (36.09 mg/g DW), total BA (4.94 mg/g DW), and therefore total OA + BA (41.02 mg/g DW) of four seasons. However, the highest ratio of OA to BA (9.34) was detected in that growing on C. betulus, which was 1.3-fold higher than in the mistletoe on P. persica. (Table 2).
Moreover, the total average concentration of each TTA, extracted from mistletoes on both host species, varied in different seasons as follows: 8.38 (spring), 10.47 (summer), 8.36 (autumn), and 6.53 (winter) mg/g DW for OA, as well as 0.88 (spring), 1.3 (summer), 1.1 (autumn), and 0.87 (winter) mg/g DW for BA. These fluctuations of OA and BA levels revealed that TTAs accumulation gradually increased from winter to spring, peaked in summer, then decreased to autumn, and subsequently reached a minimum concentration in winter. Table 1. Analysis of variance of OA and BA contents in the mistletoes growing on P. persica and C. betulus in different seasons. **Significant difference at 1% probability level.

Discussion
Triterpene acids, OA, BA, and UA, are a group of important bioactive compounds in V. album L., which are famous due to their apoptosis induction in cancer cell lines and cytotoxic anti-cancer property 10,13 . Viscum album L. is an evergreen semi-parasitic plant, and its phytochemical components can be affected by the host species and its vegetative period 13,18 . As described before, in the current study, the effect of P. persica and C. betulus host species was assessed on the quality and quantity of Iranian V. album TTAs at different harvest times. The findings of this investigation revealed that the quality and type of TTAs in mistletoe samples from both host species during different seasons were identical. All samples included OA and BA, while OA was the main TTA, but UA was not discovered in any of them. This trend of TTAs composition was in agreement with the Wójciak-Kosior et al. 18 report, which, to the best of our knowledge, is the only study about the variations of TTAs content in European mistletoes growing on several host species in different seasons. However, research on other bioactive compounds of V. album also showed that host species and harvest time had no effect on the type of viscotoxin compounds in V. album subsp. album 24 and components of flavonoid glycosides in V. album var. coloratum 25 , but the quantity of these compounds varied depending on the host species. Likewise, the present results indicated a significant variation in the content of TTAs based on the type of host species and also seasonal alterations. Regarding the effect of host species, our results indicated the mistletoe from P. persica contained higher levels of OA and BA in almost all seasons, with the only exception of spring, when its OA content was lower than that in the mistletoe from C. betulus. Furthermore, the total average concentration of TTAs varied from the lowest level in winter to a higher level in spring, reached a maximum point in summer, and gradually decreased until autumn.
Therefore, the highest concentration of OA and BA was found in the mistletoe from P. persica in summer, while the minimum content of both compounds was observed in samples from C. betulus in winter. Similarly, Wójciak-Kosior et al. 18 confirmed the host-dependent OA and BA accumulation in V. album growing on deciduous Angiospermae host species, with the utmost concentration in summer. Yet, regarding V. album growing on Pinus sylvestris (Gymnospermae), they found the highest concentration of OA and BA in winter.
Mistletoe is a semi-parasitic plant that absorbs water and nutrients from host species. As a consequence, variations in mistletoe bioactive compounds under the effect of host species are expected 5 and have already been confirmed for other phytochemicals of V. album, such as phenolic compounds 26 , viscotoxins, and lectins 5 , as well as some small molecules as important biomarkers 6 . Also, different seasons can influence the mistletoe biochemical compounds through environmental factors or particular vegetation periods 20,27 . In this regard, Barbasz et al. 27 reported significant changes in concentrations of some other mistletoe compounds like sugars, lipids, and polyamines in different periods of the year. Likewise, Vicaş et al. 20 found variations in total phenolic content and antioxidant activities of V. album depending on the harvest times.
Several studies have shown that medicinal plants contain the highest content of secondary metabolites at the full flowering stage [28][29][30][31] . Mistletoe flowering time is during the winter months, from February to March, until April 32 , which coincides with dormancy and the absence of leaves in deciduous host species. Whereas in the present study and the Wójciak-Kosior et al. 18 experiment, the highest level of TTAs in V. album from deciduous host trees was found in the summer. According to this finding, it seems that mistletoe TTA biosynthesis is more dependent on the growth periods of the host species.
Triterpenes are one of the largest and most varied plant bioactive compounds, derived from a linear 30-carbon precursor, 2,3-oxidosqualene 33 . The production of pharmaceutical terpenoids in medicinal plants can be improved by regulating the primary metabolism. Carbohydrates are the main metabolites of primary metabolism, and their synthesis is affected by the photosynthesis rate 34 . Thus, the production of terpenoids in plants is directly linked to photosynthesis and, consequently, the biosynthesis of related precursor metabolites 35 . Zhang et al. 35 demonstrated that the increase in the photosynthetic capacity of Glechoma longituba leaves under the influence of blue and green lights enhanced TTAs accumulation in this plant. Also, other studies indicated the crucial role of carbohydrates as precursor metabolites in the biosynthesis of plant terpenoid compounds. In this regard, feeding Euphorbia lathyris seedlings with various precursor compounds showed that sucrose and, to a lower extent, glucose are the main metabolites in the latex triterpene synthesis of the seedlings and produced the highest amount of triterpenes per seedling per day 36 . Likewise, the addition of higher concentrations of sucrose in cell suspension cultures of Uncaria tomentosa 37 and Salvia fruticosa 38 led to an increase in TTAs contents. This positive effect of sucrose on TTA accumulation could be attributed to the chemical structure of these secondary metabolites 38 .
Mistletoe, in addition to water and mineral salts, receives 22.6 to 45% of its carbon from the host species 39 . The transfer of carbohydrate from the host to the mistletoe considerably depends on the carbohydrate content of the host xylem sap 40 . Escher et al. 40 have shown that the seasonal variations of carbohydrate concentration and composition in the xylem sap of V. album are similar to their patterns in the respective host tree xylem sap. In Table 2. Comparing the total value of OA, BA, OA + BA, and their ratio (OA/BA) in V. album from P. persica and C. betulus during four seasons. Each value is a mean ± SE (n = 3). Different letters denote significant differences at *P ≤ 0.05, **P ≤ 0.01, and ns non-significant using Duncan's test.

Host species
Total OA ns (mg/g DW) Total BA** (mg/g DW) Total OA + BA* (mg/g DW) OA/BA ratio** www.nature.com/scientificreports/ their experiment, the carbohydrate content of V. album on a deciduous host tree, Populus × euamericana, changed from the highest concentration in spring to a low concentration in summer and autumn 40 . Moreover, mistletoe is a partially heterotrophic plant that appears to be able to meet some of its carbon demand through a high transpiration rate. However, the transpiration rate of mistletoe on deciduous trees reduces during the leafless period of the host tree 32 .
In the present study, the concentration of OA and BA in mistletoe from P. persica gradually increased in spring and reached a significant peak in summer. However, in samples from C. betulus, a significant increment of OA content was detected in spring, summer, and autumn, respectively, whereas the highest level of BA was observed in summer and autumn. It seems that the enhancement of mistletoe TTAs content is synchronous with the beginning of active growth of the host tree in spring, when the concentration of carbohydrates increases in the host and subsequently in the mistletoe. Carbohydrate accumulation as a precursor primary metabolite for TTAs biosynthesis may cause an increase in mistletoe TTAs content in spring and summer. In correspondence with our assumption, Lázaro-González et al. 41 demonstrated that Viscum album subsp. austriacum obtains metabolites mostly through the primary metabolism of the host tree and produces its defensive chemicals. Besides, in agreement with our findings, they reported a higher concentration of a few amino acids, carbohydrates, and defense compounds of V. album subsp. austriacum in the summer 41 .
From another point of view, the increase of secondary metabolite production in plants is a response to stress conditions such as high temperatures 42 . As a result, the increase in temperature and daylight length in spring and especially summer can stimulate mistletoe defense responses, leading to a rise in TTAs levels during these seasons. This hypothesis is in line with Alqahtani et al. 43 findings on triterpene accumulation in Centella asiatica, which increased in warm climates with long daylight hours in summer.
Taken as a whole, it seems that the improved primary metabolite content of mistletoe, along with environmental stresses during spring and summer, caused the highest TTAs content in the summer mistletoe samples, but further experiments are required to provide more accurate explanations for this subject. The altitude gradient of the study area is 700-1100 m a.s.l. The region has a semi-humid climate, with a mean annual temperature of 17 °C and a mean annual precipitation of 668 mm. The host plants were selected in such a way as to have homogeneous ecological conditions as well as the same diameter (at breast height, DBH), height, and morphology without other stresses such as pests, diseases, etc. For sampling, five branches of the last three generations of V. album foliage (stems and leaves) were harvested from each mistletoe on three individuals of each host species during four seasons. At the harvest times, the mistletoe was in full flowering (winter), beginning of fruiting (spring), green berry (summer), and ripe white berry (autumn) phenological stages (Fig. 3). Mistletoe samples, after being placed in a liquid nitrogen tank for a few seconds, were stored at − 80 °C until extraction. All methods were carried out under the relevant guidelines in the method section.  www.nature.com/scientificreports/ isocratic mode consisted of methanol-water-phosphoric acid (87:12.95:0.05, v/v/v) at a 1 ml/min flow rate. For each injection, 20 µL of the extract was used. The amount of TTAs, including OA, BA, and UA, was determined based on the calibration curves of the standard compounds in 50, 100, 150, and 250 μg/mL concentrations (Sigma Co.).

Statistical analysis.
First, the normality of data distribution was checked and confirmed using the Kolmogorov-Smirnov test (P > 0.05). Levene's test was also used to evaluate the assumption of data variance homogeneity. The data were analyzed by one-way and two-way ANOVA to determine the significant levels. The mean values were compared using Duncan's test at the 5% probability level. All statistical analyses were performed by SPSS 23.0 software.

Conclusion
The interaction between host species and parasites is very complex, and the role of host species in this interaction requires considerable research effort. This study was the first evaluation of the effect of host species and season on the TTAs content of V. album in the Hyrcanian forests of Iran. Iranian V. album from P. persica and C. betulus contained two kinds of TTAs, including OA and BA in all seasons, and OA was the dominant TTA. Ursolic acid was not found in any samples. Mistletoe from P. persica demonstrated the maximum concentration of OA and BA in summer, while mistletoe growing on C. betulus contained the highest amount of OA in spring and the highest content of BA in summer and autumn. Overall, to achieve the highest content of TTAs, harvesting the mistletoe growing on P. persica in summer was emphasized by the findings of this research.