Study of the avonoids of Anemone obtusiloba in the alpine meadow and the adaptation mechanism to the environment based on metabolic pathways

In this study, the main pigment composition of Anemone obtusiloba was analysed by UV-vis spectrophotometry, HPLC, and LC-MS. The results showed that there were two kinds of pigmentsin A. obtusiloba, carotenoids, and avonoids The absorbance spectra regularly changed with the depth of ower colour. Based on HPLC and LC-MS analyses, seven kinds of avonoids were inferred, including luteolin-3-7-O-glucoside, quercetin-3-O-rutoside, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-sophoroside, and myricetin-3-O-rhamnose, and the derivative of quercetin was identied as the main component in A. obtusiloba. Besides, according to the analysis of the avonoid metabolism pathway, it could be determined that avonoid 3'-hydroxylase (F3′H) was the key enzyme to increase the content of quercetin. This paper speculates that A. obtusiloba increases the content of quercetin by regulating avonoid F3′H, so that it can deepen the ower colour to adapt to the environment.


Introduction
Anemone obtusiloba, commonly known as Padar, Rattanjog, or Kawashud, is a owering plant of the family Ranunculaceae and a densely tuftedperennial herb [1].It is native to the Himalayan and mountainous regions of Myanmaroccurring in the Alpine Himalaya from Kashmir to Sikkim at 2100-4200 m altitude and in the Nilgirihills at an altitude above 1800 m [2]. It bears buttercup hermaphrodite ower and the stem is short and tufted with a single terminal ower, generally producing one to three individual stems with yellow, light-yellow, or white colour. At present, research mainly focuses on the medicinal value of A. obtusiloba, whose ethanolic extract contains saponins (obtusilobinin, obtusilobin [3], and obtusilobicinin [4]). It also contains protoanemonin, which is an irritating acrid oil and is an enzymatic breakdown product of the glycoside ranunculin [5]. A. obtusiloba is used as a purgative and in the treatment of rheumatic joints, jaundice, spleen disorders, and anxiety neurosis. Besides, it is also used as an antidote to snakebite, while its seed oil is used to cure arthritis [6,7]. However, there are few studies on its petal pigment.
In the Tibetan Plateau, A. obtusiloba has its own adaptive mechanism to the local environment. During the past years, there have been signi cant environmental changes in the plateau area, with an obvious decrease of white owers and an increase of yellow and light-yellow owers of A. obtusiloba. It is speculated that this phenomenon may be related to its adaptability to the environment and biological evolution. Flower colour is one of the most important traits of plants and is attributed to various pigments that are composed of three major classes of compounds including avonoids, carotenoids, and anthocyanin [8]. Among these compounds, avonoids are responsible for the yellow colour. At the same time, the number and position of phenolic groups in the chemical structure have a certain in uence on the ower colour. The colour of the ower will change from yellow to orange and to red by the hydroxylation at C 3 [9].However, increasing the hydroxylation of the B ring will change the colour to blue, and the methylation of the B ring will make it red [10]. Carotenoids are responsible for colours ranging from yellow to red [11]. Cyanidin, which controls red, blue, purple, and other colours, can be divided into three types: pelargonidin, delphinidin, and anthocyanins [12].
[28]. There are six genes involved in anthocyanin synthesis in different tissues of grape, CHS, CHI, F3′H, DFR, leucoanthocyanidin reductase (LAR), and avonoids glycosyltransferase (UFGT), while the accumulation of anthocyanidin is absent in white grape. Therefore, the accumulation of anthocyanidin is not only related to tissue, but also related to the alternative gene expression. The study aimed to qualitatively and quantitatively identify the chemical composition using different analytical methods including ultraviolet-visible (UV-vis) spectroscopy, HPLC, and LC-MS, and to further analyse the key enzyme synthesizing this pigment.

Materials
The experimental materials light-yellow and yellow A. obtusiloba were collected in early July at the peak of owering from Maqu County, Gansu Province of China (101.52E, 33.40N). The owers were rinsed, and the stamens were removed. After xating at 80°C for 30 min, they were dried at 60°C and crushed into powder with a pulveriser (Tianjin Taisite Instrument Co. Ltd., Tianjin, China). Then the powder sample was stored in a sealed bag and kept in a dark place before using.

Spectral analysis of avonoids
The avonoid extraction was identi ed according to the method of Murugesan et al. [29] with some modi cations. To extract avonoids, 0.1 g of ower powder was treated with methanol/formic acid (98:2, v/v) for 24 h in a Soxhlet apparatus under dark conditions. The lter residue was repeatedly extracted by the solvent. After the ltrate was combined and diluted to 10 mL, the spectral scanning was carried out by a UV spectrophotometer (UV-3000, Shanghai JiaPeng technology Co., Ltd., Shanghai, China) with a range of 200-700 nm.

Spectral analysis of carotenoids
The carotenoids of A. obtusiloba were extracted based on the method reported by Cai Xuan et al. [30] with necessary modi cations. Brie y, each sample of ower powder (0.1 g) was mixed with acetone/ethanol (1:1, v/v), and the mixture was repeatedly extracted 24 h in a Soxhlet apparatus under dark conditions. After the ltrate was combined and diluted to 10 mL, the spectral scanning was carried out with a range of 200-700 nm.

Qualitative and quantitative analysis of petal pigments
Flavonoids in petals of A. obtusiloba were extracted with methanol/formic acid (98:2, v/v) in darkness at 4°C for 24 h. Then the mixture was centrifuged at 12,000 rpm for 5 min at 4°C to remove the precipitants.
The supernatant was collected and ltered through a 0.45 µm micropore membrane, and the as-prepared sample was stored for qualitative and quantitative analysis. The experiment was repeated three times for each sample [31]. µL of the sample was injected for HPLC analysis. The chromatogram showed horizontal coordinate and vertical coordinate, corresponding to the retention time (min) and response value (mAU), respectively. The ow rate was 0.2 mL/min, and the column temperature was 30°C. Since avonoids are expected to be observed at a 300 nm wavelength by UV detector, the spectra were scanned in the range of 190-700 nm.
The MS conditions were set as follows: High purity nitrogen (99.999%) was used as a nebulizing (60 psi) and drying gas at a ow rate of 9.0 L/min. The vaporizer temperature was set at350°C. Other parameters were rationally set including an Ionspray voltage of 70-205 Vand a scanning range of 100-1000 m/z.

Statistical analysis
The data were processed and presented as mean ± standard deviation (SD). Experiments were performed in triplicates and the statistical analysis was executed by one-way analysis of variance (ANOVA). Signi cant differences between groups were discerned at p≤0.05. Statistic software GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, USA) was used for all the graphical and statistical evaluations.

Result
3.1 Spectral analysisusing UV-vis spectrometer 3.1.1Spectral analysis of avonoids As shown in the UV-vis spectrum of the avonoid fraction of A. obtusiloba ( Figure 1A), the maximum absorption peaks were observed at 268 nm (band II) and 330 nm (band I), respectively. There are two typical absorbance bands of avonoids, band B (310-350 nm) for avones and band A (250-290 nm) for avonols [32,33,34]. Generally speaking, band II absorption could be caused by ring A-cyclobenzoic acid system, while band I was caused by ring B-cyclocinnamic acid system [35,36]. As shown in Figure  1A, the absorption peak at 268 nm indicated the presence of the A-cyclobenzoyl system, while the absorption peak at 300 nm suggested that there was a B-cyclocinnamoyl system in the avonoid fraction of the ower extract. Based on the observation that band II was the main peak while band I was weak, we speculated that the cinnamoyl system might be destroyed [37]. This result showed that the extract from A. obtusiloba had the basic structure of avonoids C6-C3-C6.

Spectral analysis of carotenoids
According to the UV-vis spectrum of the carotenoid sample from A. obtusiloba ( Figure 1B), the maximum absorption peak was at 268 nm, which was consistent with the characteristic absorption peak of the carotenoid. With the deeper colour, the absorption peak of the carotenoid sample gradually increased. The total content changed regularly with the shading of the colour ( Figure 1B). The maximum light absorption value of the pale-yellow sample was 18.36% less than that of the yellow sample (4.112 and 3.357 of the optical density (OD),respectively).

Qualitative analysis of petal pigments from Maqu
Based on the HPLC-PDA analysis of the avonoid extract of yellow owers of A. obtusiloba (Figure 2), 7 kinds of compounds were detected at 300 nm with a retention time ranging from 30 to 50 min.
Meanwhile, there were 8 kinds of compounds detected in light-yellow of avonoid fraction of A. obtusiloba. Zhaoet al. [38] reported that the different colours of owers were not determined by the structure of the compound, but by the change in the content of some pigment molecules. Studies have also found that the red colour of cotton is mainly caused by avonoid accumulation. Except for the avonoid-related enzymegenes in red cotton such as CHS and F3'H, other genes that regulate avonoid biosynthesis have higher transcription levels than those in white cotton [39]. In the HPLC chromatogram (Figure 2A), peaks 3 and 8 were close to each other, suggesting that they might be isomers. Therefore, the avonoid components of the two coloured owers are supposed to be the same. Mostly, the peak intensity (response value) of yellow owers (B)was higher than that of the light yellow(B) ones. Therefore, it could be suggested that the colour intensity of A. obtusiloba was caused by the content of pigment molecules. The components (Figure 2A) at peaks 3 (1.775 mg/g), 6 (1.56 mg/g), and 7 (2.687 mg/g) were more dominant in light-yellow owers, whereas peaks 3 (4.124 mg/g), 4 (2.754 mg/g), and 7 (2.651mg/g) were more dominant in yellow owers ( Figure 2B).

Quantitative analysis of petal pigments from Maqu
To further determine the speci c type of pigment molecules, HPLC-MS analysis was carried out on yellow owers with high total contents. The MS peaks of four standards (Quercetin, Kaempherol, Luteolin, and Rutin) were shown in Figure 3. According to the total ion chromatogram and positive ion mode of avonoid from the yellow ower ( Figure 4 and Figure 5), the analysis results in Table1 were obtained.
The molecular ion peak was observed at m/z 645.18 [M+H]+ of peak 7. The fragment at m/z 465 represented the loss of one glucose molecule. Then one glucose molecule was removed to obtain a fragment m/z 287, which was luteolin aglycone. It was speculated to be luteolin-3-7-O-glucoside since it was consistent with the result reported by Waage S.K et al. [40]. the glycosyl ion at m/z 146, the data were found to be consistent with the result of myricetin-3-Orhamnose [47]. The content of the avonoids was measured ( Table 2).The content of quercetin-3-Ogalactoside (4.124 mg/g) was found to be relatively higher than the other components. As a result, quercetin derivatives were dominant in avonoids of A. obtusiloba owers. The result is the same as the Hezuo sample plot [49]. Table 2 The content of main components of anthocyanin in Maqu Anemone obtusiloba (mg/g)

The key enzyme analysis in the regulation of anthocyanin in A. obtusiloba
Although the anthocyanins of A. obtusiloba are mainly composed of four sugar derivatives of luteolin, kaempferol, myricetin and quercetin, In these major avonoids, quercetin with the highest relative abundance may be one of the main active ingredients. According to the metabolic pathway of avonoids ( Figure 6) [48], quercetin could only be synthesized from dihydroquercetin and catalysed by avonol synthetase (FLS). There were two pathways of dihydroquercetin synthesis: one was eriodictyol catalysed by avanone-3-hydroxylase (F3H), and the other was dihydrokaempferol catalysed by avonoid-3'hydroxylase (F3'H). However, eriodictyol could be also catalysed by avone synthase II (FNSII) to form luteolin. In the study, the derivatives of luteolin (2.754 mg/g) were observed. Therefore, luteolin pathway was not prohibited, and the activity of FNSII might be reduced or the gene expression of FNSII was down regulated to reduce the decomposition of luteolin. F3'H catalysed the synthesis of dihydrokaempferol and dihydroquercetin from senkyolin and naringenin, respectively. According to the reactions in Figure 6, F3'H showed ahigher a nity with naringenin. So it was speculated that the a nity of FLS with dihydrophlorin was enhanced in the pathway of the synthesis of luteolin, which competed with dihydrokaempferol and dihydromyricetin to increase the content of quercetin. According to the content of avonoids in A. obtusiloba, it was speculated that the dihydrokaempferol pathway improved the translation of F3'Hto increase the content of dihydroquercetin. And the a nity between FLS and dihydroquercetin was enhanced, so quercetin content could be eventually increased.
Therefore, enhancing the translation of F3'H gene could not only increase the content of dihydroquercetin, but also increase the content of eriodictyol, which could indirectly increase the content of dihydroquercetin and nally achieve the purpose of increasing the content of quercetin.

Discussion
In recent years, with changes in the environment in the alpine region, the pigment of the unique plant A. obtusiloba has also been changed. In the Hezuo plot with lower altitudes, the pigment of A. obtusiloba has undergone signi cant changes to adapt to the environment, the pigment of the A. obtusiloba is obviously darkened. Compared with low altitude Hezuo plot, the higher the altitude, the deeper the color, In the Maqu plot with higher altitudes, and even no white owers are observed (Figure 7). The main anthocyanins in the two places are the same, and the change trend of main pigments was consistent with the change of ower color (Table3) [49].  Table 3 The content of main components of anthocyanin in Hezuo Anemone obtusiloba (mg/g) [49] Peak The key enzyme to adapt to the environment was discovered to be F3'H. The main environmental factor causing pigment change may be light intensity. This paper speculates that A. obtusiloba increases the content of quercetin by regulating the expression or activity of F3'H to deepen the ower colour so that it could adapt to the environment. In this study, the evolution of A. obtusiloba response to the environment was analysed. This conjecture is also applicable to the adaptation to altitude changes. It provides a theoretical basis for the study of the evolution mechanism of plateau plants responding to the environment and provides a reference for the relationship between plateau environmental change and vegetation.

Declarations
Ethical approval: This article does not contain any studies with human participants performed by any of the authors.
Ethical approval: This article does not contain any studies with animals performed by any of the authors.
Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Data availability/Availability of Data and Materials Statements
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Collect Anemone obtusiloba Premissions Statement
School of life sciences, Lanzhou University of Technology and College of grassland agricultural science and technology, Lanzhou University had given a permission to collect Anemone obtusiloba on the sample plot.
Collection of Anemone obtusiloba comply with relevant institutional, national, and international guidelines and legislation.
All these voucher specimens are deposited in the public collection providing access and Author Lv wanling and Professer Liu zuojun identi ed it.  The metabolic pathway of avonoid