Nutritional Composition and Mineral Elements Content Analyses of Lonicera fulvotomentosa Hsu et S. C. Cheng Grown in China

Lonicera fulvotomentosa Hsu et S. C. Cheng (L. fulvotomentosa), a vine shrub found in Southwestern China, is used for treating epidemic fever and infectious diseases, such as SARS and Avian Inuenza. Here, we investigated the chemical composition and nutritional content of dried owers of L. fulvotomentosa grown in yellow loam and Karst landform soil in Guizhou, China. The moisture content in all samples varied from 3.25 to 3.63%, lipids from 7.76 to 9.93%, ber from 6.93 to 7.34%, ashes from 12.32 to 12.76%, crude protein from 7.85 to 8.53%, and carbohydrates from 56.21 to 59.77%. Using inductively coupled plasma-mass spectroscopy (ICP-MS), the predominant mineral elements in the dried owers were found to be calcium (297.34-351.26 mg/kg), potassium (132.56-140.37 mg/kg), iron (37.77–41.25 mg/kg), and magnesium (9.47–11.36 mg/kg). Also, HPLC identied avonoids (kaempferol, rutin, quercetin, luteolin, and apigenin) and phenolic acids (caffeic acid, gallic acid, and chlorogenic acid). Thus, the chemical composition of L. fulvotomentosa was similar to that of Lonicera japonica Thunb. (L. japonica). Thus, it could be used as an alternative to L. japonica. Our results showed that the dried ower of L. fulvotomentosa had an extremely high content of chlorogenic acids and caffeic acid, which could be developed as a candidate molecule as HIV inhibitors. on the and the values, 3, 5-di-O-caffeoylquinic acid (3, 5-diCQA) has a higher anity for the bacterial cell than 3, 4-di-O-caffeoylquinic acid (3, 4-diCQA) and 4, 5-di-O-caffeoylquinic acid (4, 5-diCQA). Different positions of the caffeoyl ester groups on the cyclohexane ring at C-3, C-4, and C-5 result in different anti-bacterial for these diCQAs in derivatives,


Introduction
For thousands of years, traditional Chinese medicine has been used for treating diseases. L. japonica (add author name) that belongs to Lonicera is an essential medicinal plant widely used in Chinese medicine. The dried owers of L. japonica are widely used with other traditional Chinese medicines to treat epidemic fever and infectious diseases, such as SARS and Avian In uenza 1 . L. fulvotomentosa is one of the most important variants in the Lonicera family, whose chemical composition and pharmacological effects are similar to L. japonica. It is commonly used as a substitute for L. japonica 2 . The numerous phytochemicals impart medicinal value to these plants. Previous research indicated that the ower buds of Lonicera species contain various compounds, such as phenolic acids, avonoids, triterpenoid saponins, and iridoids 3 . Also, trace elements, such as iron (Fe), zinc (Zn), magnesium (Mg), selenium (Se), etc. are also found in plants, which regulate various metabolic processes and oxidative disorders 4,5 . Based on the quantity-activity relationships and the IC 50 values, HAN Jin et al. 6 illustrate that 3, 5-di-O-caffeoylquinic acid (3, 5-diCQA) has a higher a nity for the bacterial cell than 3, 4-di-O-caffeoylquinic acid (3, 4-diCQA) and 4, 5-di-O-caffeoylquinic acid (4,. Different positions of the caffeoyl ester groups on the cyclohexane ring at C-3, C-4, and C-5 result in different anti-bacterial activities for these diCQAs in L. japonica. Caffeoylquinic acid derivatives, as the major active components of the herb, are well-known as potential antioxidants. Furthermore, they have also been reported to possess signi cant antitumor activity 7 and potential anti-human immunode ciency virus (HIV) activity 8 . Thus, the components impart medicinal value to the plants, and thus, scientists are trying to evaluate the nutritional composition of medicinal plants to apply to the eld of disease prevention and treatment.
The southwest karst region in China is characterized by fragile ecological environments and strong soil erosion 9 . Features that are exclusive to karst include high concentrations of Ca, Mg, and K, the lack of surface water, and a very slow rate of soil formation, which pose several challenges for colonizing plants 10 . All these led to the development of a specialized ora with special growth characteristics and nutritional composition. L. fulvotomentosa can grow in karst landform soil and tolerate high calcium, alkaline, and barren soil conditions. It is an important restoration species in the karst landform environment. Wang et al. 11 determined nine compounds from the acid hydrolysate of the L. fulvotomentosa ower buds. These compounds were identi ed as caffeic acid (CA), ethyl(3β)-3,23-dihydroxyolean-12-en-28-oate, ethyl caffeate, 5,5-0-dibutoxy-2,2-0-bifuran, βsitosterol, nonacosane-10-ol, oleanolic acid, isovanillin, and hederagenin. The inhibitory activity of these compounds against HIV protease was also evaluated, and only ethyl caffeate, CA, and isovanillin exhibited inhibitory effects against human immune de ciency virus (HIV) protease 11 . However, there are no published reports on the detailed composition of the L. fulvotomentosa owers. Additionally, the nutritional composition and mineral elements contents present in owers vary depending on the type of soil, the geographical location of the cultivation area, water, and fertilizers. This study aimed to evaluate the nutritional composition and mineral elements contents in L. fulvotomentosa owers grown in the karst landform environment.

Reagents and Standards.
We obtained HPLC grade methanol (MeOH) and acetonitrile (MeCN) from Thermo Fisher Scienti c. Chlorogenic acid (CGA) and gallic acid (GA), quercetin, apigenin, luteolin, rutin, and kaempferol were purchased from Sigma-Aldrich. All other reagents were of analytical grade. The Milli-Q Plus water puri cation system (Millipore, USA) was used to obtain ultrapure water. The multielement standard mixture solutions contained Ca, P, Fe, K, Mn, Zn, B, Mg, Mo, and Cu. Also, HNO 3 (70% w/v), which was used for digestion and decomposition were purchased from Aladdin Reagent (Shanghai) Co., Ltd.
The owers were cleaned and processed according to the method of Ting et al. (2015) with modi cations 12 . We randomly collected six samples from each geographical location. All materials were air-dried, powdered, and kept in the dark in an acclimatization room for further analysis. We used a dry-ground sample (30 g) and 80% MeOH (300 mL) to prepare the extract by successive maceration and magnetic stirring for 18 h at room temperature. Next, the extract was ltered (Whatman No.1), concentrated, frozen for 24 h, and lyophilized. All steps were repeated thrice, and the supernatants were stored at -40℃.

ICP-MS Analysis.
We used the MARS-Xpress microwave oven for digestion, followed by elemental analysis via ICP-MS using a XSeries 2 reaction cell (Thermo Scienti c). After homogenization, the sample (0.5 g) was mixed with HNO 3 (65% w/v; 5 mL) and heated in a microwave oven at 50% power for 10 min at 150℃, then at 70% power for 20 min at 220℃, and at 10% power for 15 min at 100℃. We transferred the digested extract into an acid-washed volumetric ask (50 mL) containing deionized water and stored.
Each sample batch had a blank water sample. The modi ed Kjeldahl procedure was used to determine the total nitrogen content 17 . 2.6. HPLC-DAD analysis.
Eight phenolic standards and samples (25 g/mL) were ltered (0.45 m). We used an Agilent 1200 HPLC system coupled with a diode array detector (DAD) and a C18 RP analytical column (150 mm × 4.6 mm and a 3.5 m) at 25℃. We used MeOH and water as the mobile phase with an injection volume of 20 µL. An aqueous solution of 0.1% TCA, pH 2.4, served as the buffer. The sample was eluted at 0.6 mL/min via gradient elution: 0-30 min: 20 to 50% methanol; 30-60 min: 50-70% methanol. The absorbance was recorded at 268 and 354 nm. The data were acquired and analyzed using Agilent 1200 Chemstation software and reported as means ± standard deviations of three independent analyses.

Statistical Analysis.
We used the Holm-Sidak test and one-way ANOVA to analyze the data that were represented as means ± SD. A SigmaPlot program v. 11.0 (Windows) was used for analysis. P < 0.05 indicated statistically signi cant difference.

Elemental Analysis.
The soil properties (clay, mineralogy, pH) are known to be closely related to its mineral composition and the bioavailability of the constituent trace elements. Eleven elements were determined to be present in L. fulvotomentosa collected from karst soil and yellow loam. The concentrations of N, P, K, Ca, and Mg were determined as g/kg for dry owers, and that of Mn, Fe, Zn, Cu, B, and Mo, as mg/kg ( Table 3). The results of the elemental analysis showed signi cantly higher contents of Ca, Mg, and Mn in karst soil, while N, P, K, Fe, and Zn were signi cantly higher in yellow loam. The content differences of Cu, B, and Mo were nonsigni cant between yellow loam and karst soil. Data represented as mean ± SD (n = 3). * * P < 0.001; * P < 0.05.

Quanti cation of phenolic acids and avonoids
We performed HPLC with DAD detection to identify and quantify the main phenolic acids and avonoids compounds in the methanolic extracts. We compared the peak areas and retention times among the samples and the standards to quantify the compounds (µg/g dry ower), followed by the construction of the calibration curves of the standard compounds. Figure 1 shows the chromatogram of the standard mixture under optimal conditions, demonstrating e cient puri cation of the peaks of the phenolic acids and avonoids standards. The eight compounds were effectively separated within 70 minutes. The eluting peaks were monitored at 268 nm and 354 nm between 5 and 65 min. These two wavelengths were used as they covered an extensive range of phenolic compounds: phenolic acids/iso avonoids at 268 nm and avonoids at 354 nm. All phenolic compounds showed a high degree of linearity. We could identify all extracts from the ower of L. fulvotomentosa grown in karst soil and yellow loam (Figs. 2-3). The results exhibited a similar pattern of separation with different peak sizes, which indicate the amount/proportion of each compound in the extract. We identi ed eight phenolic compounds: CGA, quercetin, CA, kaempferol, GA, luteolin, rutin, and apigenin.
For all samples, among phenolic acids, CGA and CA had the highest abundance. Among avonoids, rutin was the most abundant avonoid. Table 4 shows the results of the quantitative analysis. The plant cultivated in karst soil contained higher luteolin and kaempferol than those in yellow loam. Besides, the plant cultivated in yellow loam showed larger quantities of rutin and apigenin than in karst soil. The content of CGA and CA are the main indicators to evaluate the quality of honeysuckle; they did not show any signi cant difference.

Discussion
The medicinal plants contain various macronutrients, micronutrients, and unique phytochemicals that might be useful for human health. Our results showed that there were no signi cant changes in the concentrations of ash, moisture, lipid, ber, protein, and carbohydrates between the two different soils. Additionally, the high ash content (> 12%) was indicative of owers being a good source of inorganic minerals. The availability of micronutrients in the soil can strongly affect the production and quality of crops.
Even though essential minerals are generally su ciently abundant in soils, most of them are in forms that are not easily available to plants. We found that Ca, Mg, and Mn were the most abundant mineral elements in the samples from karst soil. In contrast, the Fe and Zn contents were higher in the samples from yellow loam. There is a coordination relationship between the absorption of zinc and iron in plants. The content of N, P, and K in samples from karst soil was lower than those from yellow loam. Gawalko et al. (2001) found that soil factors appear to be the major controlling in uences on the trace elements 18 . The same cultivar may absorb different amounts of minerals in different soils 19 . Recently, it has been documented in a greenhouse experiment that the N nutritional status of the plant is a critical tool for agronomic bioforti cation of wheat with Zn and Fe 20 . Soil organic matter exerts a signi cant and direct effect on the availability of Zn, Fe, and Mn but has little in uence on the availability of soil Cu, so the Cu content of samples from yellow loam was lower than karst soil. Rice Zn, Fe, and Ca concentrations initially increased and then decreased with a continuous increase in the dose of N. Shortage of fertilizer K increased plant Zn and Fe concentrations 21 . The K content in samples from yellow loam was lower than that from karst soil, which was probably related to the lower content of Zn and Fe in samples from yellow loam. Increased knowledge of metal homeostasis is an urgent step needed right now to su ciently understand plant mineral acquisition and storage. This knowledge is expected to e ciently improve crop yield, crop nutritional value, and food safety.
Thus, as previously mentioned, the therapeutic e cacy of L. fulvotomentosa could be attributed to the abundance and diversity of avonoids 11 . Studies have shown the high antioxidant potential of the methanolic extract of the owers of Lonicera species 12 could be due to polyphenols and other antioxidants. CGA and its isomers are esters of quinic and CA can inhibit oxidation and promote a reduction in obesity, liver lipids, as well as inhibition of acute lung injury 22 . Also, L. fulvotomentosa owers contain considerable amounts of rutin, which also performs a broad range of physiological activities 23 .

Conclusion
Thus, we found that L. fulvotomentosa was rich in minerals and phenolic components, including natural antioxidants, such as avonoids and phenolic acids, and thus, might contribute to the therapeutic potential of L. fulvotomentosa. Further clinical and epidemiological studies are required to understand the link between avonoids and suppression of bacterial growth.