DOI: 10.5061/dryad.7h44j0ztx
title: Effects of rocky desertification habitat on main secondary metabolites of Akebia trifoliata

description: In recent years, Akebia trifoliata used to restore rocky desertification
 environment. We first discovered that the medicinal content of A.
 trifoliata will increase in rocky desertification habitats, but its
 mechanism of action is not clear. In this study, A. trifoliata was planted
 in normal habitats and rocky desertification habitats, and changes in the
 content of secondary metabolites and related enzyme activities were
 analyzed. The results showed that: (1) the biomass of the roots, stems and
 leaf of A. trifoliata reduced significantly, but the content of secondary
 metabolites increased significantly in rocky desertification habitats. It
 is mainly reflected in the content of tannins in leaves, flavonoids in
 roots, and total phenols in roots, stems and leaves. (2) A. trifoliata
 changed the enzyme activities of PAL (Phenylalanine ammonialyase), C4H
 (Cinnamate-4-Hydroxylase) and 4CL (4-Coumarate: Coenzyme A Ligase),
 thereby regulated the increase in the content of secondary metabolites in
 rocky desertification habitat. (3) the content of medicinal components of
 A. trifoliata increased significantly in rocky desertification habitat.
 The highest content of oleanolic acid in the roots from July to August,
 and the highest content of α-hederagenins in the stems in July; (4)
 principal component analysis showed that the main response index of A.
 trifoliata secondary metabolites and related enzymes in rocky
 desertification habitats was total phenols. This study revealed the
 response mechanism of A. trifoliata secondary metabolites and related
 enzymes in rocky desertification habitats. It not only provided a new
 choice for the exploiting of medicinal resources of A. trifoliata, but
 also provided a new theoretical basis for A. trifoliata to restore rocky
 desertification environment.
description: <strong>Water
 content</strong>: Take a representative fresh soil sample with soil
 drill in the field, crush about 20g of soil at the required depth in the
 middle of the soil drill, quickly put it into a large aluminum box with
 known accurate quality, cover it tightly, put it into a wooden box or
 other container, take it back to the room, wipe the surface of the
 aluminum box, weigh it immediately and measure the moisture as soon as
 possible. Weigh the large aluminum box containing fresh soil samples on
 the balance to the accuracy of 0.01g. Open the box cover, put it under the
 box and bake in an oven preheated to 105 ± 2 ℃. Take it out, cover it,
 cool it to room temperature in a dryer and weigh it again. So far, the
 quality of water bearing soil and non water bearing soil can be obtained,
 and the difference between the two is the water content in the soil. The
 ratio of moisture content to the mass of dry soil is the moisture content
 of this soil. <br> <strong>Porosity
 and Bulk</strong>: Useing ring knife method. <br> <strong>Soil pH
 value</strong>: Soil pH detector <br> <strong>Total
 nitrogen / phosphorus / potassium:</strong>  Total nitrogen: at room
 temperature, the nitrite nitrogen in the sample is oxidized to nitrate
 nitrogen with potassium permanganate solution before the sample is boiled,
 and then all nitrate nitrogen is reduced to ammonium nitrogen with reduced
 iron powder. Finally, a temperature controlled digester is used to boil
 with concentrated sulfuric acid in the presence of accelerator selenium
 powder copper sulfate potassium sulfate. Various nitrogen-containing
 organic compounds are decomposed at high temperature, All converted to
 ammonium nitrogen. The digestion solution was automatically distilled and
 titrated with Kjeldahl nitrogen meter to calculate the total nitrogen
 content of soil. Total phosphorus: add potassium
 persulfate solution to phosphate standard solutions of different
 concentrations, put it in a 120℃ electric thermostatic drying oven for
 digestion, take it out after 30 minutes, cool to room temperature, then
 add 1.00mL ascorbic acid solution, add 2.00mL molybdate solution mix after
 30 seconds, and leave it at room temperature for 15 minutes. At 700nm
 wavelength, with water as a reference, measure the absorbance and draw a
 standard curve. Then take the mixed experimental soil sample, follow the
 standard curve drawing steps, add the measured absorbance to the standard
 curve regression equation to obtain the phosphorus content (μg).
 Total potassium: Grind the
 experimental soil sample, pass a 100-mesh sieve, dry and weigh, add 5 mL
 of HNO3:HCLO4=4:1, seal with plastic wrap, place it at room temperature,
 and digest for 24 hours. Put it into the electrothermal digestion
 apparatus, and proceed to 80℃ 30min, 100℃ 30min, 110℃ 30min, 120℃ 60min,
 140℃ 60min and 160℃ to dry powder. After cooling, add 5, 10, and 15 mL of
 2.5% HNO3 respectively, seal with cling film, re-dissolve in a 70℃ water
 bath with a vortexer for 30 minutes, and let the soil stand for 12 hours,
 take it out, and pour it into a centrifuge tube for
 determination. <strong>Biomass</strong>: Separate the plant into three parts: root, stem and leaf. Rinse with water to remove impurities adhering to the plant sample, then rinse with deionized water 2 to 3 times and absorb moisture with absorbent paper. The sample was then treated at 105 °C for 30 minutes and then dried at 70 °C to constant weight. <strong>Tanin content</strong>：A 1 g sample of powdered air-dried leaf from M. piperita and C. roseus was placed in a conical flask can combined with 100 mL of distilled water. This was boiled gently for 1 h on an electric hot plate and then filtered through Whatman 42 filter paper (125 mm) into a 100 mL volumetric flask. A 5.0 mL volume of Folin-Denis reagent and 10 mL of saturated Na2CO3 solution were added to 50 mL of distilled water in a 100 mL conical flask, and 10 mL of diluted extract was added for color development. The solution was left to react for 30 min in a water bath at 25 ℃ after thorough agitation. Optical density was read at 700 nm and compared against a standard curve of tannic acid:<br> Tannic acid (mg/100 g) =（C × extract volume × 100）/（Aliquot volume × weight o f sample）<br> where C is the concentration of tannic acid read off the grap <br> <strong>Total Phenolic Content </strong><br> After extraction with ethanol (80%, v/v), the supernatant was reacted with Folin and Ciocalteau’s reagent，and the optical density of the mixture was read at 750 nm. The concentrations were estimated from a standard curve of pyrogallol. <br> <strong>Flavonoid Content </strong><br> Using catechin as a standard. Samples were extracted in methanol and the absorbance was recorded at 510 nm. The flavonoid content was expressed as mg per g fresh weight (FW). <strong>PAL(Phenylalanine ammonialyase) activity</strong>: Using Phenylalanine Ammonia Lyase (PAL) Detection Kit (Phenylalanine Colorimetry) (Leigen Beijing CHINA). Plant samples were mashed in an ice bath and ground into a slurry. The assay mixture included PAL Lysis buffer.<br> Set up a control tube and a measuring tube. The control tube mixture includes 1.5 mL of distilled water and 0.5 mL of the sample to be tested. The mixture in the measuring tube includes 1.0 mL of distilled water, 0.5 mL of the sample to be tested, and 0.5 mL of the PAL Assay buffer. Detect with a spectrophotometer at a wavelength of 290 nm. Definition of PAL activity unit: Under the experimental conditions, the amount of enzyme required for an hourly absorbance change of 0.01 is an activity unit. Tissue sample PAL(U)={(A<sub>1</sub>-A<sub>0</sub>) × Vr)/(W × Vs × 0.01 × t) In the formula: A<sub>1</sub> = the absorbance value of the test tube after 1h incubation A<sub>0</sub> = the absorbance value of the measuring tube measured immediately after adding PAL Asay buffer W=weight of tissue sample (g) Vr=Total volume of extracted enzyme solution (mL) Vs=volume of enzyme solution used in measurement (mL)<br>   <strong>C4H(Cinnamate-4-Hydroxylase) activity</strong>: Using cinnamic acid-4-hydroxylase (C4H) activity detection kit (UV spectrophotometry) (Solarbio Beijing CHINA).<br> Weigh 0.1 g of the plant sample, add 1 mL of extract to homogenize in an ice bath, centrifuge at 12000 g at 4°C for 15 min, take the supernatant, and place on ice for testing.<br> The assay mixture includes reagent one 700μL, reagent two 100μL, reagent three 100μL and sample 100μL. After fully mixing and measuring for 10 seconds, the absorbance at 340nm is A1, then quickly put it in a 37℃ water bath for 3 minutes, and then quickly take it out and wipe it for measurement. The absorbance at 190s, counted as A2, calculate ΔA=A1-A2.<br> Unit definition: The amount of enzyme that reduces lnmolNADPH per minute in the reaction system per g of tissue is defined as 1 unit of enzyme activity. C4H enzyme activity (U/g mass)=[ΔA÷(ε×d) ×109×V<sub>total</sub>]÷(W÷V<sub>extraction</sub>×V<sub>sample</sub>)÷T =535.91×ΔA÷W V<sub>total</sub>: total reaction volume, 2×10-4L; ε: molar extinction coefficient of NADPH, 6.22×103 L/mol/cm; V<sub>sample</sub>: added sample volume, 0.02mL; W: sample mass, g; V<sub>extraction</sub>: extract volume, 1mL; T: reaction time, 3min; 109: unit conversion factor, 1mol=109 nmol <strong>4CL(4-Coumarate: Coenzyme A Ligase)</strong>: Using 4-coumaric acid-CoA ligase (4CL) detection kit (coumaric acid colorimetry) (Leigen Beijing CHINA). The plant samples were mashed in an ice bath, ground into a slurry, and 4CL Lysis buffer was added.<br> Set up a control tube and a measurement tube. The mixture in the control tube includes 4CL control 0.175mL, test sample 0.35mL, and CoA-SH Solution 1.575mL. The mixture in the assay tube includes 0.35mL of the sample to be tested, 0.175mL of 4CL Assay buffer, and 1.575mL of CoA-SH Soulution. Detect with a spectrophotometer at a wavelength of 333nm.<br> Definition of 4CL activity unit: Under the experimental conditions, the amount of enzyme required for every 1 minute change in absorbance is an activity unit. Tissue sample 4CL(U)=((A<sub>1</sub>-A<sub>0</sub>)×V<sub>T</sub>)/(W×V<sub>s</sub>×0.01×t) In the formula: A<sub>1</sub> = the absorbance value of the measuring tube after 5min incubation A<sub>0</sub>=The absorbance value of the measuring tube measured immediately after adding CoA-SH Solution W=weight of tissue sample (g) V<sub>T</sub>=Total volume of extracted enzyme solution (mL) V<sub>s</sub>=Volume of enzyme solution used in measurement (mL)