Study on Optimization of Ethanol Reflux Extraction of Phenolic Acids from Salvia miltiorrhiza

The extraction technology of phenolic acid compounds from Salvia miltiorrhiza by ethanol reflux was studied. In this experiment, salvianolic acid B standard was used to make the standard curve. Single factor experiment and orthogonal experiment were used to study the extraction of different ethanol concentrations, reflux times and material-to-liquid ratios. The OD value of salvianolic acid compounds was measured with a spectrophotometer. The extraction rate of phenolic acid compounds under different extraction conditions was calculated through a regression equation, so as to obtain the optimal conditions for the ethanol reflux extraction process of Salvia miltiorrhiza. The experimental data can provide a reference for the ethanol reflux extraction process of salvianolic acids in the industry. According to the experiment, the extraction rate of phenolic acids in Salvia miltiorrhiza was the highest when the ethanol concentration was 60%, the reflux time was 1.5 hours, and the ratio of material-to-liquid was 1:10.

A change of phenolic acids content in poplar leaves induced by methyl sali-cylate and methyl jasmonate

The contents of seven different phenolic acids such as gallic acid, catechinic acid, pyrocatechol, caffeic acid, coumaric acid, ferulic acid and benzoic acid in the poplar leaves （Populus Simonii×Populus Pyramibalis c.v and Populus deltoids） suffocated by Methyl jasmonate （MeJA） and Methyl salicylate （MeSA） were monitored for analyzing their functions in interplant communications by using high-pressure liquid chromatography （HPLC）.The results showed that the contents of phenolic acids had obviously difference in leaves exposed to either MeSA or MeJA.When P.deltoides leaves exposed to MeJA or MeSA, the level of gallic acid, coumaric acid, caffeic acid, ferulic acid and benzoic acid was increased, gallic acid in leaves treated with MeJA comes to a peak at 24 h while to a peak at 12-d having leaves treated with MeSA.When P.Simonii ×P.Pyramibalis c.v leaves were exposed to MeJA or MeSA, the level of gallic acid, pyrocatechol and ferulic acid was increased; The catechinic acid and benzoic acid had a little drop; The caffeic acid and coumaric acid were undetected in both suffocated and control leaves.This changed pattern indicated that MeJA and MeSA can act as airborne signals to induce defense response of plants.

Effects of Phenolic Acids on Growth and Activities of Membrane Protective Enzymes of Cucumber Seedlings

Two phenolic acids P-hydroxy benzoic acid and cinnamic acid were designated as four concentrations (0, 50 μmol/L, 100 μmol/L, 150 μmol/L) to investigate the effects of phenoic acids on the growth and the activities of membrane protective enzymes of cucumber seedlings. The results showed that both phenolic acids inhibited the seedlings growth. The inhibitory effects were increased with the concentration of phenolic acids increasing and the time of treatment prolonging. Seedlings treated with A150 (P-hydroxy benzoic acid, 150 μmol/L), B50 (cinnamic acid, 50 μmol/L), B100 (cinnamic acid, 100 μmol/L), B150 (cinnamic acid, 150 μmol/L) showed significantly shorter in plant height , smaller in leaf area. and lighter in fresh weight. The inhibitory effect of cinnamic acid was comparatively stronger than that of P-hydroxy benzoic acid. For protective enzymes system, compared to control , the POD activity increased at all concentrations of P-hydroxy benzoic acid during the treatment but increased at first then decreased before increased again at last at all concentrations of cinnamic acid .In the case of CAT, its activity increased at first, then decreased, and increased again at lower concentrations of phenolic acids. However, at higher concentrations the activities decreased at first, then increased a little, decreased continuously at last. In addition, the treatments of phenolic acids led to an increase then a decreaseof SOD activity and an increase of MDA content in the seedlings. All above indicated the accumulating of free radicalsand destruction of protective enzymes at higher concentrations of phenolic acids.

Application of HPLC and ESI-MS techniques in the analysis of phenolic acids and flavonoids from green leafy vegetables (GLVs)

Diets containing high proportions of fruits and vegetables reduce the risk of onset of chronic diseases. The role of herbal medicines in improving human health is gaining popularity over the years, which also increases the need for safety and efficiency of these products. Green leafy vegetables （GLVs） are the richest source of phenolic compounds with excellent antioxidant properties. Increased consumption of diets containing phenolic compounds may give positive and better results to human health and significantly improves the immune system. Highly selective, susceptible and versatile analytical techniques are necessary for extraction, identifica- tion, and quantification of phenolic compounds from plant extracts, which helps to utilize their important biological properties. Recent advances in the pre-treatment procedures, separation techniques and spectro- metry methods are used for qualitative and quantitative analysis of phenolic compounds. The online coupling of liquid chromatography with mass spectrometry （LC-MS） has become a useful tool in the metabolic profiling of plant samples. In this review, the separation and identification of phenolic acids and flavonoids from GLVs by LC-MS have been discussed along with the general extraction procedures and other sources of mass spectrometer used. The review is devoted to the understanding of the structural configuration, nature and accumulation pattern of phenolic acids and flavonoids in plants and to highlighting the recent developments in the chemical investigation of these compounds by chromatographic and spectroscopic techniques. It concludes with the advantages of the combination of these two methods and prospects.

Temperature and Acidified Solvent Effect on Total Anthocyanins and RP-HPLC Phenolic Acids Determination in Selected Spices

Total anthocyanins of spices (Syzygium aromaticum L., Coriadrum sativum L., Cuminum cyminum L., Zingiber officinale Rosc., Elettaria cardamomum, Curcuma longa, Rhus coriaria L., Cinnamomum zeylanicum Blume, Foeniculum vulgare Mill and Laurus nobilis L.) were determined using acidified (1% HCl) solvents (methanol, ethanol and acetone) at three temperatures (20℃, 40℃ and 60℃). Also phenolic acids were separated and identified by RP-HPLC. Results showed that sumac and cinnamon had the highest levels of anthocyanins, while for the acetone the cinnamon indicated the highest amount of anthocyanins when methanol and ethanol were used as extracting solvents at 20℃. At 40℃ using ethanol, sumac showed the highest level of anthocyanins whereas acetone solvent yielded the highest anthocyanin contents for cinnamon. At 60℃, cinnamon showed the highest level of anthocyanins when methanol and acetone were the solvents, while sumac had the highest anthocyanins level using ethanol as solvent. HPLC results showed ten phenolic acids found in those spices and varied in their concentrations. Gallic acid had the highest level (1642.3 mg/100g) (cloves). Gentisic acid had the lowest level (1.2 mg/100g) in ginger. Also sumac showed the highest level of chlorogenic acid (1528.7 mg/100g). Some acids were not found in some spices, for instance, benzoic acid was not found in coriander, cumin, ginger, green cardamom, cinnamon and sweet laurel.

Phenolic Acids Content and Nutritional Quality of Conventional, Organic and Biodynamic Cultivations of the Tomato CXD271BIO Breeding Line (Solanum lycopersicum L.)

This study was undertaken to evaluate the influence of growing systems (conventional, organic and biodynamic performed with two types of manuring) on some nutritional traits of the tomato breeding line CXD271BIO (Solanum lycopersicum L.). Experimental fields were in the same geographical area and the cultivations were performed over five years. The content of macronutrients, minerals (Ca, K, Mg, P, Na), trace elements (Fe, Zn, Cu, Mn) and phenolic acids (chlorogenic, caffeic, p-coumaric and ferulic acids) was determined. The biodynamic A growing system showed the highest protein and carbohydrates content compared to the other growing systems. Data on minerals and trace elements content showed significant differences (mainly in Ca, Na, Fe and Zn content) between conventional and biodynamic tomatoes, whereas no major impact of the fertilization among organic and the two biodynamic growing systems was observed. Appreciable differences in phenolic acids biosynthesis were observed, with the year of harvest showing a marked effect especially on chlorogenic, p-cumaric and ferulic acids content. Conversely, the cultivation system did not show major influence. Principal Components Analysis (PCA), performed on mineral and phenolic acids content, substantiated the large effect of the year of harvest, also suggesting an effect of the conventional cultivation system.

Review on Comparative Analyses of Different Phenolic Acids Content in Different Beers

Beer can be a rich source of phenolic acids in the diet. The present review summarizes the current state of work on phenolic acids in beer presented in the cited papers. In an overview, 10 hydroxybenzoic acids, three related aldehydes, two phenylacetic acids and eight hydroxycinnamic acids in beer are being reported. This review compares the phenolic acids content in alcoholic and non-alcoholic beers from Czech Republic, Germany, Italy, Austria and Poland, which had been examined by several authors. The quantification was achieved by HPLC with ultraviolet, diode array, coulometric array or fluorimetric detection. Ferulic acid is the main phenolic acid in all beers that had been studied, followed by p-coumaric, vanillic, caffeic, p-OH-benzoic and sinapic acids. Most of the phenolic acids in beer are present as bound forms and only a small portion can be detected as free compounds. The content of the phenolic acids in the non-alcoholic beers is lower than in the alcoholic beers mainly due to differences in the production process, yeast strains used, dealcoholization of beer etc..

High Performance Liquid Chromatographic Determination of Phenolic Acids in Fruits and Vegetables

A simple isocratic HPLC technique has been developed for the quantitative analysis of phenolic acids (PAs) in fruits and vegetables. Nine benzoic and cinnamic acid derivatives were separated in less than 30 min, and the resolution was all more than 1.23. The ranges of linearity for PAs standards were 0.2-100 ng, even up to 600 ng (r = 0.983-1.000) and the detection limits were 0.02-0.24 mg/kg. Samples of fresh vegetables and fruits were extracted with 80% mcthanol and ethyl acetate, then purified with C18 Sep-Pak cartridge and determined by HPLC. This method was applied to the determination of PAs in 7 kinds of fruits and vegetables, i.e., apple, pear, Chinese cabbage, cauliflower, turnip, soybean sprout and white grape wine. The content of the 9 PAs varied widely in the 7 kinds ol'foods studied. The average concentrations ofchlorogenic acid in apple (100.2 mg/kg) and pear (30.8 mg/kg) were quite high, and sinapinic acid was remarkable (42.5 mg/kg) in Chinese cabbage, and protocatechuic acid had the highest concentration of all the PAs in white wine.

Use of Ultrasound Bath in the Extraction and Quantification of Ester-Linked Phenolic Acids in Tropical Forages

A method was developed for the analysis of ester-linked phenolic acids in forage samples using extraction by an ultrasound-assisted treatment and quantification by HPLC with a UV-VIS detector. A reversed-phase C18 column was used for developing the method and the optimal condition was established with isocratic elution using acetonitrile/methanol/H3PO4 pH 2.08 (13:12.5:74.5) as the mobile phase. To reduce the time of sample processing, the extraction of ester-linked phenolic acids was studied using ultrasound bath and the results were then compared with those from an extraction usual using alkaline hydrolysis (20°C for 24 h). The method was valued through external and internal calibration. Internal calibration using o-coumaric acid as internal standard and m-coumaric acid as surrogate internal standard showed better results. The detection limits were of 0.09 and 0.04 mg●L-1 for p-coumaric and ferulic acids, respectively. The proposed method showed a good linear dynamic range (3.00 - 30.00 mg●L-1) for the analytes. The usefulness of the methodology was demonstrated by addition-recovery experiments using forage samples and values were in the 83 to 99% range. The extraction of ester-linked phenolic acids by 120 minutes of ultrasound bath was faster and more reproducible than alkaline hydrolysis (20°C for 24 h).

Induced Effects of Exogenous Phenolic Acids on Allelopathy of a Wild Rice Accession(Oryza longistaminata,S37)

Four exogenous phenolic acids, including salicylic acid, fumalic acid, p-coumaric acid and p-hydroxybenzonic acid, were used to investigate the regulatory effects on allelopathy of a wild rice accession of S37 （Oryza Iongistaminata）, which is a known allelopathic rice. The four exogenous phenolic acids induced the enhancement of the allelopathic potential of wild rice S37 in target weeds though the weed-suppressive activities were low, and the inducible effects were dependent on the specific phenolic acid, concentration and treatment time. After foliar application of exogenous phenolic acids, the inhibition rates for plant height, root length and fresh weight of barnyard grass （Echinochioa crus-galli） were significantly higher than those of the control. Especially at the concentration of 100 mg/L, the inhibition rates for plant height and fresh weight of barnyard grass by fumalic acid were 38.12% and 26.31% higher than those of the control, showing that fumalic acid was more effective compared with other phenolic acids in inhibiting monocotyledon weed growth. Furthermore, the weedsuppressive activity of aqueous extract from the leaves of wild rice S37 treated with exogenous phenolic acids was increased, and it peaked at 48 h after the treatment with the aqueous extract, and then gradually declined.

Effects of Exogenous Phenolic Acids on Allelopathy of Potted Soybean Seedlings

In this study, several general y reported exogenous phenolic acids were selected as regents for potting experiments, aiming at revealing the effects of differ-ent concentrations of exogenous phenolic acids on the growth and development of soybean seedlings. The growth and development indicators of soybean seedlings treated with five different concentrations of exogenous phenolic acids, such as plant height, leaf area, root nodule number and root-shoot ratio, were determined and compared. According to the experimental results, three exogenous phenolic acids with different concentrations exhibited slight effects on plant height of potted soy-bean seedlings. The leaf area of potted soybean seedlings treated with 1.0 g/kg benzoic acid and cinnamic acid varied extremely significantly compared with the control. Root nodule number of potted soybean seedlings treated with 1.0 g/kg ben-zoic acid and vanil ic acid increased remarkably, but 1.0 g/kg cinnamic acid had lit-tle effects on root nodule number. When the concentration of phenolic acids was 1.0 g/kg, three phenolic acids exhibited the maximum effects on root-shoot ratio of potted soybean seedlings. Overal , low concentrations of exogenous phenolic acids exhibited no significant effects on the growth and development of potted soybean seedlings; high concentrations of exogenous phenolic acids inhibited the growth and development of potted soybean shoots, increased root nodule number and improved root-shoot ratio.

Improvement of Selectivity with Boric Acid in Separating Phenolic Acids by Capillary Zone Electrophoresis

It was shown that in capillary electrophoresis (CE) the addition of boric acid as a complexing agent in phosphate buffer can modulate the separation selectivity of phenolic acids.

Theoretical and experimental cocrystal screening of temozolomide with a series of phenolic acids, promising cocrystal coformers

The virtual cocrystal screening approach based on molecular electrostatic potential surface(MEPS)maps is a fast and feasible computational method to estimate the probability of cocrystal formation by calculating the difference in the interaction site pairing energies of monomers and that of their assemblies prior to experimental screening.In this paper,we report 12 cocrystal forms of temozolomide with mono-,di-,and trihydroxy benzoic acids,namely,3-hydroxy-,2,4-dihydroxy-,2,5-dihydroxy-,2,6-dihydroxy-,3,4-dihydroxy-,and 3,4,5-trihydroxy-benzoic acids,as well as benzoic acid,as pharmaceutical coformers for the first time.10 single crystals out of the 12 cocrystal forms were obtained and unequivocally determined by single-crystal X-ray diffraction,which clarified spatial arrangements,molecular conformations,and supramolecular synthons.MEPS further gains some insights into the sites of hydrogen bonding interactions for exploring combination patterns in these assemblies.Modulated stability of TMZ was successfully achieved by cocrystallization with these acids.

Distribution of antioxidants and phenolic compounds in flour milling fractions from hard red winter wheat

Mature wheat kernels contain three main parts:endosperm,bran,and germ.Flour milling results in multiple streams that are chemically different;however,the distribution of antioxidants and phenolic compounds has not been well documented in terms of conventional milling by-product streams.In this study,multiple analytical methods were used to investigate antioxidant activity and phenolic compound compositions of hard red winter wheat(whole ground wheat),the parts of a wheat kernel(bran,flour,germ),and wheat by-product streams(mill feed,red dog,shorts)for the first time.For each mill stream,phenolic compounds(total,flavonoid,and anthocyanin contents)were determined and antioxidant activities were evaluated with 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical-scavenging activity,ferric reducing/antioxidant power(FRAP),and total antioxidant capacity assays.Significant differences(P<0.05)were observed in phenolic concentrations among fractions of bran,flour,and germ milled from the same kernels and noted that germ accounts for the majority of antioxidant properties,whereas bran contains a substantial portion of phenolic compounds and anthocyanins.Mill feed was high in phenolic content(5.29 mg FAE/g),total antioxidant capacity(866 mg/g),and antioxidant activity(up to 75% DPPH inhibition and 20.26μmol FeSO_(4)/g).The comprehensive information on distribution of antioxidants and phenolic compounds provides insights for future human consumption of commonly produced co-products from flour milling,and for selecting and using different milling fractions to make foods with improved nutritional properties.

Glucuronic acid metabolites of phenolic acids target AKT-PH domain to improve glucose metabolism

Objective:Phenolic acids widely exist in the human diet and exert beneficial effects such as improving glucose metabolism.It is not clear whether phenolic acids or their metabolites play a major role in vivo.In this study,caffeic acid(CA)and ferulic acid(FA),the two most ingested phenolic acids,and their glucuronic acid metabolites,caffeic-4’-O-glucuronide(CA4G)and ferulic-4’-O-glucuronide(FA4G),were investigated.Methods:Three insulin resistance models in vitro were established by using TNF-a,insulin and palmitic acid(PA)in HepG2 cells,respectively.We compared the effects of FA,FA4G,CA and CA4G on glucose metabolism in these models by measuring the glucose consumption levels.The potential targets and related pathways were predicted by network pharmacology.Fluorescence quenching measurement was used to analyze the binding between the compounds and the predicted target.To investigate the binding mode,molecular docking was performed.Then,we performed membrane recruitment assays of the AKT pleckstrin homology(PH)domain with the help of the PH-GFP plasmid.AKT enzymatic activity was determined to compare the effects between the metabolites with their parent compounds.Finally,the downstream signaling pathway of AKT was investigated by Western blot analysis.Results:The results showed that CA4G and FA4G were more potent than their parent compounds in increasing glucose consumption.AKT was predicted to be the key target of CA4G and FA4G by network pharmacology analysis.The fluorescence quenching test confirmed the more potent binding to AKT of the two metabolites compared to their parent compounds.The molecular docking results indicated that the carbonyl group in the glucuronic acid structure of CA4G and FA4G might bind to the PH domain of AKT at the key Arg-25 site.CA4G and FA4G inhibited the translocation of the AKT PH domain to the membrane,while increasing the activity of AKT.Western blot analysis demonstrated that the metabolites could increase the phosphorylation of AKT and downstream glycogen synthase kinase 3βin the AKT signaling pathway to increase glucose consumption.Conclusion:In conclusion,our results suggested that the metabolites of phenolic acids,which contain glucuronic acid,are the key active substances and that they activate AKT by targeting the PH domain,thus improving glucose metabolism.

Simultaneous determination of five bioactive phenolic acids in Salvia yunnanensis and Salvia miltiorrhiza by HPLC

A reverse-phase HPLC method was developed for the simultaneous separation and determination of five bioactive phenolic acids,yunnaneic acid E,rosmarinic acid,lithospermic acid,salvianolic acid B and salvianolic acid A in eight different samples of Salvia yunnanensis collected in Yunnan Province.For comparison,the sample of Salvia miltiorrhiza was included. All the samples were extracted for 60 min with 50%methanol in an ultrasonic bath.The optimal separation was achieved on a YMC-Pack Pro C18 column,with a gradient of 0.1%（v/v） phosphoric acid and acetonitrile,at a flow rate of 1.0 mL/min and at a detection wavelength of 280 nm.The separation was obtained within 65 min for five bioactive phenolic acids.All calibration curves showed good linearity（r^2〉0.999） within test ranges.The relative standard deviation of the method was less than 5%for intra- and inter-day assays.The mean recovery of the method was in the range from 97%to 104%,with RSD less than 5%.This assay was successfully applied to the quantitative determination of five bioactive phenolic acids in nine resource samples. The results showed that the developed HPLC assay was suitable for the quality control of S.yunnanensis and it can be used to differentiate S.yunnanensis from S.miltiorrhiza.

Impact of Continuous Cropping on Soil Phenolic Acid Substances and Research Progress on Continuous Cropping Obstacle Reduction Techniques

At present,long-term continuous cropping in agricultural production has formed a relatively common development trend.With the increase of continuous cropping years,soil phenolic acids are also affected to varying degrees.This paper summarized the effects of continuous cropping on soil phenolic acids and the research progress of continuous cropping obstacle reduction techniques,aiming at providing theoretical basis and technical support for the research of continuous cropping obstacle reduction techniques and promoting the healthy and sustainable development of modern agriculture.

Phenolic acids isolated from the fungus Schizophyllum commune exert analgesic activity by inhibiting voltage-gated sodium channels

The present study was designed to search for compounds with analgesic activity from the Schizophyllum commune(SC), which is widely consumed as edible and medicinal mushroom world. Thin layer chromatography(TLC), tosilica gel column chromatography, sephadex LH 20, and reverse-phase high performance liquid chromatography(RP-HPLC) were used to isolate and purify compounds from SC. Structural analysis of the isolated compounds was based on nuclear magnetic resonance(NMR). The effects of these compounds on voltage-gated sodium(NaV) channels were evaluated using patch clamp. The analgesic activity of these compounds was tested in two types of mouse pain models induced by noxious chemicals. Five phenolic acids identified from SC extracts in the present study included vanillic acid, m-hydroxybenzoic acid, o-hydroxybenzeneacetic acid, 3-hydroxy-5-methybenzoic acid, and p-hydroxybenzoic acid. They inhibited the activity of both tetrodotoxin-resistant(TTX-r) and tetrodotoxin-sensitive(TTX-s) NaV channels. All the compounds showed low selectivity on NaV channel subtypes. After intraperitoneal injection, three compounds of these compounds exerted analgesic activity in mice. In conclusion, phenolic acids identified in SC demonstrated analgesic activity, facilitating the mechanistic studies of SC in the treatment of neurasthenia.

Simultaneous quantification of ten phenolic acids in Lonicerae Japonicae Flos by HPLC-DAD

A reverse-phase HPLC-DAD method was developed for simultaneous quantification of ten phenolic acids （caffeic acid, chlorogenic acid, neochlorogenic acid, 4-O-caffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid methyl ester, 3,4-O-dicaffeoylquinic acid methyl ester, and 4,5-0- dicaffeoylquinic acid methyl ester） in the dried flower buds of Lonicera japonica Thunb. （Lonicerae Japonicae Flos; LJF）. An optimal sample preparation method was established as 30-min ultrasonication with 100 times 50% （v/w） ethanol aqueous solution based on the orthogonal test results. The chromatographic separation of the ten phenolic acids was achieved with an AQ-C18 column （4.6 mm x 250 mm, 5 p.m） and a gradient elution of acetonitrile, methanol and 0.1% formic acid aqueous solution within 55 rain. All calibration curves showed good linearity （r2〉0.999） within test ranges. The average recoveries were in the range of 98.57%-103.22% with RSD less than 3%. The method developed was accurate, sensitive and reproducible for determination of ten phenolic acids in LJF.

bHLH transcription factor SmbHLH92 negatively regulates biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza

Objective:Salvia miltiorrhiza is a valuable herbal medicine with tanshinone and phenolic acid as the main biological active ingredients.The biosynthetic regulation of these bioactive compounds is controlled by a set of transcription factors(TFs).The basic helix-loop-helix(bHLH)transcription factor plays an important role in various physiological and biochemical processes in plants.However,research on bHLH TFs regulating phenolic acid or tanshinone biosynthesis in S.miltiorrhiza is limited.Methods:qRT-PCR was used for gene expression analysis.The subcellular localization of SmbHLH92 was detected by SmbHLHg2-GFP transient transformation into tobacco leaves,and its fluorescence was observed using a confocal laser scanning microscope.The transcriptional activity of SmbHLH92 was confirmed in the AH109 yeast strain.RNA interference hairy roots of SmbHLH92-RNAi transgenic lines were obtained through Agrobacterium-mediated genetic transformation.Ultra performance liquid chromatography(UPLC)was used to detect the changes of phenolic acids and tanshinones.Results:SmbHLH92 is a bHLH transcription factor that is highly expressed in the root and phloem of S.miltiorrhiza.The subcellular localization and transcriptional activity of SmbHLH92 indicated that SmbHLH92 was located in the nucleus and may be a transcription factor.RNA interference(RNAi)of SmbHLH92 in hairy roots of S.miltiorrhiza significantly increased the accumulation of phenolic acid and tanshinone.Quantitative RT-PCR(RT-qPCR)analysis showed the transcription level of genes encoding the key enzymes involved in the phenolic acid and tanshinone biosynthetic pathways was increased in the hairy roots of the SmbHLH92-RNAi transgenic line,comparing with the control line.Conclusion:These data indicate that SmbHLH92 is a negative regulator involved in the regulation of phenolic acid and tanshinone biosynthesis in S.miltiorrhiza.