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J. Crop Sci. Biotech. 2010 (September) 13 (3) : 183 ~ 188 DOI No. 10.1007/s12892-010-0040-3 RESEARCH ARTICLE
Salicylic Acid acts as Potent Enhancer of Growth, Photosynthesis and Artemisinin Production in Artemisia annua L. Tariq Aftab*, M. Masroor A. Khan, Mohd. Idrees, M. Naeem, Moinuddin Plant Physiology Section, Department of Botany, Aligarh Muslim University, Aligarh- 202 002, India Received: March 16, 2010/ Revised: Jun 7, 2010/ Accepted: September 8, 2010 Ⓒ Korean Society of Crop Science and Springer 2010
Abstract Plant secondary metabolites constitute the most important class of natural products with diverse and valuable chemical properties and biological activities. Artemisinin, isolated from Artemisia annua L., is potentially a drug that could be effective against multidrug-resistant strains of the malarial parasite, Plasmodium. Salicylic acid (SA) acts as a potential plant growth regulator and plays an important role in regulating a number of plant physiological and biochemical processes. The present study was conducted to assess the alterations in plant growth, photosynthetic capacity, enzyme activities, and content and yield of artemisinin in Artemisia annua L. in response to foliar application of SA. Four levels of SA (0.00, 0.25, 0.50, and 1.00 mM SA) were applied on the aboveground plant parts. Plant height and dry weight were altered significantly as the level of SA increased. Besides, application of SA positively improved chlorophyll and carotenoid contents. Furthermore, significant enhancement in net photosynthetic rate (31.7%) and the activity of nitrate reductase (17.2%) and carbonic anhydrase (10.9%) was noticed as the level of SA was increased from 0.00 to 1.00 mM SA. Most importantly, the content and yield of artemisinin was positively regulated by the SA. In comparison to no SA application (control), SA at 1.00 mM increased the content and yield of artemisinin by 25.8 and 50.0%, respectively. Key words: Artemisia annua L., artemisinin, carbonic anhydrase, net photosynthetic rate, nitrate reductase, salicylic acid
Introduction Artemisia annua L., also known as qinghao or sweet wormwood, is an annual herb native to Asia and most probably China (McVaugh 1984). Artemisinin, a sesquiterpene lactone with a peroxide group, has been held responsible for antimalarial activity (Klayman 1985). Artemisinin, along with taxol, is considered as one of the novel discoveries in recent medicinal plant research, and its isolation and characterization have increased the interest of plant scientists in A. annua worldwide (Ferreira et al. 2005). It is effective against all Plasmodium species, including P. vivax and P. falciparum, two of the four species that cause malaria in humans (WHO 2002). Being the world's most severe parasitic infection, malaria causes more than a million Tariq Aftab ( ) Department of Botany, Aligarh Muslim University Aligarh- 202 002, India Email:
[email protected] Tel: +91-99976 16302
The Korean Society of Crop Science
deaths out of 500 million cases annually. The World Health Organization (WHO) now recommends the use of artemisininbased combination therapies (ACT) for the treatment of malaria (WHO 2002; Kindermans et al. 2007). Plant growth regulators (PGRs) stimulate growth and terpenoid biosynthesis in various aromatic plants, which can result in beneficial changes in quality as well as quantity of terpenoids (Shukla et al. 1992). Biosynthesis of terpenoids is dependent on primary metabolism, e.g. photosynthesis and oxidative pathways, for carbon and energy supply (Singh et al. 1990). Out of a variety of PGRs, SA has been shown to influence diverse plant developmental processes such as stomatal regulation (Arfan et al. 2007), photosynthesis, growth (Khan et al. 2003; Arfan et al. 2007), and a variety of metabolic events (Raskin 1992; Lee et al. 1995). Moreover, SA inhibits the breakdown of ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO (Pancheva and Popova 1998), altering Hill activity, and kinetics of O2 evolution (Maslenkova and Toncheva 1998) in association with the
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changes in net photosynthetic rate, and content of carotenoids and sugars (Chandra and Bhatt 1998). The increase in the productivity of this immensely important antimalarial-drug plant is the need of the hour because of the low artemisinin content in plants all over the world. This study was undertaken to assess the physiological and biochemical changes in A. annua in response to SA, and to examine whether this plant growth regulator is involved in the induction of crop productivity, photosynthetic capacity, enzyme activities, and content and yield of artemisinin.
Materials and Methods Plant material and sampling procedures The seedlings of Artemisia annua L. were obtained from the seeds which were initially sown in November in the seedbeds. The seedlings were transplanted into the field in January with 45 x 45 cm spacing. Physico-chemical characteristics of the soil were: texture sandy loam, pH (1:2) 7.2, E.C. (1:2) 0.46 m mhos cm-1, available N, P, and K 98.84, 6.83, and 142.9 mg kg-1 soil, respectively. Twelve seedlings were transplanted in each plot and the plots were spaced 1 m apart to abolish the effect of SA treatments. Each SA treatment was replicated five times, using a randomized block design. Before transplantation, a uniform basal dose of N, P, and K was applied at the rate of 80, 40, and 40 kg ha-1, respectively. The experiment was conducted for the two consecutive crop seasons (2007/08 and 2008/09). The plants were sprayed with various concentrations of SA (0.00, 0.25, 0.50, and 1.00 mM) using a hand sprayer. Three foliar sprays of SA were given at 10-day intervals starting from 30 days after planting (DAP). Fresh leaves were sampled at 90 DAP for physiological and biochemical parameters. The crop was irrigated as required. Plant height was measured using a meter scale. The plants from each treatment were carefully harvested with the roots intact. They were dried at 80 °C for 48 h, and dry weight of shoot was recorded.
Physiological and biochemical analyses Determination of net photosynthetic rate, stomatal conductance, and internal CO2 Net photosynthetic rate (PN), stomatal conductance (gs), and internal CO2 concentration (ci) were measured on sunny days at 1100 hours using the youngest fully developed leaves of A. annua using IRGA (Infra Red Gas Analyzer, LI-COR 6400 Portable Photosynthesis System, Lincoln, Nebraska, USA). Before recording the measurements, the IRGA was calibrated and zero was adjusted approximately every 30 min during the measurement period. Each leaf was enclosed in a 1 liter gas exchange chamber for 60 s. All the parameters measured by IRGA were recorded three times for each treatment.
Estimation of total chlorophyll and carotenoid content Total chlorophyll and carotenoid contents in fresh leaves were estimated by the method of Mac Kinney (1941) and
MacLachlan and Zalik (1963), respectively. A total of 100 mg of fresh tissue from interveinal leaf-areas was ground using a mortar and pestle containing 80% acetone. The absorbance of the leaf extract was recorded at 645 and 663 nm for chlorophyll estimation and at 480 and 510 nm for carotenoid estimation using a spectrophotometer (Shimadzu UV-1700, Tokyo, Japan).
Determination of nitrate reductase (NR) and carbonic anhydrase (CA) activity Nitrate reductase (E.C. 1.6.6.1) activity in the fresh leaves was determined by the intact tissue assay method of Jaworski (1971). Chopped leaf pieces (200 mg) were incubated for 2 h at 30 °C in a 5.5 mL reaction mixture, which contained 2.5 mL of 0.1 M phosphate buffer, 0.5 mL of 0.2 M potassium nitrate, and 2.5 mL of 5% isopropanol. The nitrite formed subsequently was colorometrically determined at 540 nm after azocoupling with sulphanilamide and naphthylene diamine dihydrochloride. The NR activity was expressed as nM NO2 g-1 FW h-1. Carbonic anhydrase (E.C. 4.2.1.1) activity was measured in fresh leaves using the method described by Dwivedi and Randhawa (1974). Two hundred mg of fresh leaf pieces were transferred to Petri plates. The leaf pieces were dipped in 10 mL of 0.2 M cystein hydrochloride solution for 20 min at 4 °C. To each test tube, 4 mL of 0.2 M sodium bicarbonate solution and 0.2 mL of 0.022% bromothymol blue were added. The reaction mixture was titrated against 0.05 N HCl using methyl red as indicator. The enzyme activity was expressed as μM CO2 kg-1 leaf FW s-1.
Yield and quality parameters For the yield attributes, 12 plants from each treatment were collected at 90 DAP. The leaves were dried using a hot air oven for 24 h. The dried leaves were weighed for measuring the leafyield accordingly. Artemisinin yield was calculated by multiplying corresponding leaf-yield and artemisinin content.
Extraction and estimation of artemisinin Dry leaf material (1 g) was used for the estimation of artemisinin using the method of Zhao and Zeng (1986). As artemisinin lacks any chromophore for UV detection in HPLC, it was chemically modified to a compound Q260 and then quantified using HPLC method (Zhao and Zeng 1986). Standard curve was prepared using 1 mg of standard artemisinin dissolved in 1 mL of HPLC-grade methanol to prepare the stock solution. One g dry leaf-powder was used for artemisinin extraction. The extraction was carried out with 20 mL petroleum ether using a shaker run at 70 rpm for 24 h. Later, the solvent was decanted and pooled, adding 20 mL of petroleum ether again. This step was repeated three times. Petroleum ether fractions were pooled and concentrated under reduced pressure and the residue was defatted with CH3CN (10 mL x 3). The precipitated fat was filtered out and the filtrate was concentrated under reduced pressure. The residue was dissolved in 1 mL of methanol. To a 100 µL aliquot of each sample, 4 mL of 0.3% NaOH solution was added. The samples were incubated in shaking water bath at 50
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°C for 30 min. Thereafter, the content was cooled and neutralized with glacial acetic acid (0.1 M in 20% MeOH). The pH of the solution was maintained at 6.8. Derivatized artemisinin was analyzed and quantified through reverse phase column (C18; 5 µm; 4.6 mm; 250 mm) using premix methanol: 10 mM potassium phosphate buffer (pH 6.5) in the ratio of 60:40 as mobile phase at constant flow rate of 1 mL min-1, with the detector set at 260 nm. Artemisinin was quantified using HPLC against the standard curve of artemisinin obtained from Sigma-Aldrich, USA.
Statistical analysis Each plot was treated as one replicate and all the treatments were replicated five times. The data of the two crop seasons were pooled together and statistically analyzed using SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA) according to one way ANOVA. Mean values were statistically compared by Duncan's Multiple Range Test (DMRT) at P < 0.05 level using different letters.
Fig. 1. Effect of different concentrations of SA on net photosynthetic rate in leaves of Artemisia annua L. Values showing the same letter are not significantly different at P ≤ 0.05 as determined by Duncan's multiple range test. Vertical bars show SE.
Results Application of SA significantly enhanced the growth of the plants. The shoot height was increased by 27.8% at 1.00 mM SA as compared to the control (Table 1). The shoot dry weight was also significantly affected by various concentrations of SA. The greatest increase in shoot dry weight (20.7%) was found at 1.00 mM concentration of SA (Table 1). Foliar application of SA significantly enhanced the net photosynthetic rate in the leaves. At 1.00 mM SA, the photosynthetic rate was 31.7% higher than that in the control (Fig. 1). The stomatal conductance and internal CO2 concentration were also altered significantly in accordance with the photosynthetic rate as SA was applied to the plants (Fig. 2). Content of chlorophyll and carotenoid increased as the level of applied SA increased. At the highest SA concentration (1.00 mM), chlorophyll and carotenoid content was 17.2 and 10.9% higher, respectively, as compared to the control (Fig. 3). Activities of nitrate reductase and carbonic anhydrase were also dose-dependent, SA-treated plants had 20.9 and 19.3% higher values, respectively, as compared to the untreated plants (Fig. 4). A dose-dependent elevation was also noted in the leaf artemisinin content of SA-treated plants. Application of SA at 1.00 mM proved most efficient in enhancing the
Fig. 2. Effect of different concentrations of SA on stomatal conductance and internal CO2 in leaves of Artemisia annua L. Values showing the same letter are not significantly different at P ≤ 0.05 as determined by Duncan's multiple range test. Vertical bars show SE.
Table 1. Effect of different concentrations of SA on shoot height and shoot dry weight per plant of Artemisia annua L. Means within a column followed by the same letter are not significantly different (p ≤ 0.05). The data shown are means of five replicates ± SE. Treatments 0.00 mM SA 0.25 mM SA 0.50 mM SA 1.00 mM SA
Shoot height (cm) d
145.6 ± 1.43 157.1 ± 1.92c 165.4 ± 2.21b 186.1 ± 1.67a
Shoot dry weight (g) 166.2 ± 3.64c 173.8 ± 4.04b 181.2 ± 3.98b 197.8 ± 3.23a
Fig. 3. Effect of different concentrations of SA on total chlorophyll and carotenoid contents of Artemisia annua L. Values showing the same letter are not significantly different at P ≤ 0.05 as determined by Duncan's multiple range test. Vertical bars show SE.
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artemisinin content, exceeding the control by 25.8% (Fig. 5). Compared to the control, application of SA at 1.00 mM increased the artemisinin yield by 50.0% (Fig. 6). The dry leaf-yield per plant increased to the maximum extent when the SA was applied to the plants at 1.00 mM, surpassing the control by 18.4% (Fig. 6).
Discussion
Fig. 4. Effect of different concentrations of SA on nitrate reductase and carbonic anhydrase activities of Artemisia annua L. Values showing the same letter are not significantly different at P ≤ 0.05 as determined by Duncan's multiple range test. Vertical bars show SE.
Fig. 5. Effect of foliar sprays of different concentrations of SA on artemisinin content of Artemisia annua L. Values showing the same letter are not significantly different at P ≤ 0.05 as determined by Duncan's multiple range test. Vertical bars show SE.
Fig. 6. Effect of foliar sprays of different concentrations of SA on leaf-yield and artemisinin yield per plant of Artemisia annua L. Values showing the same letter are not significantly different at P ≤ 0.05 as determined by Duncan's multiple range test. Vertical bars show SE.
Salicylic acid has been reported to play an important role in the growth and development processes of plants (Raskin et al. 1989), improving the quality and quantity of proteins ( Jung et al. 1993; Canakci 2003), the endurance against stress (Agnes et al. 2005; Hayat and Ahmad 2007), and the rate of photosynthetic and enzyme activities (Fariduddin et al. 2003). As revealed by the data presented in Table 1, it is clear that increasing levels of SA brought about a significant increase in the growth of the plants. The SA-enhanced growth of the plants might be associated with the regulatory effects of SA on cell growth and division (El-Tayeb 2005). The increase in shoot height and dry weight with increasing concentrations of SA in the present investigation is in agreement with the finding reported by Rajasekaran and Blake (1999). Moreover, the enzyme activities viz. NR and CA were significantly increased by the gradual increase in the applied levels of SA, with 1.00 mM proving the best foliar application. Our results are in line with the findings of Fariduddin et al. (2003). The increase in the uptake of various nutrients, including NO3, and the resultant activation of NR, is well established under normal growth conditions (Campbell 1999). Nevertheless, the beneficial interaction of SA with the NR inhibitors as reported by Shrivastava (1980) might result in the increased activity of NR in SA-treated plants. Alternatively, the increased activity of NR can be attributed to the fact that SA stabilizes the plasma membrane, hence preventing damage, as evidenced by the SA-increased membrane stability index in wheat (Agarwal et al. 2005). This membrane stabilization could have facilitated the increased uptake of nutrients including the nitrate (NR activity inducer), thereby, increasing the NR activity in the leaves (Campbell 1999). Foliar application of SA increased CA activity, with 1.00 mM concentration proving the best. Such a response of the plants to the applied SA is expected because SA increased the stomatal conductance (Fig. 2) that might have facilitated the diffusion of carbon dioxide into the stomata. In turn, the CO2 might have been acted upon by CA. Finally, the CO2 could be reduced by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in the chloroplast stroma. A probable reason for the enhancement of CA activity due to SA treatment might be the de novo synthesis of CA, which involves translation/transcription of the associated genes (Okabe et al. 1980). Application of SA alleviated the photosynthetic pigments (chlorophyll and carotenoid contents) in the present study
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(Fig. 3). Such an increase in the content of these pigments was also observed by Zhao et al. (1995) and Chandra and Bhatt (1998) as a result of SA treatment. Presumably, SAtreated plants might trap more sunlight to increase the rate of photosynthesis as compared to the control plants. The SAimproved contents of photosynthetic pigments might have resulted in the increased photosynthetic rate accordingly as shown in the present study (Fig. 1). Similar results have been declared by Zhao et al. (1995), Fariduddin et al. (2003) and Khodary (2004) in other studies. Furthermore, beneficial effects of SA on photosynthetic rate could be attributed by its stimulatory effects on RuBisCO activity (Khodary 2004). In the present study, it was observed that SA significantly enhanced the content and yield of leaf-artemisinin as compared to the untreated plants. According to Pu et al. (2009), SA regulates gene transcription in the artemisinin biosynthetic pathway and thereby increases artemisinin concentration in A. annua. They reported that 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) gene catalyzes the first step in the biosynthesis of isoprenoids which ultimately culminates in the biosynthesis of sesquiterpenoids. Stermer and Bostock (1987) also emphasized the importance of HMGR in the regulation of sesquiterpenoid phytoalexin accumulation. Terpenoid biosynthesis occurs in oil glands through a mevalonate-isoprenoid pathway. These oil glands are present in the leaves of many aromatic plants. In fact, the leaves are the major site of trichomes in which biosynthesis of artemisinin occurs, and the increase in artemisinin content by the leafapplied PGRs has been documented in various studies (Shukla et al. 1992; Ferriera et al. 2005; Aftab et al. 2010). Here we report the first study about the influence of SA on A. annua under field conditions for two consecutive seasons. In both crop seasons, foliar application of SA significantly stimulated the growth, photosynthesis, and artemisinin production in A. annua. Thus, SA can be utilized as a valuable source for the cultivation of A. annua with increased artemisinin content and yield.
Acknowledgements The authors would like to thank Mr. Mauji Ram of Jamia Hamdard (Hamdard University), New Delhi, for his kind help in the HPLC analysis of artemisinin.
References Aftab T, Khan MMA, Idrees M, Naeem M, Singh M, Ram M. 2010. Stimulation of crop productivity, photosynthesis and artemisinin production in Artemisia annua L. by tria contanol and gibberellic acid application. J. Plant Interact. doi: 10.1080/1742914100364 7137 Agarwal S, Sairam FK, Srivastava GC, Tyagi A, Meena RC. 2005. Role of ABA, salicylic acid, calcium and hydrogen
peroxide an antioxidant enzymes induction in wheat seedlings. Plant Sci. 169: 559-570 Agnes S, Jolan C, Moria LS, Irma T. 2005. Role of salicylic acid pre-treatment on acclimation of tomato plants to salt and osmotic stress. Acta Biol. Szeged. 49: 123-125 Arfan M, Athar HR, Ashraf M. 2007. Does exogenous appli cation of salicylic acid through the rooting medium modu late growth and photosynthetic capacity in two differently adapted spring wheat cultivars under salt stress. J. Plant Physiol. 6: 685-694 Campbell HW. 1999. Nitrate reductase structure, function and regulation: Bridging the gap between biochemistry and physiology. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 277-303 Canakci S. 2003. Effects acetalsalicylic acid on fresh weight pigment and protein content of bean leaf discs (Phaseolus vulgaris L.). Acta Biol. Hung. 54: 385-391 Chandra A, Bhatt RK. 1998. Biochemical and physiological response to salicylic acid in relation to the systemic acquired resistance. Photosynthetica 35: 255-258 Dwivedi RS, Randhawa NS. 1974. Evaluation of rapid test for hidden hunger of Zinc in plants. Plant Soil. 40: 445451 El-Tayeb MA. 2005. Response of barley grains to the inter active effect of salinity and salicylic acid. Plant Growth Regul. 45: 215-224 Fariduddin Q, Hayat S, Ahmad A. 2003. Salicylic acid influ ences net photosynthetic rate, carboxylation efficiency, nitrate reductase activity, and seed yield in Brassica juncea. Photosynthetica 41: 281-284 Ferreira JFS, Laughlin JC, Delabays N, Magalhaes PM, de Magalhaes PM. 2005. Cultivation and genetics of Artemisia annua L. for increased production of the anti malarial artemisinin, Plant Genet. Resour. 3: 206-229 Hayat S, Ahmad A. 2007. Salicylic acid: A Plant Hormone. Springer, Netherlands Jaworski EG. 1971. Nitrate reduc tase assay in intact plant tissue. Biochem. Biophys. Res. Commun. 43: 1274-1279 Jung JL, Friting B, Hahne G. 1993. Sunflower (Helianthus annua L.) pathogenesis-proteins: Induction by aspirin (acetylsalicylic acid) and characterization. Plant Physiol. 101: 873-880 Khan W, Prithiviraj B, Smith D. 2003. Photosynthetic response of corn and soybean to foliar application of sali cylates. J. Plant Physiol. 160: 485-492 Khodary SEA. 2004. Effect of salicylic acid on the growth, photosynthesis and carbohydrate metabolism in saltstressed maize plants. J. Agric. Biol. 6: 5-8 Kindermans JM, Pilloy J, Olliaro P, Gomes M. 2007. Ensuring sustained ACT production and reliable artemisinin supply. Malaria J. 6: 125-130 Klayman DL. 1985. Qinghaosu (artemisimin): an antimalari al drug from China. Science 228: 1049-1055 Lee H, Leon J, Raskin I. 1995. Biosynthesis and metabolism of salicylic acid. Proc. Natl. Acad. Sci. USA 92: 40764079
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Salicylic acid as a potent enhancer
Mac Kinney G. 1941. Absorption of light by chlorophyll solutions. J. Biol. Chem. 140: 315-322 MacLachlan S, Zalik S. 1963. Plastid structure, chlorophyll concentration, free amino acid composition of chlorophyll mutant of barley. Can. J. Bot. 41: 1053-1062 Maslenkova L, Toncheva S. 1998. Salicylic acid induced changes in photosystem II reduction in barley plants. Proc. Bulg. Acad. Sci. 51: 101-104 McVaugh R. 1984. Compositae. In WR Anderson, ed, Flora Novo-Galiciana. A descriptive account of the vascular plants of Western Mexico, University of Michigan Press, Ann Arbor Okabe K, Lindlar A, Tsuzuki M, Miyachi S. 1980. Carbonic anhydrase on ribulose 1, 5-biphosphate carboxylase and oxygenenase. FEBS Lett. 114: 142-144 Pancheva TV, Popova LP. 1998. Effect of salicylic acid on the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in barley leaves. Plant Physiol. 152: 381-386 Pu GB, Ma DM, Chen JL, Ma LQ, Wang H, Li GF, Ye HC, Liu BY. 2009. Salicylic acid activates artemisinin biosyn thesis in Artemisia annua L. Plant Cell Rep. 28: 11271135 Rajasekaran LR, Blake TJ. 1999. New plant growth regula tors protect photosynthesis and enhance growth under drought of jack pine seedlings. J. Plant Growth Regul. 18: 175-181 Raskin I. 1992. Role of salicylic acid in plants. Annual Rev. Plant Physiol. Plant Mol. Biol. 43: 439-463 Shrivastava HS. 1980. Regulation of nitrate reductase activi ty in higher plants. Phytochemistry 19: 725-733 Shukla A, Farooqi AHA, Shukla YN, Sharma S. 1992. Effect of triacontanol and chlormequat on growth, plant hor mones and artemisinin yield in Artemisia annua L. Plant Growth Regul. 11: 165-17 Singh N, Luthra R, Sangwan RS. 1990. Oxidative pathways and essential oil biosynthesis in the developing lemon grass (Cymbopogon flexuosus) leaf. Plant Physiol. Biochem. 28: 703-710 Stermer BA, Bostock RM. 1987. Involvement of 3-hydroxy3-methylglutaryl coenzyme-A reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato. Plant Physiol. 84: 404-408 WHO. 2002. Report: Meeting on antimalarial drug develop ment, Shanghai, China, 16-17 Nov. 2001, Manila, Philippines, WHO Zhao H, Lin X, Shi A, Chang S. 1995. The regulating effects of phenolic compounds on the physiological characteris tics and yield of soybeans. Acta Agron. Sin. 21: 351-355 Zhao SS, Zeng MY. 1986. Determination of qinghaosu in Artemisia annua L. by high performance liquid chro matography. Chinese J. Pharma. Anal. 6: 3-5
