Mohd Hafiz Ibrahim

The Relationship between Phenolics and Flavonoids Production with Total Non Structural Carbohydrate and Photosynthetic Rate in Labisia pumila Benth. under High CO2 and Nitrogen Fertilization

A factorial split plot 4 × 3 experiment was designed to examine and characterize the relationship among production of secondary metabolites (total phenolics, TP; total flavonoids, TF), carbohydrate content and photosynthesis of three varieties of the Malaysian medicinal herb Labisia pumila Benth. namely the varieties alata, pumila and lanceolata under CO2 enrichment (1,200 µmol mol-1) combined with four levels of nitrogen fertilization (0, 90, 180 and 270 kg N ha-1). No varietal differences were observed, however, as the levels of nitrogen increased from 0 to 270 kg N ha-1, the production of TP and TF decreased in the order leaves>roots>stems. The production of TP and TF was related to increased total non structural carbohydrate (TNC), where the increase in starch content was larger than that in sugar concentration. Nevertheless, the regression analysis exhibited a higher influence of soluble sugar concentration (r2 = 0.88) than starch on TP and TF biosynthesis. Photosynthesis, on the other hand, displayed a significant negative relationship with TP and TF production (r2 = -0.87). A decrease in photosynthetic rate with increasing secondary metabolites might be due to an increase in the shikimic acid pathway that results in enhanced production of TP and TF. Chlorophyll content exhibited very significant negative relationships with total soluble sugar, starch and total non structural carbohydrate.

Effects of nitrogen fertilization on synthesis of primary and secondary metabolites in three varieties of Kacip Fatimah (Labisia pumila Blume).

A split plot 3 by 4 experiment was designed to examine the impact of 15-week variable levels of nitrogen fertilization (0, 90, 180 and 270 kg N/ha) on the characteristics of total flavonoids (TF), total phenolics (TP), total non structurable carbohydrate (TNC), net assimilation rate, leaf chlorophyll content, carbon to nitrogen ratio (C/N), phenyl alanine lyase activity (PAL) and protein content, and their relationships, in three varieties of Labisia pumila Blume (alata, pumila and lanceolata). The treatment effects were solely contributed by nitrogen application; there was neither varietal nor interaction effect observed. As nitrogen levels increased from 0 to 270 kg N/ha, the production of TNC was found to decrease steadily. Production of TF and TP reached their peaks under 0 followed by 90, 180 and 270 kg N/ha treatment. However, net assimilation rate was enhanced as nitrogen fertilization increased from 0 to 270 kg N/ha. The increase in production of TP and TF under low nitrogen levels (0 and 90 kg N/ha) was found to be correlated with enhanced PAL activity. The enhancement in PAL activity was followed by reduction in production of soluble protein under low nitrogen fertilization indicating more availability of amino acid phenyl alanine (phe) under low nitrogen content that stimulate the production of carbon based secondary metabolites (CBSM). The latter was manifested by high C/N ratio in L. pumila plants.

Impact of Soil Field Water Capacity on Secondary Metabolites, Phenylalanine Ammonia-lyase (PAL), Maliondialdehyde (MDA) and Photosynthetic Responses of Malaysian Kacip Fatimah (Labisia pumila Benth)

A randomized complete block design 2 × 4 experiment was designed and conducted for 15 weeks to characterize the relationships between production of total phenolics, flavonoid, anthocyanin, leaf gas exchange, total chlorophyll, phenylalanine ammonia-lyase (PAL) and malondialdehyde (MDA) activity in two varieties of Labisia pumila Benth, namely the var. alata and pumila, under four levels of evapotranspiration replacement (ER) (100%; well watered), (75%, moderate water stress), (50%; high water stress) and (25%; severe water stress). The production of total phenolics, flavonoids, anthocyanin, soluble sugar and relative leaf water content was affected by the interaction between varieties and SWC. As the ER levels decreased from 100% to 25%, the production of PAL and MDA activity increased steadily. At the highest (100%) ER L. pumila exhibited significantly higher net photosynthesis, apparent quantum yield, maximum efficiency of photosystem II (fv/fm) and lower dark respiration rates compared to the other treatment. The production of total phenolics, flavonoids and anthocyanin was also found to be higher under high water stress (50% ER replacement) compared to severe water stress (25% ER). From this study, it was observed that as net photosynthesis, apparent quantum yield and chlorophyll content were downregulated under high water stress the production of total phenolics, flavonoids and anthocyanin were upregulated implying that the imposition of high water stress can enhance the medicinal properties of L. pumila Benth.

Primary, Secondary Metabolites, H2O2, Malondialdehyde and Photosynthetic Responses of Orthosiphon stimaneus Benth. to Different Irradiance Levels

The resource availability hypothesis predicts an increase in the allocation to secondary metabolites when carbon gain is improved relative to nutrient availability, which normally occurs during periods of low irradiance. The present work was carried out to confirm this hypothesis by investigating the effects of decreasing irradiance on the production of plant secondary metabolites (flavonoids and phenolics) in the herbal plant Orthosiphon stamineus, and to characterize this production by carbohydrate, H2O2, and malondialdehyde (MDA) levels, net photosynthesis, leaf chlorophyll content and carbon to nitrogen ratio (C/N). Four levels of irradiance (225, 500, 625 and 900 µmol/m2/s) were imposed onto two-week old seedlings for 12 weeks in a randomized complete block design experiment. Peak production of total flavonoids, phenolics, soluble sugar, starch and total non-structural carbohydrate ocurred under low irradiance of 225 µmol/m2/s, and decreased with increasing irradiance. The up-regulation of secondary metabolites could be explained by the concomitant increases in H2O2 and MDA activities under low irradiance. This condition also resulted in enhanced C/N ratio signifying a reduction in nitrogen levels, which had established significant negative correlations with net photosynthesis, total biomass and total chlorophyll content, indicating the possible existence of a trade-off between growth and secondary metabolism under low irradiance with reduced nitrogen content. The competition between total chlorophyll and secondary metabolites production, as exhibited by the negative correlation coefficient under low irradiance, also suggests a sign of gradual switch of investment from chlorophyll to polyphenols production.

Involvement of Nitrogen on Flavonoids, Glutathione, Anthocyanin, Ascorbic Acid and Antioxidant Activities of Malaysian Medicinal Plant Labisia pumila Blume (Kacip Fatimah)

A split plot 3 by 4 experiment was designed to characterize the relationship between production of gluthatione (GSH), oxidized gluthatione (GSSG), total flavonoid, anthocyanin, ascorbic acid and antioxidant activities (FRAP and DPPH) in three varieties of Labisia pumila Blume, namely the varieties alata, pumila and lanceolata, under four levels of nitrogen fertilization (0, 90, 180 and 270 kg N/ha) for 15 weeks. The treatment effects were solely contributed by nitrogen application; there was neither varietal nor interaction effects observed. As the nitrogen levels decreased from 270 to 0 kg N/ha, the production of GSH and GSSG, anthocyanin, total flavonoid and ascorbic acid increased steadily. At the highest nitrogen treatment level, L. pumila exhibited significantly lower antioxidant activities (DPPH and FRAP) than those exposed to limited nitrogen growing conditions. Significant positive correlation was obtained between antioxidant activities (DPPH and FRAP), total flavonoid, GSH, GSSG, anthocyanin and ascorbic acid suggesting that an increase in the antioxidative activities in L. pumila under low nitrogen fertilization could be attributed to higher contents of these compounds. From this observation, it could be concluded that in order to avoid negative effects on the quality of L. pumila, it is advisable to avoid excessive application of nitrogen fertilizer when cultivating the herb for its medicinal use.

Photosynthetic capacity, photochemical efficiency and chlorophyll content of three varieties of Labisia pumila Benth. exposed to open field and greenhouse growing conditions

Three varieties (Alata, Pumila and Lanceolata) of Malaysian indigenous medicinal herb Labisia pumila Benth. grown in greenhouse and open field were tested to evaluate and compare their photosynthetic and maximal quantum efficiency of photosystem II (Fv/Fm) reactions. Every variety grown in greenhouse demonstrated higher light-saturated photosynthetic capacity than in the open field. The diurnal net photosynthesis (A) curve in the open field also displayed dual peaks with lower daily average A compared to the greenhouse. Varieties Alata and Pumila were found to acclimatize better under both growing conditions. The diurnal patterns of Fv/Fm indicated that plants grown under greenhouse encountered less photoinhibition than in open field condition. A decrease in chlorophyll (chl) a/b ratio in leaves of greenhouse plants with significant increase in chl b was observed. This study indicates that var. Alata and var. Pumila have the capacity to acclimatize to greenhouse growth condition.

Phenolics and Flavonoids Compounds, Phenylanine Ammonia Lyase and Antioxidant Activity Responses to Elevated CO2 in Labisia pumila (Myrisinaceae)

A split plot 3 × 3 experiment was designed to examine the impact of three concentrations of CO2 (400, 800 and 1,200 µmol·mol−1) on the phenolic and flavonoid compound profiles, phenylalanine ammonia lyase (PAL) and antioxidant activity in three varieties of Labisia pumila Benth. (var. alata, pumila and lanceolata) after 15 weeks of exposure. HPLC analysis revealed a strong influence of increased CO2 concentration on the modification of phenolic and flavonoid profiles, whose intensity depended on the interaction between CO2 levels and L. pumila varieties. Gallic acid and quercetin were the most abundant phenolics and flavonoids commonly present in all the varieties. With elevated CO2 (1,200 µmol·mol−1) exposure, gallic acid increased tremendously, especially in var. alata and pumila (101–111%), whilst a large quercetin increase was noted in var. lanceolata (260%), followed closely by alata (201%). Kaempferol, although detected under ambient CO2 conditions, was undetected in all varieties after exposure. Instead, caffeic acid was enhanced tremendously in var. alata (338~1,100%) and pumila (298~433%). Meanwhile, pyragallol and rutin were only seen in var. alata (810 µg·g−1 DW) and pumila (25 µg·g−1 DW), respectively, under ambient conditions; but the former compound went undetected in all varieties while rutin continued to increase by 262% after CO2 enrichment. Interestingly, naringenin that was present in all varieties under ambient conditions went undetected under enrichment, except for var. pumila where it was enhanced by 1,100%. PAL activity, DPPH and FRAP also increased with increasing CO2 levels implying the possible improvement of health-promoting quality of Malaysian L. pumila under high CO2 enrichment conditions.

Enhancement of leaf gas exchange and primary metabolites under carbon dioxide enrichment up-regulates the production of secondary metabolites in Labisia pumila seedlings.

A split plot 3 by 3 experiment was designed to investigate and distinguish the relationships among production of primary metabolites (soluble sugar and starch), secondary metabolites (total phenolics, TP; total flavonoids, TF) and leaf gas exchange of three varieties of the Malaysian medicinal herb Labisia pumila Blume, namely the varieties alata, pumila and lanceolata, under three levels of CO2 enrichment (400, 800 and 1,200 µmol mol−1) for 15 weeks. The treatment effects were solely contributed by CO2 enrichment levels; no varietal differences were observed. As CO2 levels increased from 400 to 1,200 µmol mol−1, the production of carbohydrates also increased steadily, especially for starch more than soluble sugar (sucrose). TF and TP content, simultaneously, reached their peaks under 1,200 µmol exposure, followed by 800 and 400 µmol mol−1. Net photosynthesis (A) and quantum efficiency of photosystem II (fv/fm) were also enhanced as CO2 increased from 400 to 1,200 µmol mol−1. Leaf gas exchange characteristics displayed a significant positive relationship with the production of secondary metabolites and carbohydrate contents. The increase in production of TP and TFs were manifested by high C/N ratio and low protein content in L. pumila seedlings, and accompanied by reduction in cholorophyll content that exhibited very significant negative relationships with total soluble sugar, starch and total non structural carbohydrate.

Impact of Organic and Inorganic Fertilizers Application on the Phytochemical and Antioxidant Activity of Kacip Fatimah (Labisia pumila Benth)

A study was conducted to compare secondary metabolites and antioxidant activity of Labisia pumila Benth (Kacip Fatimah) in response to two sources of fertilizer [i.e., organic (chicken dung; 10% N:10% P2O5:10% K2O) and inorganic fertilizer (NPK green; 15% N, 15% P2O5, 15% K2O)] under different N rates of 0, 90, 180 and 270 kg N/ha. The experiment was arranged in a randomized complete block design replicated three times. At the end of 15 weeks, it was observed that the application of organic fertilizer enhanced the production of total phenolics, flavonoids, ascorbic acid, saponin and gluthathione content in L. pumila, compared to the use of inorganic fertilizer. The nitrate content was also reduced under organic fertilization. The application of nitrogen at 90 kg N/ha improved the production of secondary metabolites in Labisia pumila. Higher rates in excess of 90 kg N/ha reduced the level of secondary metabolites and antioxidant activity of this herb. The DPPH and FRAP activity was also highest at 90 kg N/ha. The results indicated that the use of chicken dung can enhance the production of secondary metabolites and improve antioxidant activity of this herb.

Impact of Elevated Carbon Dioxide on Primary, Secondary Metabolites and Antioxidant Responses of Eleais guineensis Jacq. (Oil Palm) Seedlings

A split plot 3 by 3 experiment was designed to investigate the relationships among production of primary metabolites (soluble sugar and starch), secondary metabolites (total flavonoids, TF; total phenolics, TP), phenylalanine lyase (PAL) activity (EC 4.3.1.5), protein and antioxidant activity (FRAP) of three progenies of oil palm seedlings, namely Deli AVROS, Deli Yangambi and Deli URT, under three levels of CO2 enrichment (400, 800 and 1,200 µmol·mol−1) for 15 weeks of exposure. During the study, the treatment effects were solely contributed by CO2 enrichment levels; no progenies and interaction effects were observed. As CO2 levels increased from 400 to 1,200 µmol·mol−1, the production of carbohydrate increased steadily, especially for starch more than soluble sugar. The production of total flavonoids and phenolics contents, were the highest under 1,200 and lowest at 400 µmol·mol−1. It was found that PAL activity was peaked under 1,200 µmol·mol−1 followed by 800 µmol·mol−1 and 400 µmol·mol−1. However, soluble protein was highest under 400 µmol·mol−1 and lowest under 1,200 µmol·mol−1. The sucrose/starch ratio, i.e., the indication of sucrose phosphate synthase actvity (EC 2.4.1.14) was found to be lowest as CO2 concentration increased from 400 > 800 > 1,200 µmol·mol−1. The antioxidant activity, as determined by the ferric reducing/antioxidant potential (FRAP) activity, increased with increasing CO2 levels, and was significantly lower than vitamin C and α-tocopherol but higher than butylated hydroxytoluene (BHT). Correlation analysis revealed that nitrogen has a significant negative correlation with carbohydrate, secondary metabolites and FRAP activity indicating up-regulation of production of carbohydrate, secondary metabolites and antioxidant activity of oil palm seedling under elevated CO2 was due to reduction in nitrogen content in oil palm seedling expose to high CO2 levels.