Kacip Fatimah (Labisia pumila Blume), one of the most famous and widely used herbs, especially in Southeast Asia, is found to have interesting bioactive compounds and displays health promoting properties. In this study, the antioxidant activities of the methanol extracts of leaves, stems and roots of three varieties of L. pumila (var. alata, pumila and lanceolata) were evaluated in an effort to compare and validate the medicinal potential of this indigenous Malaysian herb species. The antioxidant activity determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, as well as the total amount of phenolics and flavonoids were the highest in the leaves, followed by the stems and roots in all the varieties. A similar trend was displayed by the ferric reducing antioxidant potential (FRAP) activity, suggesting that the L. pumila varieties possess high foliar antioxidant properties. At low FRAP activity concentrations, the values of the leaves' inhibition activity in the three varieties were significantly higher than those of the stems and roots, with var. alata exhibiting higher antioxidant activities and total contents of phenolics and flavonoids compared to the varieties pumila and lanceolata. The high production of secondary metabolites and antioxidant activities in var. alata were firmly related to low nitrogen content and high C/N ratio in plant parts. The study also demonstrated a positive correlation between secondary metabolite content and antioxidant activities, and revealed that the consumption of L. pumila could exert several beneficial effects by virtue of its antioxidant activity.
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 (f(v)/f(m)) 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.
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 CO(2) 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 (r(2) = 0.88) than starch on TP and TF biosynthesis. Photosynthesis, on the other hand, displayed a significant negative relationship with TP and TF production (r(2) = -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.
A randomized complete randomized design (RCBD) 3 by 3 experiment was designed to investigate and distinguish the relationships among production of secondary metabolites (total phenolics, TP; total flavonoids, TF), gluthatione (GSH), oxidized gluthatione (GSSG), soluble carbohydrate and antioxidant activities of the Malaysian medicinal herb Labisia pumila Blume under three levels of CO₂ enrichment (400, 800 and 1,200 µmol mol⁻¹) for 15 weeks. It was found that the treatment effects were solely contributed by interaction of CO₂ levels and secondary metabolites distribution in plant parts, GSH, GSHH and antioxidant activities (peroxyl radicals (ROO), superoxide radicals (O₂), hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH). The records of secondary metabolites, glutahione, oxidized gluthathione and antioxidant activities in a descending manner came from the leaf enriched with 1,200 µmol/mol CO₂ > leaf 800 µmol/mol CO₂ > leaf 400 µmol/mol CO₂ > stem 1,200 µmol/mol CO₂ > stem 800 µmol/mol CO₂ > stem 400 µmol/mol CO₂ > root 1,200 µmol/mol CO₂ > root 800 µmol/mol CO₂ > root 400 µmol/mol CO₂. Correlation analyses revealed strong significant positive coefficients of antioxidant activities with total phenolics, flavonoids, GSH and GSHH indicating that an increase in antioxidative activity of L. pumila under elevated CO₂ might be up-regulated by the increase in production of total phenolics, total flavonoids, GSH, GSHH and soluble sugar. This study implied that the medicinal potential of herbal plant such as L. pumila can be enhanced under elevated CO₂, which had simultaneously improved the antioxidative activity that indicated by the high oxygen radical absorbance activity against ROO, O₂, H₂O₂, and OH radicals.
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.
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 (f(v)/f(m)) 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.
A preliminary study was conducted to study the effects of different types and concentrations of co-solvents based on yield, composition and antioxidants capacity of extract prior to optimization studies of supercritical fluid extraction (SFE) of Labisia pumila (locally referred to as 'kacip fatimah'). The following co-solvents were studied prior to the optimization of supercritical carbon dioxide (SC-CO2) technique: ethanol, water, methanol, as well as aqueous solutions of ethanol-water and methanol-water (50% and 70% v/v). By using the selected co-solvents, identification of phenolic acids (gallic acid, methyl gallate and caffeic acid) was determined by using High-Performance Liquid Chromatography (HPLC). Then, the antioxidant capacity was evaluated by using three different assays: total phenolic content (TPC), ferric reducing/antioxidant power (FRAP) and free radical-scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl (DPPH). SC-CO2 with 70% ethanol-water co-solvent was superior in terms of a higher combination of phenolic compounds extracted and antioxidants capacity. Overall, SC-CO2 with co-solvent 70% ethanol-water technique was efficient in extracting phenolic compounds from L. pumila, and thus the usage of this solvent system should be considered for further optimization studies.
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.
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.
A randomized complete block design was used to characterize the relationship between production of total phenolics, flavonoids, ascorbic acid, carbohydrate content, leaf gas exchange, phenylalanine ammonia-lyase (PAL), soluble protein, invertase and antioxidant enzyme activities (ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) in Labisia pumila Benth var. alata under four levels of potassium fertilization experiments (0, 90, 180 and 270 kg K/ha) conducted for 12 weeks. It was found that the production of total phenolics, flavonoids, ascorbic acid and carbohydrate content was affected by the interaction between potassium fertilization and plant parts. As the potassium fertilization levels increased from 0 to 270 kg K/ha, the production of soluble protein and PAL activity increased steadily. At the highest potassium fertilization (270 kg K/ha) L. pumila exhibited significantly higher net photosynthesis (A), stomatal conductance (g(s)), intercellular CO(2) (C(i)), apparent quantum yield (ξ) and lower dark respiration rates (R(d)), compared to the other treatments. It was found that the production of total phenolics, flavonoids and ascorbic acid are also higher under 270 kg K/ha compared to 180, 90 and 0 kg K/ha. Furthermore, from the present study, the invertase activity was also found to be higher in 270 kg K/ha treatment. The antioxidant enzyme activities (APX, CAT and SOD) were lower under high potassium fertilization (270 kg K/ha) and have a significant negative correlation with total phenolics and flavonoid production. From this study, it was observed that the up-regulation of leaf gas exchange and downregulation of APX, CAT and SOD activities under high supplementation of potassium fertilizer enhanced the carbohydrate content that simultaneously increased the production of L. pumila secondary metabolites, thus increasing the health promoting effects of this plant.
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.