Allocation of Secondary Metabolites, Photosynthetic Capacity, and Antioxidant Activity of Kacip Fatimah (Labisia pumila Benth) in Response to CO2 and Light Intensity

A split plot 3 by 4 experiment was designed to investigate and distinguish the relationships among production of secondary metabolites, soluble sugar, phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity, leaf gas exchange, chlorophyll content, antioxidant activity (DPPH), and lipid peroxidation under three levels of CO2 (400, 800, and 1200 μmol/mol) and four levels of light intensity (225, 500, 625, and 900 μmol/m2/s) over 15 weeks in Labisia pumila. The production of plant secondary metabolites, sugar, chlorophyll content, antioxidant activity, and malondialdehyde content was influenced by the interactions between CO2 and irradiance. The highest accumulation of secondary metabolites, sugar, maliondialdehyde, and DPPH activity was observed under CO2 at 1200 μmol/mol + light intensity at 225 μmol/m2/s. Meanwhile, at 400 μmol/mol CO2 + 900 μmol/m2/s light intensity the production of chlorophyll and maliondialdehyde content was the highest. As CO2 levels increased from 400 to 1200 μmol/mol the photosynthesis, stomatal conductance, f v/f m (maximum efficiency of photosystem II), and PAL activity were enhanced. The production of secondary metabolites displayed a significant negative relationship with maliondialdehyde indicating lowered oxidative stress under high CO2 and low irradiance improved the production of plant secondary metabolites that simultaneously enhanced the antioxidant activity (DPPH), thus improving the medicinal value of Labisia pumila under this condition.


Introduction
Labisia pumila, locally known as Kacip Fatimah, is a herbaceous plant belonging to the family Myrsinaceae. It is widely scattered in the lowland and hilly rain forests of Malaysia, Thailand, Indochina, The Philippines, and New Guinea [1][2][3]. Three varieties of L. pumila are identified in Malaysia, classified as L. pumila var. alata, L. pumila var. pumila, and L. pumila var. lanceolata [2]. It is usually used as a tonic drink among females, and its indication for women's health may be related to its phytoestrogen effects and having similar chemical structure to estrogen [4]. The varieties most universally utilized by the traditional healers are L. pumila var. alata and L. pumila var. pumila. Kacip Fatimah has been generally used by generations of Malay women by boiling it in water. It is believed that the decoction drink can induce and facilitate childbirth, as well as being a postpartum medication to help contract the birth channel, to tone the abdominal muscles, and to regain body strength [5,6]. The other uses of this herb include treatments for dysentery, dysmenorrheal, flatulence, and gonorrhea [7][8][9][10].
Recently, it was found that Labisia pumila extract contains flavanoids, phenolics, and various bioactive volatile compounds [11]. These compounds include demethylbelamcandaquinone B, fatimahol, dexyloprimulanin, epoxyoleanane glycosides, alkenated phenolics, cerebroside, glycerogalactolipids, and lipids [12]. These compounds have been identified as natural antioxidants that reduce oxidative damage to the human body [13][14][15]. The concentration of plant secondary metabolites was found to be influenced by environmental conditions such as light intensity, CO 2 levels, temperature, fertilization, and biotic and abiotic factors which 2 The Scientific World Journal can change the concentration of these active constituents [16,17]. Changes in the accumulation pattern of secondary metabolites due to environmental factors are, however, the result of changes in both the rate of synthesis and the rate of catabolism [18,19]. The activity of L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) determines the extent to which phenylalanine is withdrawn from primary metabolism and enters the general phenylpropanoid pathway [17]. Lately, it was found that the enrichment of L. pumila with high levels of CO 2 increased the secondary metabolite production (phenolics and flavonoids) of this plant [20][21][22][23][24][25]. Similar results were observed in ginger (Zingiber officianale) [24].
Atmospheric CO 2 concentration has increased by about a third over preindustrial levels and is continuing to increase at around 0.4% per year. By the year 2100, the atmospheric partial pressure of CO 2 is projected to be 700 parts per million (ppm) or higher [26]. Increased CO 2 concentration in the air promotes growth and accelerates physiologic processes in young trees because it causes an increase in internal carbon availability and changes partitioning of assimilates among plant parts (roots, stems, and leaves) and primary and secondary metabolites [27]. Plant responses to CO 2 will likely be influenced by other environmental factors [28], including nutrient and water availability [29,30] and climate change [31]. Irradiance also directly affects plant growth and phytochemistry [31] and is expected to mediate effects of elevated CO 2 on plants. Both irradiance and CO 2 are required resources for photosynthesis and their levels will interactively affect primary and secondary plant metabolism [31]. However, studies on interactive effects between irradiance and CO 2 levels on phytochemistry of herbal plants were limited. Recently, researchers have reported that environmental factors such as irradiance and CO 2 concentration can significantly alter secondary metabolite synthesis and production in plants [32][33][34][35][36]. The synthesis of medicinal components in the plant is affected by CO 2 and light intensity with changes observed in plant morphology and physiological characteristics [37,38]. Both factors were known to adjust not only plant growth and development but also the biosynthesis of primary and secondary metabolites [39].
Two theories that best describe plant phyto-chemical responses to elevated CO 2 and different irradiance have received much attention. The carbon/nutrient balance (CNB) theory [40,41] proposes that allocation to storage and secondary metabolites is determined by the balance between the availability of carbon and nutrients, such that carbon in excess of growth demands is allocated to carbon-based secondary metabolites or storage compounds. The growthdifferentiation balance (GDB) theory [42] is premised upon general patterns for relative responses of net assimilation and growth to resource availability (e.g., water, nutrients, and light), such that conditions limiting growth more than assimilation allow resources to be available for differentiation processes such as secondary metabolism.
There have been several studies that investigated the effects of elevated CO 2 on plant primary and secondary metabolites [25,[43][44][45], but only a few covered the responses of secondary metabolites under increasing CO 2 and irradiance. Although secondary metabolites constitute a significant sink for assimilated carbon, to date the picture is unclear as to how these compounds respond to different levels of CO 2 and irradiance. However, the interaction of CO 2 enrichment with different irradiance levels is expected to enhance the medicinal value of this plant. Hence, the objective of this study was to examine the effects of different CO 2 and irradiance levels on the secondary metabolites (total flavonoids, phenolics, anthocyanins, and ascorbic acid), soluble sugars, leaf gas exchange characteristics, phenylalanine ammonia lyase (PAL) activity, lipid peroxidation, and antioxidant activity (DPPH) of L. pumila seedlings. The study also aims to investigate at which CO 2 and irradiance levels the production of secondary metabolites was enhanced. The relationships between all these parameters were also investigated.

Experimental Location, Plant Materials, and Treatments.
The experiment was carried out in the glasshouse complex at Field 2, Faculty of Agriculture, Universiti Putra Malaysia (longitude 101 ∘ 44 N and latitude 2 ∘ 58 S, 68 m above sea level), with a mean atmospheric pressure of 1.013 kPa. Threemonth-old L. pumila var. alata seedlings were left for a month in the nursery to acclimatize until they were ready for the treatments. CO 2 enrichment treatments were initiated when the seedlings reached four months of age, and when plants were exposed to 400, 800, or 1200 mol/mol CO 2 . Labisia pumila plants were grown under four levels of shade (0, 20, 40, or 60% shade) using black net. The average light intensity passing through each of the 0, 20, 40, and 60% shade treatments was 900, 625, 500, and 225 mol/m 2 /s, respectively. The photosynthetic photon flux density was measured using LICOR-1412 quantum sensors. When the seedlings had reached 4 months of age, they were fertilized with NPK Blue Special at 15 g per plant. The seedlings were planted in a soilless medium containing coco-peat, burnt paddy husk, and well composted chicken manure in a 5 : 5 : 1 (v/v) ratio in 25 cm diameter polyethylene bags. Day and night temperatures in the glasshouse were maintained at 27-30 ∘ C and 18-21 ∘ C, respectively. The relative humidity was maintained between 50 and 60%. All seedlings were irrigated using overhead mist irrigation, which was given four times a day or when necessary. Each irrigation session lasted for 7 min. The factorial experiment was arranged in a split plot using a randomized complete block design with the three CO 2 levels (400, 800, and 1200 mol/mol) as the main plots and four levels of light intensity (900, 625, 500, and 225 mol/m 2 /s) as the subplots replicated three times. Each experimental unit consisted of seven seedlings, with a total of 252 seedlings in the experiment. All plants were harvested at the end of 15 weeks of treatments.

Growth Chamber Microclimate and CO 2 Enrichment.
The seedlings were raised in specially constructed growth chambers receiving 12 h photoperiod and average photosynthetic photon flux density of 330 mol/m 2 /s. Day and night temperatures were recorded at 30 ± 1.0 ∘ C and 20 ± 1.5 ∘ C, The Scientific World Journal 3 respectively, with a relative humidity of about 70 to 80%. Vapor pressure deficit ranged from 1.11 to 2.32 kPa. Carbon dioxide at 99.8% purity was supplied from a high-pressure CO 2 cylinder and injected through a pressure regulator into the fully sealed 2 × 3 m growth houses at 2 h daily and applied continuously from 08:00 to 10:00 a.m. The CO 2 concentrations of the different treatments were measured using Air Sense CO 2 sensors designated to each chamber during CO 2 exposition period. Plants were watered three to four times a day at 5 min per session to ensure normal growth of plants using drip irrigation with emitter capacity of 2 L/h [46].

Total Phenolics and Flavonoids Quantification.
The extraction and quantification of total phenolics and flavonoids followed the method described by Ibrahim et al. [47]. An amount of ground tissue samples (0.1 g) was extracted with 80% ethanol (10 mL) on an orbital shaker for 120 minutes at 50 ∘ C. The mixture was subsequently filtered (Whatman Number 1), and the filtrate was used for the quantification of total phenolics and total flavonoids. Folin-Ciocalteu reagent (diluted 10-fold) was used to determine the total phenolics content of leaf samples. Two hundred L of the sample extract was mixed with Follin-Ciocalteau reagent (1.5 mL) and allowed to stand at 22 ∘ C for 5 minutes before adding NaNO 3 solution (1.5 mL, 60 g L −1 ). After two hours at 22 ∘ C, absorbance was measured at 725 nm. The results are expressed as mg g −1 gallic acid equivalent (mg GAE g −1 dry sample). For total flavonoids determination, a sample (1 mL) was mixed with NaNO 3 (0.3 mL) in a test tube covered with aluminium foil and left for 5 minutes. Then 10% AlCl 3 (0.3 mL) was added followed by addition of 1 M NaOH (2 mL). The absorbance was measured at 510 nm using a spectrophotometer with rutin as a standard (results expressed as mg/g rutin dry sample).

Anthocyanin Content.
Anthocyanin content was determined according to Bharti and Khurana [48]. Fresh leaves (1 g) were added in 10 mL acidic methanol (1% v/v HCl) and incubated overnight. Anthocyanin was partitioned from chlorophyll with 10 mL chloroform, followed by addition of 9 mL of double deionised water. The test tubes containing the samples were shaken gently and the mixture was allowed to settle. The absorbance was read at 505 nm. Petunidin was used as a standard. Anthocyanin content was recorded as mg/g petunidin fresh weight.

Ascorbic Acid Content.
The ascorbic acid content was measured using the modified method of Davies and Masten [49]. Fresh leaf samples (1 g) were extracted in 1% of phosphate-citrate buffer (pH 3.5) using a chilled pestle and mortar. The homogenate was filtered. The filtrate was added to 1 mL of 1.7 mM 2,6-dichloroindophenol (2,6-DCPIP) in a 3 mL cuvette. The absorbance at 520 nm was read within 10 min of mixing the reagents. The extraction buffer was used as a blank. L-Ascorbic acid was used as a standard. Ascorbic acid was recorded as mg/g L-ascorbic acid fresh leaves.

Total Soluble Sugar Determination.
Total soluble sugar was measured spectrophotometrically using the method of Ibrahim and Jaafar [50]. Samples (0.5 g) were placed in 15 mL conical tubes, and distilled water was added to make up the volume to 10 mL. The mixture was then vortexed and later incubated for 10 min. Anthrone reagent was prepared by dissolving anthrone (Sigma Aldrich, St Louis, MO, USA, 0.1 g) in 95% sulphuric acid (Fisher Scientific, USA, 50 mL). Sucrose was used as a standard stock solution to prepare the standard curve for the quantification of sucrose in the sample. The mixed sample of ground dry sample and distilled water was centrifuged at a speed of 3400 rpm for 10 min and then filtered to get the supernatant. A sample (4 mL) was mixed with anthrone reagent (8 mL) and then placed in a water bath set at 100 ∘ C for 5 min before the sample was measured at an absorbance of 620 nm using a spectrophotometer (model UV160U; Shimadzu Scientific, Kyoto, Japan). The total soluble sugar in the sample was expressed as mg/g sucrose dry sample.

Phenylalanine Ammonia Lyase (PAL) Activity.
Phenylalanine ammonia lyase (PAL) activity was measured using the method described by Martinez-Tellez and Lafuente [51]. The enzyme activity was determined spectrophotometrically by measuring the production of trans-cinnamic acid from Lphenylalanine. Enzyme extract (10 L) was incubated at 40 ∘ C with 12.1 mM L-phenylalanine (90 L, Sigma) prepared in 50 mM Tris-HCl, (pH 8.5). After 15 min of reaction, transcinnamic acid yield was estimated by measuring increase in the absorbance at 290 nm. The standard curve was prepared using trans-cinnamic acid as standard (Sigma) and the PAL activity was expressed as nM trans-cinnamic acid/ g protein/h.

Leaf Gas Exchange Measurement.
The measurement was obtained using a closed infrared gas analyzer (LICOR 6400 Portable Photosynthesis System; IRGA, Licor Inc. Nebraska, USA). Prior to use, the instrument was warmed for 30 minutes and calibrated with the ZERO IRGA mode. Two steps are required in the calibration process: first the initial zeroing process for the built-in flow meter and second zeroing process for the infrared gas analyzer. The measurements used optimal conditions set at 400 mol mol −1 CO 2 30 ∘ C cuvette temperature, 60% relative humidity with air flow rate set at 500 cm 3 min −1 , and modified cuvette condition of 800 mol m −2 s −1 photosynthetically photon flux density (PPFD). The measurements of gas exchange were carried out between 09:00 and 11:00 a.m. using fully expanded young leaves numbered three and four from the plant apex to record net photosynthesis rate ( ). The operation was automatic and the data were stored in the LI-6400 console and analyzed using "Photosyn Assistant" software (Version 3, Lincoln Inc, USA). Several precautions were taken to avoid errors during measurement. Leaf surfaces were cleaned and dried using tissue paper before being enclosed in the leaf cuvette. The light response curve was produced following procedures described in Ibrahim and Jaafar [6] to generate the apparent quantum yield and dark respiration rate.

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Maximum Quantum Efficiency of Photosystem II ( V / ).
Measurements of chlorophyll fluorescence were recorded on fully expanded second leaves. Leaves were darkened for 15 minutes by attaching light-exclusion clips to the central region of the leaf surface. Chlorophyll fluorescence was measured using a portable chlorophyll fluorescence meter (Handy PEA, Hansatech Instruments Ltd., Kings Lynn, UK). Measurements were recorded for up to 5 seconds [52]. The fluorescence responses were induced by emitting diodes. Measurements of 0 (initial fluorescence), (maximum fluorescence), and V (variable fluorescence) were obtained.
The V values were derived as the differences between and 0 .

DPPH Radical Scavenging
Assay. The DPPH free radical scavenging activity of each sample was determined according to the method described by Joyeux et al. [53]. A solution of 0.1 mM DPPH in methanol was prepared. The initial absorbance of the DPPH in methanol was measured at 515 nm. An aliquot (40 L) of an extract was added to 3 mL of methanolic DPPH solution. The change in absorbance at 515 nm was measured after 30 min. The antiradical activity (AA) was determined using the following formula: The optic density of the samples, the control, and the empty samples were measured in comparison with methanol. The synthetic antioxidant, BHT (butylhydroxytoluene), andtocopherol were used as positive controls.

Malondialdehyde (MDA) Determination.
Lipid peroxidation of plant parts was estimated by the level of malondialdehyde (MDA) production using the thiobarbituric acid (TBA) method as described by Alexieva et al. [54]. One gram of ground (0.25 mm) plant sample was homogenized with a mortar and pestle in 0.5% trichloracetic acid (TCA, 1 mL). The homogenate was centrifuged at 9,000 rpm for 20 min. The supernatant (0.5 mL) was mixed with 20% TCA (2.5 mL) containing 0.5% TBA and heated in a boiling water bath for 30 min and allowed to cool in an ice bath quickly. The supernatant was centrifuged at 9,000 rpm for 10 min, and the resulting supernatant was used for determination of MDA content. Absorbance values at 532 nm were recorded.

Statistical
Analysis. Data were analyzed using the analysis of variance procedure in SAS version 17. Means separation test between treatments was performed using Duncan multiple range test. The standard error of differences between means was calculated with the assumption that data were normally distributed and equally replicated [55,56].

Results and Discussion
3.1. Total Phenolics and Flavonoids. Accumulation of total phenolics and flavonoids in L. pumila was influenced by the interaction effect between CO 2 and irradiance ( ≤ 0.01; Table 1). Generally, total phenolics and flavonoids was observed to be the highest at 1,200 mol/mol CO 2 + 225 mol/m 2 /s (3.25 mg gallic acid/g dry weight) and was the lowest at 400 mol/mol CO 2 + 900 mol/m 2 /s (0.92 mg gallic acid/g dry weight). Total flavonoids content followed the same trend as total phenolics where the highest total flavonoids content was observed at 1,200 mol/mol CO 2 + 225 mol/m 2 /s irradiance which registered 2.12 mg rutin/g dry. The lowest weight was recorded at 400 mol/mol CO 2 + 900 mol/m 2 /s irradiance which contained only 0.46 mg rutin/g dry weight. The present results are in contrast with a previous study by McDonald et al. [57] who found that the highest bioactive compound was accumulated under elevated CO 2 and high light conditions in Populus tremuloides, Betula papyrifera, and Acer saccharum. In the present study, the enrichment with high CO 2 (1200 mol/mol) under low irradiance (225 mol/m 2 /s) was found to increase the production of L. pumila secondary metabolites. Enhanced production of secondary metabolites under high levels of CO 2 is usually reported [47][48][49]. The present results indicate that enrichment of L. pumila with high CO 2 (1200 mol/mol) with 225 mol/m 2 /s irradiance can upregulate the production of total phenolics and flavonoids in the plant. The increased production of secondary metabolites under high CO 2 and low irradiance is attributed to the enhanced availability of soluble carbohydrates under this condition that upregulates the production of total phenolics and flavonoids [58]. Furthermore, the phenolics and flavonoids contents have high significant positive correlationships with soluble carbohydrates (total phenolics 2 = 0.901; ≤ 0.05; total flavonoids 2 = 0.783; ≤ 0.05; Table 2) which indicates that the up-regulation of secondary metabolites in L. pumila under this condition might be contributed by the increase in soluble carbohydrate content. The increase in carbohydrate content might increase the production of L. pumila secondary metabolites due to increase in substrates to the shikimic acid pathway [59]. The high total phenolics and flavonoids content under high CO 2 and low irradiance in the plant has been shown to have anticancer properties and also to have applications for the use as antibiotics, antidiarrhea, antiulcer, and anti-inflammatory agents, as well as in the treatment of diseases such as hypertension, vascular fragility, allergies, and hyperchlolesterolemia [60,61]. The results imply that CO 2 enrichment under low irradiance can enhance the medicinal properties of this herb.

Soluble
Carbohydrates. The accumulation of soluble carbohydrates in L. pumila followed the same trends with total phenolics and flavonoids. The soluble sugar was influenced by the interaction between CO 2 and irradiance ( ≤ 0.05; Table 3). The soluble sugar content was found to be higher as CO 2 levels increased from 400 to 1200 mol/mol and as the irradiance decreased from 900 to 225 mol/m 2 /s with the highest accumulation of soluble sugar observed at 1200 mol/mol CO 2 + 225 mol/m 2 /s irradiance which registered 40.71 mg/g sucrose dry weight, and it was the The Scientific World Journal 5   All results are expressed as means ± standard error of mean (SEM). = 21. Means not sharing a common letter within a column were significantly different at ≤ 0.05.

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The Scientific World Journal lowest at 400 mol/mol CO 2 + 900 mol/m 2 /s irradiance that only registered 18.34 mg/g sucrose dry weight. The present results suggest that at high levels of CO 2 (1200 mol/mol) under low irradiance (225 mol/m 2 /s), the production of flavonoids soluble sugars was enhanced. Ghasemzadeh et al. [24] described the accumulation of carbohydrates as a signal of an increase in production of secondary metabolites that enhanced the medicinal quality of plants. The extra carbohydrates accumulated in L. pumila seedlings in the present study might be channeled for the production of secondary metabolites (total phenolics and flavonoids) [62][63][64][65][66][67][68]. Carbohydrates are the basic compounds required to produce phenolic compounds through the shikimic acid pathway where extra carbohydrates derived from glycolysis and the penthose phosphate pathway are converted into aromatic amino acids [69]. The upregulation in carbon-based secondary metabolites (CBSM) in the present study might be related to the balance between carbohydrate source and sink, as the greater the source-sink ratio, the greater the production of secondary metabolites that might occur [70]. The present findings are in agreement with Guo et al. [71] who found that an increase in sucrose content corresponded with the enhanced production of ascorbic acid, glucosinolates, sulforaphane, anthocyanins, and total phenolics and increased the antioxidative activities in broccoli sprouts. The current results indicate that the exposure of L. pumila to high CO 2 under low irradiance can enhance the health promoting effects of this plant.

Anthocyanin Content.
In the present study, anthocyanin content was found to be influenced by the interaction effects between CO 2 and irradiance levels ( ≤ 0.05; Table 3). The accumulation of anthocyanin exhibited a similar pattern with total phenolics, flavonoids, and soluble sugars. In the leaves, the imposition of 225 mol/m 2 /s irradiance increased anthocyanin production to a maximum at 1200 mol/mol (0.97 mg/g fresh weight), whilst the lowest anthocyanin content of 0.69 mg/g fresh weight was recorded at ambient CO 2 levels (400 mol/mol) under 900 mol/m 2 /s irradiance. The same patterns that were observed for total phenolics, flavonoid, and soluble sugar accumulation suggest that enhancement of secondary metabolites production in L. pumila was optimized under the highest level of CO 2 (1200 mol/mol) under low irradiance (225 mol/m 2 /s). The increase in anthocyanins under high CO 2 was also observed in Populus euramericana [72] and strawberry [73]. Anthocyanins are naturally occurring phenolic compounds responsible for the color of many flowers, fruits, and berries [63]. They are the most important group of water soluble pigments in plants and they have beneficial health effects as antioxidant and anti-inflammatory agents [74,75]. Tamura and Yamagami [76] reported that anthocyanins possess positive therapeutic value, mainly associated with their antioxidant activity. Improvement in the anthocyanin content in the current study with elevated CO 2 and low irradiance suggests a possible mechanism for increasing the medicinal quality of L. pumila.

Ascorbic Acid Content.
Ascorbic acid was found to be influenced by the interaction effects of elevated CO 2 and irradiance levels ( ≤ 0.05; Table 3). The imposition of 1200 mol/mol CO 2 + 225 mol/m 2 /s irradiance significantly increased the ascorbic acid content compared to the other treatments. At the end of 15 weeks, the ascorbic acid content with 800 mol/mol CO 2 + 225 mol/m 2 /s irradiance, 400 mol/mol CO 2 + 225 mol/m 2 /s, 1200 mol/mol CO 2 + 500 mol/m 2 /s, 800 mol/mol CO 2 + 500 mol/m 2 /s, 400 mol/mol CO 2 + 500 mol/m 2 /s, 1200 mol/mol CO 2 + 675 mol/m 2 /s, 800 mol/mol CO 2 + 675 mol/m 2 /s, 400 mol/mol CO 2 + 675 mol/m 2 /s, 1200 mol/mol CO 2 + 900 mol/m 2 /s, 800 mol/mol CO 2 + 900 mol/m 2 /s, and 400 mol/mol CO 2 + 900 mol/m 2 /s was 0.0065, 0.0064, 0.0059, 0.0053, 0.0050, 0.0047, 0.0043, 0.0041, 0.0036, 0.0030, and 0.0024 mg/g, respectively, compared to 0.0071 mg/g with 1200 mol/mol CO 2 + 225 mol/m 2 /s irradiance. There were no previous reports on the combined impact of elevated CO 2 and irradiance on the accumulation of ascorbic acid. However, previous research had shown that exposure to elevated CO 2 can enhance the production of ascorbic acid in Capsicum anuum [77], tomato [78], and potato [79]. Ascorbic acid had a significant positive correlation with soluble sugar ( 2 = 0.887; ≤ 0.05) which indicated that the increase in production of ascorbic acid under high CO 2 and low irradiance can be attributed to the increased carbohydrate content that upregulated the production of this component. The increase in ascorbic acid content in L. pumila seedlings is attributed to the high production of TNC under high CO 2 and low irradiance. TNC (d-glucose) is a precursor for ascorbic biosynthesis in plants, and the more the availability of TNC was, the more the ascorbic acid would be produced in the L-galactose pathway [80,81].

Phenylalanine Ammonia Lyase (PAL) Activity.
The phenyl alanine ammonia lyase (PAL) activity was influenced by interaction effects between CO 2 and irradiance levels ( ≤ 0.05; Figure 1). In general it was observed that increase in light intensity reduced the activity of this enzyme in Labisia pumila. The highest PAL activity was found to be under 1200 mol/mol CO 2 + 225 mol/m 2 /s irradiance that recorded 47.21 nM transcinnamic mg/protein/hour and the lowest was at 400 mol/mol CO 2 + 900 mol/m 2 /s irradiance, which only registered 15.71 nM transcinnamic mg/protein/hour. The increase in secondary metabolites content in the present study is attributed to the increase in PAL activity under high CO 2 and low irradiance. Correlation analysis revealed that PAL activity had a significant positive correlation with total phenolics ( 2 = 0.921; ≤ 0.05) and total flavonoids ( 2 = 0.895; ≤ 0.05), suggesting an increase in secondary metabolites production with increasing PAL activity. The increase in PAL activity under high CO 2 and low irradiance might be due to the reduction in nitrogen content under high CO 2 and low irradiance that enhanced growth and reduced the nitrogen pool in the plant; this restricted the protein production and thus more phenylalanine was available for secondary metabolites production [82,83].  3.6. Total Chlorophyll Content. Similar to PAL activity, the chlorophyll content was also influenced by the interaction between CO 2 and irradiance levels ( ≤ 0.01; Figure 2). The chlorophyll content was found to be the highest at ambient CO 2 under 225 mol/m 2 /s irradiance which registered 30.71 mg/g fresh weight and the lowest at 1200 mol/mol CO 2 + 900 mol/m 2 /s irradiance which recorded only 14.21 mg/g fresh weight. The decrease in chlorophyll content with increasing CO 2 levels has been reported by Porteaus et al. [84] in wheat. The correlations revealed ( Table 2) that total chlorophyll were significantly ( ≤ 0.05) and negatively related to total phenolics ( 2 = −0.892), flavonoids ( 2 = −0.899), anthocyanins ( 2 = −0.867), and ascorbic acid ( 2 = −0.846). Competition between secondary metabolites and chlorophyll content fitted well with the prediction of the protein competition model (PCM) and indicated that the secondary metabolites content was controlled by the competition between protein and secondary metabolites biosynthesis pathway and its metabolites regulation [85]. The negative relationship between the secondary metabolites and chlorophyll was a sign of gradual switch of investment from protein to polyphenols production [86]. The present results indicate that under high CO 2 and low irradiance the production of total chlorophyll content was downregulated in L. pumila that also indicated the upregulation on production of secondary metabolites.

Leaf Gas Exchange
Properties. The photosynthesis rate, stomatal conductance, and maximum efficiency of photosystem II ( V / ratio) were influenced by CO 2 levels ( ≤ 0.05; Figure 3). No irradiance and interaction effects were observed. Leaf net photosynthesis, stomatal conductance, and V / rate increased with increasing CO 2 from 400 to 1200 mol/mol. The highest net photosynthesis was obtained in L. pumila exposed to 1200 mol/mol CO 2 (11.45 mol/m 2 /s), followed by 800 mol/mol (8.66 mol/m 2 /s) and 400 mol/mol (4.44 mol/m 2 /s; Figure 3(a)). A similar trend was observed for stomatal conductance, where the stomatal conductance for 1200, 800, and 400 mol/mol CO 2 was 30.15, 25.12, and 12.40 mmol/m 2 /s, respectively (Figure 3(b)). The V / ratio was 16 and 25% higher in treatments with 800 and 1200 mol/mol CO 2 , respectively, compared to the ambient CO 2 level (Figure 3(c)). These findings exhibit the importance of CO 2 in further enhancing properties of leaf gas exchange and photosystem II efficiency of L. pumila plants. The increase in photosynthesis and photosystem II efficiency in the present work could have stimulated the production of plant secondary metabolites, as suggested by the positive correlation coefficients in Table 2 between photosynthesis and secondary metabolites of total phenolics ( 2 = 0.987; ≤ 0.05) and flavonoids ( 2 = 0.786; ≤ 0.05), and also between V / and total phenolics ( 2 = 0.887; ≤ 0.05) and flavonoids ( 2 = 0.846; ≤ 0.05). Thus, an increase in photosynthetic and photochemical efficiency had increased the shikimic acid pathway and enhanced the production of plant secondary metabolites, and this was in turn due to an increase in the concentration of soluble sugars [87,88]. Some studies had reported that when production of secondary metabolites increased, the photosynthesis would decrease due to feedback control of the secondary metabolites production [89][90][91]; however, such an effect was not observed in the present study.
3.8. DPPH Radical Scavenging Activity. DPPH activity was influenced by the interaction between elevated CO 2 and irradiance levels ( ≤ 0.05; Figure 4). The highest DPPH antioxidant activity was recorded with 1200 mol/mol CO 2 followed by 800 mol/mol CO 2 , and it was lowest in the treatment with 400 mol/mol CO 2 . As the level of irradiance was reduced from 900 to 225 mol/m 2 /s, the DPPH activity was increased. At 320 g/mL, the DPPH antioxidant activity recorded the highest inhibition value when exposed to 1200 mol/mol CO 2 + 225 mol/m 2 /s irradiance (65.44%), followed by the 800 mol/mol + 225 mol/m 2 /s (63.21%), 400 mol/mol + 225 mol/m 2 /s (60.21%), 1200 mol/mol + 500 mol/m 2 /s (57.21%), 800 mol/mol + 500 mol/m 2 /s (56.23%), 400 mol/mol + 500 mol/m 2 /s (52.11%), 1200 mol/mol + 625 mol/m 2 /s (49.21%), 800 mol/mol + 625 mol/m 2 /s (48.32%), 400 mol/mol + 625 mol/m 2 /s (45.27%), 1200 mol/mol + 900 mol/m 2 /s (40.21%), 800 mol/mol + 900 mol/m 2 /s (39.21%), and the least in the 400 mol/mol CO 2 + 900 mol/m 2 /s irradiance (32.21%) treatment. The results of the current work also showed that the DPPH radical scavenging ability of the plant extracts at 320 g/mL was less than those of the reference antioxidants butylated hydroxytoluene (BHT, 66.41%) and -tocopherol (80.38%). This study showed that L. pumila methanolic extract had good free radical scavenging activity, and, hence, it can be used as a radical scavenging agent acting possibly as a primary antioxidant. The results also imply that exposure to high CO 2 concentrations under low irradiance could significantly enhance the DPPH radical scavenging activity of the medicinal plant. It must be noted that the DPPH assay principally measures the activity of water-soluble antioxidants [92]. Results of the current work suggest that high CO 2 supply under low irradiance is advantageous to L. pumila in the improvement of antioxidant activity of the water-soluble antioxidants. In our previous study, besides phenolics and flavonoid compounds, other water-soluble antioxidants of the extracts such as ascorbic acid and anthocyanins were suggested to exert additive effects on DPPH radical scavenging activity. This was evident from the current results where DPPH had a significant positive correlationship with total phenolics ( 2 = 0.923; ≤ 0.05), total flavonoid Bars represent standard errors of differences between means (SEM). that the increase in secondary metabolites production under high CO 2 and low irradiance increases the activity of DPPH.
3.9. Lipid Peroxidation Activity. Malondialdehyde (MDA) is a highly reactive three carbon dialdehyde by-product of polyunsaturated fatty acid peroxidation and arachidonic acid metabolism. The production of MDA was influenced by CO 2 levels imposed onto irradiances ( ≤ 0.05; Figure 5). It was observed that as the level of CO 2 was enhanced from 400 to 1200 mol/mol CO 2 the production of MDA was decreased. The MDA content was also found to decrease with increasing irradiance (from 900 to 225 mol/m 2 /s). This suggests that as CO 2 levels and irradiance were reduced, the oxidative stress in L. pumila cells and tissues was decreased, thus implying occurrence of lipid peroxidation in L. pumila under low CO 2 and high irradiance. Yu et al. [93] working on P. subcordiformis had also observed that the reduction in MDA at high concentrations of CO 2 was due to reduced photorespiration that simultaneously reduced photoreduction of dioxygen. In this case, fewer electrons were transported to dioxygen during photosynthesis and in effect, reducing the potential damage of active oxygen to the membrane system (MDA). The MDA production had established significant negative correlations with total phenolics ( 2 = 0.876; ≤ 0.05) and total flavonoids ( 2 = 0.821; ≤ 0.05), indicating that reduction in MDA might be involved in the upregulation of secondary metabolite production under high CO 2 and low irradiance in L. pumila ( Table 2). The formation of MDA was considered as a measure of lipid peroxidation that was induced by a high stress level [92]. MDA, a decomposition product of polyunsaturated fatty acid hydroperoxides, has been utilized very often as a suitable biomarker for oxidative stress. Usually the increase in lipid peroxidation is simultaneously accompanied by an increase in hydrogen peroxide levels, but in the present study hydrogen peroxide content was not measured. Hydrogen peroxide may function as a signal for the induction of plant defense systems and this could enhance secondary metabolite production [94]. Hence, the results of the present study suggest that oxidative stress was reduced and was not a prerequisite for secondary metabolite synthesis in L. pumila under high CO 2 and low irradiance.

Conclusions
The present work has demonstrated that high levels of CO 2 and low irradiance are able to change the synthesis and allocation of secondary metabolites in Labisia pumila. The production of total phenolics, flavonoids, anthocyanins, and ascorbic acid was enhanced as CO 2 levels increased from 400 to 1200 mol/mol CO 2 and irradiance was reduced from 900 to 225 mol/m 2 /s. The increase in the production of secondary metabolites was followed by an increase in the soluble sugar content. The DPPH antioxidant activity was found to be the highest at 1200 mol/mol CO 2 + 225 mol/m 2 /s irradiance and the lowest under 400 mol/mol CO 2 + 900 mol/m 2 /s irradiance. Elevated CO 2 significantly improved the leaf gas exchange properties of Labisia pumila. The high production of secondary metabolites under high CO 2 is attributed to increased PAL activity under these conditions. MDA had a significant negative correlation with production of secondary metabolites, indicating that reduction in lipid peroxidation under high CO 2 and low irradiance in L. pumila might improve secondary metabolites production.