Correlation of Leaf NPK and Leaf Pigments of Coleus atropurpureus L. Benth during Vegetative and Generative Phases

Coleus atropurpureus L. Benth is a annual plant that has a distinctive leaf aroma and bitter taste. C. atropurpureus leaves contain phenolic compounds and antioxidants that can capture free radicals; free radicals play an important role in preventing various human diseases. A study was conducted to determine the correlation between leaf position (1st to 4th) at the vegetative and generative phases with leaf pigments, N, P, K, and total fl avonoid concentrations. The results showed that leaf chlorophyll a, chlorophyll b, total chlorophyll, carotenoid, anthocyanin, nitrogen, and total fl avonoids were higher in the vegetative phase. Therefore, C. atropurpureus is better harvested in the vegetative phase, and the 2nd leaf position can be used as indicator for N, K, pigments and total fl avonoid content.


Introduction
Indonesians are well known to use medicinal plants as a treatment for health problem. One of the medicinal plants that has been widely used is jawer kotok (Coleus atropurpureus L. Benth). Coleus atropurpureus an annual herbaceous plant which can grow up to 100 cm tall (Wiart, 2006). C. atropurpureus grows upright and has branches with square rod shapes and jagged leaf edges (Figure 1). The length of the leaf stalk can reach 7.5 cm with an oval leaf shape 5-10 cm long. Flowers are purplish, white, or bluish on the terminal stalks with a shape like nails arranged 10-20 cm long. The colorful C. atropurpureus leaves make the plants to be used as ornamentals. The colors of the leaves diff er with diff erent types and cultivars. According to Osman (2013) Coleus blumei with purplish red and red to dark red leaves contains high phenolic levels, which indicates it is potential as medicinal plant. The genus of Coleus belongs to Lamiaceae or Labiatae family; many species from this family can be used in traditional medicine. C. atropurpureus leaves are usually used to overcome dermatitis, post partum, abdominal pain, coughing and muscles pains, particularly by people in West Java (Roosita et al., 2008). In addition, its uses to cure bronchitis, asthma, angina, digestive disorders, animal bites (Suva et al., 2016), for dengue fever and malaria drugs in Philippines (Gascon, 2011), for hemorrhoids, antioxidants and anti-tuberculosis (Ahmad and Massi, 2014) have been reported. C. atropurpureus contain saponins, fl avonoids, alkaloids, polyphenols, quercetin and essential oils (Moektiwardoyo et al., 2011). The compounds which have antioxidant properties can capture free radicals and play an important role in preventing various chronic diseases (Gross, 2004). One of antioxidants is fl avonoid, which has been reported to inhibit proliferation of SP-C1 tongue cancer cells (Achmad et al., 2014). This study aims to determine whether or not diff erent leaf positions contain diff erent levels of secondary metabolite. The correlation between the position of leaves (1 st to 4 th ) and the leaf nutrient content (NPK) and secondary metabolites during the vegetative and generative phases were also determined.

Plant Materials
Coleus atropurpureus leaves used are 5 MAP (months after planting) which planted in IPB University experimental station, Bogor, West Java, Indonesia. The fully open leaves from the 1 st to the 4 th position from the shoot tip were collected for analysis (fi gure2).

Leaf Nutrient and Pigment Analysis
Analysis of nutrient levels of N, P, and K was carried out in the Testing Laboratory of the Department of Agronomy and Horticulture, IPB University. Total N analysis used Kjeldahl method, P was determined using a UV-1800 UV-VIS spectrophotometer, and K analysis used AAS (Atomic Absorption Spectrophotometry).
Analysis of chlorophyll, carotenoid, anthocyanin and total fl avonoids was carried out in the Postharvest Laboratory of the Department of Agronomy and Horticulture, IPB University. Analysis of chlorophyll, carotenoid and anthocyanin used Sims and Gamon (2002) method with the following protocol: 200 mg of fresh leaves were weighed, mashed with 2 ml acetic solution, centrifuged (6000 rpm, 10 minutes), 1 ml supernatant was added 3 ml of acetic acid then measured with wavelengths of 470, 537, 647 and 663 nm. The total level of fl avonoids was analyzed using the method of Chang et al (2002): 10 mg quercetin was dissolved in 80% ethanol then diluted to 25, 50 and 100 μg/ml. The diluted 0.5 ml solution was mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of potassium acetate 1M and 2.8 ml of distilled water. This solution was incubated at room temperature for 30 minutes, absorbance was measured at 415 nm. Blanks were made by replacing the amount of aluminum chloride with distilled water. An extract in 0.5 ml of ethanol was reacted with aluminum chloride to determine the fl avonoid concentration. According to Aziz (2015) production of bioactive compounds can be carried out with the following method: bioactive compound production = leaf dry weight (g per plant) x concentration of leaf bioactive compounds (%).

Data Analysis
Data were analyzed using R for t-student test and SAS 9.4 for Pearson correlation test.

Leaf Pigment at The Vegetative and Generative Phase
The leaf clorophyll a and clorophyll b at the vegetative phases is in Figure 3, and the highest content are in the 3 rd leaf. However, chlorophyll a and chlorophyll b levels in each leaf position showed fl uctuating results. Leaves at positions 1 st , 2 nd , and 4 th had 21.61%, 23.80%, and 22.37% lower chlorophyll a than the 3 rd leaf, whereas leaves at the 1 st , 2 nd , and 4 th had 20.16%, 22.62%, and 20.98% lower chlorophyll b than the 3 rd leaf. Leaves at position 1 st , 2 nd , and 4 th have total chlorophyll levels which were 21.14%, 23.40%, and 21.92% lower than the 3 rd leaf. The carotenoid levels in the 1 st and 3 rd leaves were not signifi cantly diff erent from each other, but were signifi cantly higher than the 2 nd and 4 th leaves. The 2 nd leaf showed a markedly lower carotenoid level of 12.97% and the 4 th leaf showed a markedly lower carotenoid level of from the 1 st leaf position.
Intan Annisa Respita, Sandra Arifi n Aziz, Ani Kurniawati The level of anthocyanin in the 1 st and 2 nd leaf was not signifi cantly diff erent, but was signifi cantly higher than 3 rd and 4 th leaves. At the 4 th leaf position there was an increasing anthocyanin levels of 10.81%. The level of leaf pigment at the generative phase is described in Figure 4, and it shows fl uctuating results. Chlorophyll a and carotenoid levels showed the highest results at the 4 th leaf position. The 3 rd position leaves have anthocyanin levels that are signifi cantly diff erent from the 1 st , 3 rd and 4 th leaf.

Leaf NPK Levels at The Vegetative and Generative Phase
The levels of N and P at the vegetative and generative phases were not signifi cantly diff erent from those in the generative stage ( Figure 5) but N levels in the vegetative phase was 0.22% higher. The highest P at the vegetative and generative phase was found in the 1 st leaf. Nutrient K from the 1 st and 2 nd leaf at the generative phase is greater than the vegetative phase. N levels of the 2 nd and 3 rd leaves, as well as the 3 rd and the 4 th leaves were not signifi cantly diff erent. Leaf P decreases along the leaf position and this occurs in the vegetative and the generative phases. In the generative phase, the levels of P of the 1 st , 2 nd , 3 rd leaf was 0.08%, 0.03%, and 0.003% higher than the 4 th leaf. The 1 st and 2 nd leaves had higher potassium levels than the 3 rd and 4 th leaves, but the levels of the 3 rd and 4 th leaves were similar. In the vegetative phase, the levels of n decreased with from the 1 st to 4 th leaves, the levels of the 1 st , 2 nd , 3 rd were 0.82%, 0.30%, and 0.24% higher than in the 4 th leaf. Similar trend was noticed in the generative phase, where the levels of N of the 1 st , 2 nd , 3 rd leaves were 0.51%, 0.24%, and 0.07% higher the levels of N of the 4 th leaf. In addition, the nutrient P levels in the generative phase was higher 0.27% compared to the vegetative phase.
The fi rst leaf had the highest K level in the vegetative and generative phase, and K levels was 2.40% higher in the generative compared to the vegetative phase. K level of the 1 st , 2 nd , and 3 rd leaves were 0.27%, 0.13%, and 0.18% higher than K level of 4 th leaf. In the generative phase, there was a decreasing K levels from the position of the 1 st to 4 th leaves; K levels of the 1 st , 2 nd , 3 rd leaves were 1.48%, 0.90%, and 0.03% higher than the 4 th leaf.

Total Flavonoid Concentration and Total Flavonoid Content
The highest total fl avonoids concentration in the vegetative and generative phases were found at the 1 st leaf ( Figure 6). In the vegetative phase, total fl avonoids concentration decreased according to the position 1 st to 4 th leaves; the levels of the 1 st , 2 nd , and 3 rd leaves were 49.00%, 16.50%, and 7.76% higher than the 4 th leaf. Similar trend was noticed during the generative phase; total fl avonoids of the 1 st , 2 nd , and 3 rd leaves were at 25.58%, 5.92%, and 3.23% higher than the 4 th leaf.
There was a fl uctuating total fl avonoid content in the vegetative phase from 1 st to 4 th leaf but the 4 th leaf had highest total fl avonoid content in both vegetative and generative phase (Figure 7). The total fl avonoids content was obtained from multiplication of total fl avonoids concentration to leaf dry weight. The 3 rd and 4 th leaves were larger and heavier than other leaves. The total fl avonoids content of the 1 st , 2 nd , and 3 th leaves were 0.39%, 0.77% and 0.25% lower than the 4 th leaf in vegetative phase. Total fl avonoid content decreased from the 1 st to 4 th leaves at the generative phase, which was 0.41%, 0.14%, and 0.17% lower   Table 1 showed the correlation between leaf NPK with leaf chlorophyll a, chlorophyll b, total chlorophyll, anthocyanin, carotenoids and total fl avonoid of the fi rst to fourth leaves at the vegetative phase. N levels were positively correlated with chlorophyll a, total chlorophyll, carotenoid levels, at the 1 st , 2 nd and 3 rd leaves positions, and positively correlated with anthocyanin and total fl avonoid levels and levels in the 1 st , 2 nd , and 4 th leaf positions. In the 2 nd , 3 rd , and 4 th leaf N has a positive correlation with chlorophyll b. In the 1 st , 2 nd , and 3 rd leaf P has a positive correlation with chlorophyll a, chlorophyll b, carotenoids, total chlorophyll. In the 1 st , 2 nd and 3 rd leaf K was positively correlated with chlorophyll a, chlorophyll b, carotenoids, and positively correlated with anthocyanin in the 2 nd and 4 th leaf. A positive correlation between K and total content of fl avonoids were recorded in all leaf positions. Table 2 showed the correlation at the generative phase. N was positively correlated with levels of chlorophyll a, chlorophyll b, carotenoid, and total  chlorophyll of the 4 th leaf , but positively correlated with anthocyanin of the 1 st , 2 nd , and 3 rd leaf Leaf N also positively correlated with levels and the total content of fl avonoids in the 1 st and 3 rd leaf. Leaf P has a positive correlation with the levels of chlorophyll a, chlorophyll b, total chlorophyll in the position of the 2 nd and 3 rd leaves, while with the carotenoids in the position of the 3 rd leaf. P nutrient content was positively correlated with anthocyanin at the 4 th leaf position, while positively correlated with the levels and total fl avonoid content in the 1 st and 2 nd leaf positions. Nutrient content K was positively correlated with levels of chlorophyll a, chlorophyll b at the 2 nd and 3 rd leaf position, while positively correlated with carotenoids at the 1 st , 2 nd , and 3 rd leaf positions. K nutrient levels were positively correlated with anthocyanin in leaf position 2 nd and 4 th , while positively correlated with total chlorophyll at the 3 rd leaf position. Nutrient K has a positive correlation with the level and total content of fl avonoids in the 1 st leaf position.

Discussion
The presence of pigments in the form of chlorophyll a and chlorophyll b play a role in absorbing solar radiation during photosynthesis. Photochemical processes can act to release electrons, so that light energy is converted into chemical energy. This level of chlorophyll can aff ect photosynthesis (Richardson et al., 2002). Photosynthesis rates are directly proportional to the concentration of photosynthetic pigments. Therefore, the position of leaf that close to apex will have a low pigment concentration. Marschner (2012) reported that in mature leaves ~ 15% of the volume of all cells is occupied by chloroplasts, cytoplasm and cell walls while the remainder is by vacuoles (85%). Therefore, in mature leaves the levels of bioactive compounds had higher levels of anthocyanin, chlorophyll and fl avonoids.
In croton concentration of chlorophyll a, chlorophyll b and total chlorophyll were found to be higher in older leaves, and the carotenoid level in the generative phase is lower than the vegetative phase (Gogahu et al., 2016). Research by Tjhia et al., (2018) reported that the levels of carotenoid in Vernonia amygdalina Del at the generative phase was higher than that the vegetative phase. Chlorophyll activity plays a role in the process of organogenesis which can aff ect the generative phase (Simova et al., 2001). All pigment levels in the vegetative phase are higher than the generative phase. Similarly for leaf weights. The level of anthocyanin in the generative phase decreases because in the early phase of this development anthocyanins are required to carry out photoprotection. Anthocyanin is needed because at this stage, chlorophyll cannot develop properly to absorb excessive sunlight. Decreased anthocyanin levels in the generative phase indicate that anthocyanin function might have been replaced by the presence of carotenoids (Hughes et al., 2007).
Higher nitrogen levels aff ect chlorophyll levels in each phase because nitrogen is one of the important component of chlorophyll (Marschner, 2012). Photosynthesis has a positive relationship with the growth process of all parts of the plant (Diem et al.,   . In cotton, increasing photosynthesis rate is aff ected by increasing CO 2 uptake, while CO 2 uptake is aff ected by ion concentration including K + , NO 3 -, PO 4 - (Longstreth et al., 1980). With a decrease in the concentration of nitrogen and phosphorus, plants experience stress and will respond with an increase in anthocyanin. Anthocyanin functions as an antioxidant which will free radicals when stress occurs (Scott, 1999).
Plants have young leaf parts that act as sinks and adult leaves act as sources (Marschner, 2012). The position of the leaf can indicate the direction of the nutrient translocation path and the water for the plant sink towards the source. Usually, the young leaves have higher nutrient levels and become strong sinks. Nitrogen concentration in both vegetative and generative phases decreased from the 1 st to 4 th leaf position, and potassium concentration decreased in the generative phase but the level is still high.
A decrease in nitrogen in the tissue causes a decrease in protein and chlorophyll content. Munawar (2011) reported that nitrogen plays an important role for plants. Nitrogen is involved in the synthesis and transfer of energy, plant growth, improves leaf quality, seed and fruit production, and plays a role in the preparation of amino acids, proteins, chlorophyll, nucleic acids and co-enzymes.
There is a relationship between the availability of nutrients and the accumulation of fl avonols; reduction of nitrogen will increase fl avonol levels. But in the availability of high nitrogen, phosphate reduction can facilitate the formation of fl avonols. Nitrogen defi ciency in tomato plants produces accumulation of fl avonol in adult leaves. Conversely, when phosphorus defi ciency causes accumulation of fl avonol at the beginning of fruit ripening (Stewart et al., 2001). Karimuna et al. (2015) reported the total fl avonoids of Murraya paniculata leaves were negatively correlated with potassium at diff erent leaf ages and positions. Potassium concentrations can be categorized very high (3.59-4.10%), phosphorus was high (0.28-0.29%) or very high (0.33-0.35%).

Conclusion
Coleus atropurpureus leaf chlorophyll a, chlorophyll b, carotenoids, anthocyanin, total chlorophyll, nitrogen, total levels of fl avonoids and total fl avonoid content at the vegetative phase were higher than those at generative phase. The indicator leaf at the vegetative phase which have positive correlations with pigment and total fl avonoids are the second leaf, whereas all leaf positions at the generative phase do not have correlations with the leaf NPK, leaf pigments, and leaf total fl avonoids.