Production of Flavonoids in Pegaga (Centella Asiatica L. Urban) Cell Suspension Culture

Centella asiatica L. Urban (Umbelliferea), locally known as ‘Pegaga’ was an herbal plant that had been used in traditional medicine in Asia for many centuries. Its medicinal values are attributed to the presence of flavonoid compounds. Since there was still no information available on the flavonoid production in cultured tissues, studied have been carried out in evaluating the distribution of the flavonoid content particularly kaempherol, quercertin, luteolin and apigenin in intact plants of the four accessions of C. asiatica collected several locations in Malaysia as well as in callus and cell suspension cultures. The total flavonoid content has been determined by using spectrophotometry methods. The flavonoid compounds present in the cell cultures have been analyzed with thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) techniques. Results obtained from the studies revealed that the four accessions of C. asiatica which showed distinctive differentiation in morphological characteristic were differed in the biochemical constituents especially in their flavonoid contents. The flavonoid constituents were detected in the range of 1.93 ± 0.094 to 8.99 ± 0.346 mg/g dry weight in the whole plant tissues. Flavonoids were also successfully detected in callus (0.98 ± 0.097 to 2.46 ± 0.021 mg/g dry weight) and cell suspension culture (0.67 ± 0.056 to 0.89 ± 0.044 mg/g dry weight). The flavonoid content found in cultured tissues were lower that that produced in the intact plant tissues. In the TLC analysis for the leaf tissues of intact plant, the possible of flavonoid compound might presented were kaempherol, naringerin, luteolin, (+)-catechin and rutin. Similar results were also been observed in callus, cell suspension and cell suspension media in the TLC analysis. Further investigation using HPLC showed that leaf tissues of intact plant accession UPM03 has rutin, luteolin, quercertin and kaempherol while others accession only have rutin or/and kaempherol. An additional flavonoid compound called apigenin was detected in callus and cell suspension. As a result, culture conditions such as different cell size aggregations, pH values, light, inoculum sizes as well as interaction between plant growth regulators have been assessed to optimize and enhance the selected flavonoid production in the chosen cell suspension cultures. Cell suspension UPM03 in aggregation size of 250-500 μm with the highest cell growth rate (0.69 g fresh weight/day) was suitable to produce flavonoid in 100 mL shake flask of C. asiatica suspension cells. The cells grow better and produce more flavonoid (3.66 ± 0.13 mg/g dry weight) under the initial pH of 5.7 with the presence of light when supplemented with 3 mg/L 2,4-D and 1 mg/L kinetin. In addition, the responses of cell to selected elicitors and precursors have also been investigated. The biosynthesis of flavonoid was elicited by the addition of 1mg/L of both yeast extract (7.32 ± 0.17 mg/g dry weight) and salicylic acid (7.11 ± 0.16 mg/g dry weight). Elicition with 400 μM methyl jasmonate have increased flavonoid content to 7.82 ± 0.2 mg/g dry weight. However, the addition of 1mg/L casein hydrolysate (4.91 ± 0.15 mg/g dry weight) and chitosan (4.29 ± 0.13 mg/g dry weight) have no significant effect if compared to the control culture (5.08 ± 0.22 mg/g dry weight). The usage of 60 mg/L phenylalanine and 40 mg/L tyrosine have resulted in 14-fold and 3-fold increase in production of flavonoid, respectively. Further study on the synergistic effect of combination of precursor (60 mg/L phenylalnine) and elicitor (400 μM) showed that the cell suspension UPM03 not only produce the highest flavonoid content in cell (67.07 ± 6.47 mg/g DW) but also in the media (7.73 ± 0.12 mg/g). Thus, it was proven that the synergistic bioprocess was a very useful strategy to enhance the flavonoid production particularly luteolin in in vitro cultures of C. asiatica

Analysis of the comparative study on flower pigments with phenylalanine ammonia-lyase activity in orchid plants

Background: Orchidaceae plants have been one of the most important industrial products in the agricultural industry around the world. Since the flower petals have eye-catching pigments, they can be used as cut flowers in addition to being potted flowers. Objective: The key pigments found in orchid flower petals were studied, as well as their relationships to phenylalanine ammonia-lyase (PAL) activity. Results: Total anthocyanin content of six different orchids’ petals was determined specthrophotometrically and the value ranged from 0 mg/g (in Dendrobium Shavin white) to 2.128 mg/g (in Mokara Aranda). The petals with vivid colour have a high amount of anthocyanin content, while, they have a high amount of chlorophyll content for those with pale colour. Total anthocyanin content was found to be the highest when compare to β-carotene and chlorophyll content. PAL behaviour was found to be significantly positive associated with anthocyanin content in correlation analysis. Conclusion: The results indicate the potential for PAL enzyme as a biomarker for flower colour in orchids

Comparison on phytochemical constituents in the patchouli oil of in vitro and ex vitro pogostemon cablin leaves

Pogostemon cablin (patchouli) is a medicinal herb well known for its essential oil derived from the leaves. Patchouli oil shows excellent base note in fragrance industries for its fixatives properties and its patchouli alcohol (patchoulol) is used as quality indicator for its oil. However, the P. cablin is the only commercial source of patchoulol and cannot be obtained synthetically in the laboratory. Higher demand in the production of its essential oil gave a significant contribution for in vitro grown P.cablin to meet the market supply for industries. Hence, in this study, the essential oil in both in vitro and ex vitro P.cablin were extracted from its leaves by means of hydrodistillation method and its phytochemical constituents were identified and compared using Gas Chromatography Mass Spectrometry. The yield and quality of its essential oil from both in vitro and ex vitro P.cablin’s leaves were investigated. In vitro patchouli essential oil extraction gaves higher yield (40 ml) than the ex vitro patchouli essential oil (26 ml) under similar condition for hydrodistillation. Six major components were identified through GC-MS and was compared between two samples which are β- patchoulene, Caryophyllene, α- guaiene, α- cedrene, α- bulnesene and Patchouli alcohol. The patchoulol, which is the main constituents that is important in fixative had doubled (42.18 %) in the in vitro P.cablin essential oil compared to ex vitro (29.24%). This finding was reflected based on the peak area percentage of each substance through GC-MS. Other constituents in the in vitro P.cablin were found still competitive to the ex vitro in slightly lower values. Overall, in vitro P.cablin showed higher yield and quality compared to the ex vitro grown P.cablin

Growth media derived from solid waste for orchid Dendrobium kingianum culture

Food waste and diaper waste contribute to top three ranking solid wastes in Malaysia. Many studies show that urine and food waste can be used as plant fertilizer but less attention has been given to the recycling of used diaper. Therefore, this study was conducted to formulate Dendrobium kingianum growing media by utilizing diaper waste and selected food waste: banana peels, coffee waste and eggshells. The chemical composition in diaper waste and food waste were determined by using ICP-OES method. Biochar derived from oil palm waste, sugarcane and sawdust were tested to reduce and neutralize the high ammonium content from diaper waste. Biochar derived from oil palm waste showed the best reduction of ammonium in diaper waste. Survival and growth performance of Dendrobium kingianum was observed and recorded. Results show that orchid in diaper formulated growth media T3 showed the best adaptation and growth performance with highest percentage of healthy leaves and number of absorption roots within 3 weeks observation period. These results revealed that diaper waste could be recycled as orchid growing media by formulating with food waste and biochar

Antioxidants and antibacterial properties of extracts from leaves and stem of pink flower Impatiens walleriana

Impatiens walleriana is a vibrant and flowering plant sources cherished for its antioxidants and antimicrobial properties. This research aimed to evaluate the impact of three different drying treatments (microwave drying, oven drying, and air drying) on the antioxidant compounds of leaves and stems of pink flower I. walleriana. Besides, antibacterial efficacy of leaves extract against Gram-positive and Gram-negative bacteria were also evaluated. Microwave-dried leaves extract exhibited high phenolic (6.358±0.18 mg GAE/100g), flavonoid (0.528±0.005 mg QAE/100g) and caffeoylquinic acid contents (0.988±0.012 mg CQCA/100g), followed by oven-dried and air dried. Stem extracts of I. walleriana showed relatively less phenolic compound as compared to leaves extract probably due to the plant’s defense strategy against environmental stress. Besides, the leaves extract was chosen for antibacterial activity using Kirby-Bauer disc-diffusion method. The results revealed that I. walleriana able to suppress growth of both Gram-positive and Gram-negative bacteria. Nevertheless, I. walleriana had stronger inhibitory effect against Escherichia coli while least inhibitory effect against Pseudomonas aeruginosa, with inhibition zone of 6.6 mm and 1 mm, respectively. The antibacterial effect of I. walleriana also found superior to streptomycin especially against Gram-negative bacteria. The antibacterial effect of I. walleriana against E. coli was found to be two times stronger than the synthetic antibiotics. Hence, this indicates that I. walleriana had a good potential as an alternative source of antioxidant and antimicrobial and replacing the synthetic additives in current market

Preliminary study of pleurotus ostreatus (Jacq.) P. Kumm. spent mushroom compost as nutrient supplement on the shoot induction of fig tissue culture

Spent mushroom compost is identified as waste substrate. As the mushroom agriculture industry is growing rapidly, the production also increases and contributes to large amounts of spent mushroom compost annually. The conversion of spent mushroom compost as waste to wealth is a great concept that can give value to agricultural industry and solid waste management of mushroom cultivation. The use of SMC as nurient supplement may reduce the production cost for in vitro culture of fig plant. This research is to study the use of Pleurotus ostreatus (Jacq.) P. Kumm. spent mushroom compost (SMC) as a nutrient supplement of the media in shoot induction of fig plant. Nodal segment of fig’s stem was cultured in MS media supplemented with different concentrations of SMC. With presence of SMC, 10% SMC have the highest shoot proliferation (2.167 ± 1.169) and length of shoot (0.154 ± 0.060 cm). The study revealed that in presence of 10% of SMC could produce new shoots at a frequency which was comparable to the control (p<0.05; n=3; Tukey’s multiple range test). Among the different media combinations of plant growth regulators with SMC, 15 μM Kinetin with 10% SMC is the most effective treatment which gave maximum number of shoot proliferation (3.00 ± 1.27). 30 μM IAA with 10% SMC media have the highest leaves regenerated which were 1.33 ± 0.84. In conclusion, the SMC added in media for plant tissue culture had positive effect on shoot induction thus highlighted the potential of waste substrate of SMC to act as nutrient supplement for plant tissue culture

Effect of Gibberellic Acid and Eggshell on Hylocereus polyrhizus

Dragon fruit (Hylocereus polyrhizus) is a tropical fruit. Recently, it has gained interest from the public due to its potential beneficial effects on health. The acclimatization of micropropagated Hylocereus polyrhizus depends on the application of gibberellic acid (GA3) to increase plant growth. Eggshells are waste materials from industrial sectors, and they are composed of calcium source that is vital for the development of plant shoots and root. The objective of this research is to investigate the effect of different concentrations of GA3 and eggshell either added individually or in combination on the growth of shoot length and shoot diameter of H. polyrhizus. The result showed the shoot length of the H. polyrhizus increased by approximately 54.69%, from 0.64 ± 0.13 cm to 0.99 ± 0.26 cm, as the concentration of GA3 increased from 0 ppm to 10 ppm. Furthermore, this finding also reported that with eggshells, GA3 showed an adverse effect on the development of shoot diameter. The growth of shoot length and shoot diameter with the addition of eggshell was different, perhaps due to the gibberellic acid affecting the shoot length but not the shoot diameter. Generally, the growth of shoot length and shoot diameter with eggshells was higher in comparison with those without eggshells. With that, we can prove that eggshell is a good additive to promote the growth of H. polyrhizus

Effect of gibberellic acid and eggshell on Hylocereus polyrhizus

Dragon fruit (Hylocereus polyrhizus) is a tropical fruit. Recently, it has gained interest from the public due to its potential beneficial effects on health. The acclimatization of micropropagated Hylocereus polyrhizus depends on the application of gibberellic acid (GA3 ) to increase plant growth. Eggshells are waste materials from industrial sectors, and they are composed of calcium source that is vital for the development of plant shoots and root. The objective of this research is to investigate the effect of different concentrations of GA3 and eggshell either added individually or in combination on the growth of shoot length and shoot diameter of H. polyrhizus. The result showed the shoot length of the H. polyrhizus increased by approximately 54.69%, from 0.64 ± 0.13 cm to 0.99 ± 0.26 cm, as the concentration of GA3 increased from 0 ppm to 10 ppm. Furthermore, this finding also reported that with eggshells, GA3 showed an adverse effect on the development of shoot diameter. The growth of shoot length and shoot diameter with the addition of eggshell was different, perhaps due to the gibberellic acid affecting the shoot length but not the shoot diameter. Generally, the growth of shoot length and shoot diameter with eggshells was higher in comparison with those without eggshells. With that, we can prove that eggshell is a good additive to promote the growth of H. polyrhizus

Poly(hydroxamic acid) functionalized copper catalyzed C–N bond formation reactions

Highly active poly(hydroxamic acid) functionalized copper catalysts were synthesized by the surface modification of khaya cellulose through graft copolymerization and subsequent hydroximation processes. The prepared catalysts were well characterized by FTIR, FESEM, HRTEM, ICP-AES, UV-vis and XPS analyses. The supported catalysts effectively promoted C–N bond formation reactions and provided excellent yields of the corresponding products under mild reaction conditions. The catalysts were easy to recover from the reaction mixture and were reused several times without any significant loss of their catalytic activity

Valorisation of biomass and diaper waste into a sustainable production of the medical mushroom Lingzhi Ganoderma lucidum

Global solid waste is expected to increase by at least 70% annually until year 2050. The mixture of solid waste including food waste from food industry and domestic diaper waste in landfills is causing environmental and human health issues. Nevertheless, food and diaper waste containing high lignocellulose can easily degrade using lignocellulolytic enzymes thereby converted into energy for the development and growth of mushroom. Therefore, this study explores the potential of recycling biomass waste from coffee ground, banana, eggshell, tea waste, sugarcane bagasse and sawdust and diaper waste as raw material for Lingzhi mushroom (Ganoderma lucidum) cultivation. Using 2% of diaper core with sawdust biowaste leading to the fastest 100% mushroom mycelium spreading completed in one month. The highest production yield is 71.45 g mushroom; this represents about 36% production biological efficiency compared to only 21% as in commercial substrate. The high mushroom substrate reduction of 73% reflect the valorisation of landfill waste. The metabolomics profiling showed that the Lingzhi mushroom produced is of high quality with a high content of triterpene being the bioactive compounds that are medically important for treating assorted disease and used as health supplement. In conclusion, our study proposed a potential resource management towards zero-waste and circular bioeconomy for high profitable mushroom cultivation