D-optimal Design Optimization of Solvent Mixture for Flavonoid Extraction from Phalaenopsis Leaves with Antioxidant Activity.

Aims: This study aimed to optimize the extraction of flavonoids and antioxidants from Phalaenopsis leaves by using solvent mixtures. Method: The total flavonoid content (TFC) and antioxidant activity were evaluated using the colorimetric method and ferric-reducing antioxidant power (FRAP), respectively. Maceration extracts from fresh leaves were used for the analysis. The study used the Design Expert 13.0 program to optimize the solvents (water, acetone, and methanol) and their combined ratio. Result: The results showed that 100% acetone was the best solvent for both responses, with a desirability value of 0.884, TFC of 0.434 mg QE/g fresh weight (FW) and FRAP of 713.53 µmol TE/g FW. Screening of the most potent Phalaenopsis genotypes for obtaining the most active leaf extract showed that P. amboinensis and P. pantherina were the best genotypes for TFC (0.786-0.797 mg QE/g FW) and FRAP activity (862.25-891.48 µmol TE/g FW). Conclusion: This study demonstrates an easy and useful way to obtain flavonoids and antioxidants from Phalaenopsis materials that can be used in the flower-based industry to make new functional ingredients.

Morphological Diversity and Genetic Parameter of Okra (Abelmoschus esculentus L. Moench) Genotypes.

<b>Background and Objective:</b> Considering that the potential for okra as an anti-diabetic is very high, while okra productivity in Indonesia is still low, a plant breeding program through variety development is needed. One of the initial activities that needs to be carried out is the characterization of various genotypes, both quantitative and qualitative characters. This research aimed to obtain information on the diversity of morpho-agronomic characters in okra genotypes. <b>Materials and Methods:</b> The experiment was conducted as a randomized block design, one factor is genotype with three replications. The materials used in this research were 20 okra genotypes. The experimental units used in this research were 60 units. Each experimental unit consists of 10 sample plants. Analysis of quantitative character variations used PKBT-STAT 3.1. Cluster analysis was carried out with PBSTAT-CL 2.1.2 with the Gower dissimilarity and average linkage clustering methods. Furthermore, analysis was carried out using SAS OnDemand for Academics to see the distinguishing characteristics between clusters. <b>Results:</b> There were differences in okra genotypes based on qualitative and quantitative characteristics. The most diverse quantitative character is the yield component, which is the fruit character. Variance in genetic and heritability showed broad and high criteria, respectively. Based on cluster analysis results, okra genotypes were grouped into 3 clusters with a cophenetic distance value of 0.40. Cluster 1 consists of 9 genotypes. Cluster 2 consists of 10 genotypes. Cluster 3 consists of 1 genotype the Red Hill Country genotype. The grouping in cluster analysis was carried out based on leaf width, number of fruits, fruit weight, fruit diameter and carpel thickness character. <b>Conclusion:</b> This diversity of okra germplasm can facilitate plant breeding activities in the future by selecting genotypes to serve as parents according to the objectives carried out.

Optimized Solvents for the Maceration of Phenolic Antioxidants from Curcuma xanthorrhiza Rhizome using a Simplex Centroid Design.

Background: Curcuma xanthorrhiza Roxb., from the Zingiberaceae family, is a famous plant native to Indonesia that is highly effective in treating diseases due to the various chemical compounds it contains. Objective: This study aims to optimize the extraction process for the phenolic content, with its antioxidant activity, from the rhizome of C. xanthorrhiza using different solvent (water, acetone, methanol, and ethanol) systems based on the simplex centroid design using the Design Expert 13.0 program. Methods and Material: Total phenolic content (TPC) was analyzed by colorimetry using Follin-Ciocalteu, while the antioxidant activity was measured based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP) using a spectrophotometer. Results: To measure TPC and DPPH, a special cubic model was used; to measure FRAP, a linear model was utilized. Each model demonstrated a good match with the R2 values for TPC (0.9808), DPPH (0.9583), and FRAP (0.7872). The combination of a mixture of water (0.409), acetone (0.307), and methanol (0.284) with a desirability level of 0.723, resulted in a TPC of 34.112 mg gallic acid equivalent (GAE)/g dry weight (DW), DPPH of 26.533 µmol Trolox equivalent (TE)/g DW, and FRAP of 92.353 µmol TE/g DW. This showed a high extraction efficiency which was optimal. Conclusions: The best condition to extract the rhizomes of C. xanthorrhiza was a ternary combination of solvents including water, acetone, and methanol in the proportions of 0.409, 0.307, and 0.284, respectively, with a desirability level of 0.723.

Effects of Methods and Durations of Extraction on Total Flavonoid and Phenolic Contents and Antioxidant Activity of Java Cardamom (Amomum compactum Soland Ex Maton) Fruit.

Free radicals contribute to the pathophysiology of degenerative diseases which increase mortality globally, including mortality in Indonesia. Amomum compactum Soland. Ex Maton fruit from the Zingiberaceae family, also known as Java cardamom, contains secondary metabolites that have high antioxidant activities. The antioxidant activity of the methanol extract of Java cardamom fruit correlates with its flavonoid and phenolic compound contents, which can be affected by different methods and durations of extraction. This study aimed to measure and compare the effects of extraction methods and durations on total flavonoid and phenolic contents (TFCs and TPCs) and subsequent antioxidant activities by the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical, ferric reducing antioxidant power (FRAP), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and cupric ion reducing antioxidant capacity (CUPRAC) assays. Methanol extracts of Java cardamom were produced by continuous shaking (CSE), microwave-assisted (MAE), or ultrasonic-assisted extractions (UAE) for three different durations. CSE for 360 min resulted in the highest TFCs (3.202 mg Quercetin Equivalent/g dry weight), while the highest TPCs (1.263 mg Gallic Acid Equivalent/g dry weight) were obtained by MAE for 3 min. Out of the investigated methods, MAE for 3 min resulted in the highest antioxidant activity results for the extracts. We conclude that the polyphenolic antioxidant yield of Java cardamom depends on two parameters: the method and the duration of extraction.

Phenolic, Flavonoid and Antioxidant Capacities Evaluation of Celosia cristata Resulted from Induced Mutation Using Ethyl Methane Sulphonate.

<b>Background and Objective:</b> <i>Celosia cristata</i> L. is an ornamental plant that has the potency to be developed as a medicinal plant. The mutation breeding of <i>C. cristata</i> targets the increase of biochemical compounds that are potential as antioxidants. This study aimed to evaluate the phenolic and flavonoid contents and antioxidant activity of the putative mutants of <i>C. cristata</i> in the M1 generation. <b>Materials and Methods:</b> The samples used in this study were flowers and seeds. Induced mutation, using ethyl methane sulphonate (EMS) was used to obtain M1 generation of putative mutants and twelve putative mutants were selected for polyphenol contents analysis composed of total phenolic (TPC), total flavonoid (TFC) and antioxidant activities analysis using two approaches, i.e., 2,2-diphenyl picrylhydrazyl (DPPH) method and ferric reducing antioxidant power (FRAP). <b>Results:</b> This study showed that total phenolics were varied from 11.73-18.06 mg GAE g¹ DW and total flavonoids were varied from 2.34-3.11 mg QE g¹ DW. Meanwhile, the antioxidant activity gain using the DPPH method ranged from 16.43-19.02 µmol TE g¹ DW and the FRAP method ranged from 40.72-59.61 µmol TE g¹ DW. The clustering analysis results formed three clusters with two clusters consisting of potential mutants with higher biochemical content and antioxidant capacities. It was found that total phenolic and flavonoids highly correlated with the antioxidant FRAP. <b>Conclusion:</b> Induced mutation using EMS can increase the diversity of biochemical characters and antioxidant activity of <i>C. cristata</i> and provide potential genetic material with higher chemical content for further development.

Influence of Combined NPK and Manure on Improving Growth, Photosynthetic Characteristic and Yield of Justicia gendarussa Burm. F.

<b>Background and Objective:</b> <i>Justicia gendarussa </i>(family <i>Acanthaceae</i>) is regarded as a medicinal plant valued for its beneficial pharmacological activities. A polybag experiment has been conducted at Bogor (Indonesia) to determine the effects on growth, photosynthetic and yield of various NPK ratios and manure fertilizer application in <i>J. gendarussa</i>. <b>Materials and Methods:</b> Six treatments (control: 50 g/plant manure, NPK (1.1 g/plant N:0.7 g/plant P₂O₅ and 0.6 g/plant K), 50+50%/plant (manure+NPK), 10+50%/plant (manure+NPK) and 50+10%/plant (manure+NPK) were performed in a randomized complete block design. Data of individual plants were extracted from various treatments: plant height, number of leaves, number of branches, chlorophyll content, photosynthetic parameters, yield and sugar content were collected. <b>Results:</b> The plant treated with 10+50%/plant (manure+NPK) demonstrated a statistically significant increase in plant height, number of leaves and number of branches with the value of 59.00 cm, 85.00 per plant and 9.50 per plant, respectively. Chlorophyll a (0.538 mg g¹ FW), chlorophyll b (0.192 mg g¹ FW) and total chlorophyll (0.730 mg g¹ FW) were significantly improved by the treatment of NPK fertilizer. The application of manure and NPK at (50+50%) and (50+10%) was significantly increased stomatal conductance and transpiration rate, respectively. Leaves yield (9 g/plant DW) was improved by the application of 10+50%/plant (manure+NPK) fertilizer. The highest sugar content was obtained from <i>J. gendarussa</i> growth with 50+50%/plant (manure+NPK) followed by 10+50% plant (manure+NPK) fertilizer. <b>Conclusion:</b> The <i>J. gendarussa</i> plants could benefit from combined manure and NPK fertilizer application in growth, stomatal conductance, intercellular CO₂ concentration, transpiration rate, leaves yield and sugar content.

Genotype selection for phytochemical content and pharmacological activities in ethanol extracts of fifteen types of Orthosiphon aristatus (Blume) Miq. leaves using chemometric analysis.

Orthosiphon aristatus (Blume) Miq. of the Lamiaceae family, called as kumis kucing in Indonesia, is a valuable medicinal plant for their pharmacological properties. The present study comprised of fifteen genotypes of O. aristatus was undertaken to evaluate the genotypes based on phytochemical content and pharmacological activities of leaves ethanol extract. Chemometric analysis (correlation and principal component analysis) was also used to investigate the genetic variability based on phytochemical content and pharmacological activities of O. aristatus genotypes. Results of phytochemical characterization showed that total phenolic ranged from 1.48 to 36.08 (maximum in A15) mg GAE/g DW, total flavonoid ranged from 0.10 to 3.07 (maximum in A15) mg QE/g DW, sinensetin ranged from 0.36 to 4.02 (maximum in A11) mg/g DW, and rosmarinic acid ranged 0.06 to 7.25 (maximum in A7) mg/g DW. Antioxidant activity was tested using DPPH and FRAP assay. Antioxidant results showed that DPPH ranged from 1.68 to 15.55 (maximum in A15) µmol TE/g DW and FRAP ranged from 0.07 to 1.60 (maximum in A1 and A7) µmol TE/g DW. The genotype A8 showed the highest cytotoxic activities against HeLa (66.25%) and MCF-7 (61.79%) cell lines. Maximum α-glucosidase inhibitory activity was recorded in genotype A2 with the value of 62.84%. The genotypes A1, A2, A7, A11, and A15 were identified as superior based on their phytochemicals content and pharmacological activities coupled with chemometric analysis. This finding is important for breeding studies and also the pharmaceutical perspective of O. aristatus.

In vitro antiproliferation activity of Typhonium flagelliforme leaves ethanol extract and its combination with canine interferons on several tumor-derived cell lines.

BACKGROUND AND AIM: Tumor disorder is one of the degenerative diseases that affected human and animals and recently is tend to increase significantly. The treatment of tumor diseases can be performed through surgical, chemotherapy, radiotherapy, biological substances, and herbs medicine. Typhonium flagelliforme leaves extract known to have an antiproliferation activity, while interferons (IFNs) one of the cytokines that first used as an antiviral agent was also known to have antitumor activity. Nowadays, the treatment of tumors using a traditional way, including the use of herbal substances, becomes popular. Some limitations of the antitumor activity due to resistant development of the cell to some substances were one of the problems on why the treatment of cancer was unsuccessful. This study aimed to elaborate the synergistic effect on the antiproliferation and anti-angiogenesis activities of the combinations between T. flagelliforme leaves ethanol extract and canine natural (natural canine IFN [nCaIFN]) and recombinant (recombinant canine IFN [rCaIFN]) IFNs on tumor-derived cell lines to find the new potential antitumor substances. MATERIALS AND METHODS: The extraction of T. flagelliforme leaves was performed using the maceration method and followed by phytochemical screening assays. According to the result of LC50 by the brine shrimp lethality test, the dose used for T. flagelliforme extract was 120 ppm while the dose of IFNs was 102 U/ml. The tumor-derived cell lines (canine squamous cell carcinoma [CSCC], canine mammary gland benign mixed tumor/MCM-IPB-B3, and feline squamous cell carcinoma [FSCC]) and normal rabbit endothelial cells were cultured and maintained on Dulbecco's Modified Eagle's Medium DMEM/Ham-F12 medium supplemented with 10% fetal calf serum, antibiotic, and antifungal. The antiproliferation activity was assayed by calculated the total cell number after treated with the tested substances. The antiangiogenesis assay was performed using in vitro method on rabbit normal endothelial cells and in ovo using chicken chorioallantoic membrane (CAM). RESULTS: The phytochemical screening test of the T. flagelliforme leaves ethanol extract indicated that the compound consisted of flavonoid, steroid, and tannin. The antiproliferation activity was increased in the combination of substances compared to the single exposure of each substance on all tested tumor-derived cell lines. There was no significantly different on the antiproliferation activity between a combination of T. flagelliforme with nCaIFN or rCaIFN in every single tested cell lines, but the comparison of this activity among the three tumor-derived cell lines seem that the antiproliferation activity is more effective on CSCC cell lines compared to the canine mammary gland benign mixed tumor and FSCC cell lines. A similar pattern of synergistic effect was also detected on the anti-angiogenesis activity in vitro using rabbit endothelial cells as well as in ovo assays. The most effective of the in vitro and in ovo anti-angiogenesis activity was observed on the combination substances between T. flagelliforme extract and rCaIFN compared to other treatments. CONCLUSION: There was a synergistic effect on the antiproliferation and antiangiogenesis activities of the combination between T. flagelliforme and canine IFNs (natural and recombinant) and this result could be developed as another alternative on the cancer treatments.

Impact of composted guava leaves and neem seeds on the growth and curcuminoid- and xanthorrhizol-yields of Curcuma zanthorrhiza RoxB / Impacto de folhas de goiabeira compostada e sementes de nim no crescimento e r endimento de curcuminóides e xanthorrizil de Curcuma zanthorrhiza RoxB

ABSTRACT: The compost from the waste of pharmaceutical industries, such as guava leaves (GL) and neem seeds (NS) is used in organic agriculture. Curcuma zanthorrhiza RoxB. is a widely recognized herbal medicine that grows natively in Indonesia. Curcuminoids and xanthorrhizol are the primary bioactive components of C. zanthorrhiza. In this study, we aimed to evaluate the impact of GL and NS compost on the growth and bioactive yields of C. zanthorrhiza. Treatments consisted of cow manure, GL compost, NS compost, GL+NS compost, or a no compost control, at 2 and 4 kg per plant. Results demonstrated that the NS and GL+NS compost applied with 4 kg per plant had increased fresh rhizome yield compared with the other treatments. Composted NS at 2 kg per plant increased the plant height and pseudo stem diameter traits compared with the control treatment. The compost application of GL+NS at 2 and 4 kg per plant significantly increased the leaf length and number of shoots. All treatments showed unchanged the leaf width and number of leaves. The compost application of GL and NS (2 kg per plant) showed higher production of curcuminoidsthan the control. The compost consisting of GL (2 kg per plant), NS (4 kg per plant), and GL+NS also increased the production of xanthorrhizol compared with the control treatment. Results illustrated the practical application of GL and NS composts from industrial pharmaceutical extraction waste for the organic farming cultivation of C. zanthorrhiza.

RESUMO: O composto dos resíduos das indústrias farmacêuticas, como folhas de goiaba (GL) e sementes de nim (NS), é usado na agricultura orgânica. Curcuma zanthorrhiza RoxB. é um medicamento fitoterápico amplamente reconhecido que cresce de forma nativa na Indonésia. Os curcuminóides e o xanthorrizol são os principais componentes bioativos de C. zanthorrhiza. Neste trabalho, objetivou-se avaliar o impacto do composto GL e NS no crescimento e produtividade bioativa de C. zanthorrhiza. Os tratamentos consistiram em esterco de vaca, composto GL, composto NS, composto GL + NS ou controle sem composto, em 2 e 4 kg por planta. Os resultados demonstraram que o composto NS e GL + NS aplicado com 4 kg por planta aumentou a produção de rizoma fresco, em comparação com os outros tratamentos. A NS compostada a 2 kg por planta aumentou as características de altura da planta e diâmetro do pseudoestêmico em comparação com o tratamento controle. A aplicação de composto de GL + NS em 2 e 4 kg por planta aumentou significativamente o comprimento das folhas e o número de brotações. Todos os tratamentos apresentaram alteração na largura e número de folhas. A aplicação de composto de GL e NS (2 kg por planta) apresentou maior produção de curcuminóide do que o controle. Os compostos constituídos por GL (2 kg por planta), NS (4 kg por planta) e GL + NS também aumentaram a produção de xanthorrizol em comparação com o tratamento controle. Os resultados ilustraram a aplicação prática de compostos GL e NS de resíduos de extração farmacêutica industrial para o cultivo orgânico de C. zanthorrhiza.

Nrf2-inducing and HMG-CoA reductase inhibitory activities of a polyphenol-rich fraction of Guazuma ulmifolia leaves

To fractionate and identify polyphenols from Guazuma ulmifolia Lam. leaves, and to explore their antioxidant, 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitory, and Nrf2 modulatory activities. Methods: The 1,1-diphenyl-2-picrylhydrazyl assay was used to evaluate the antioxidant activity of a polyphenolic fraction of the extract of Guazuma ulmifolia Lam. leaves. THP-1 gene reporter cell lines constructed with a transcriptional response element specific for Nrf2 and a minimal promoter for the firefly luciferase–green fluorescent protein transgene were used to determine the effect of the polyphenolic fraction on the Nrf2 signaling pathway. Furthermore, an assay of HMG-CoA reductase inhibitory activity was performed by using a commercial enzyme kit. Polyphenolic compounds were identified by liquid chromatography-tandem mass spectrometry. Results: The polyphenolic fraction showed fairly strong antioxidant activity [IC50 = (14.90 ± 4.70) μg/mL] and inhibited HMG-CoA reductase activity by 69.10％, which was slightly lower than that by pravastatin (84.37％) and quercetin (84.25％). Additionally, the polyphenolic fraction activated the Nrf2 antioxidant signaling pathway at 500 μg/mL. Eleven subfractions resulting from the column chromatography separation of the polyphenolic fraction also showed relatively strong antioxidant activities (IC50: 17.46–217.14 μg/mL). The subfraction (F6) stimulated the Nrf2 signaling pathway and had HMG-CoA reductase inhibitory activity (65.43％). Moreover, the subfraction contained two main flavonoids: quercetin and quercimeritrin. Conclusions: The polyphenolic fraction of Guazuma ulmifolia could induce antioxidant genes via the Nrf2/antioxidant regulatory elements pathway, and is a promising candidate for an inhibitor of HMG-CoA reductase.

Xanthorrhizol contents, α-glucosidase inhibition, and cytotoxic activities in ethyl acetate fraction of Curcuma zanthorrhiza accessions from Indonesia

ABSTRACT Curcuma zanthorrhiza Roxb., Zingiberaceae, a species from Indonesia with xanthorrhizol as the major metabolite, has been used as a folk medicine in several of pharmacological activities. This work aimed to evaluate the xanthorrhizol contents, α-glucosidase inhibition, and cytotoxic activities in ethyl acetate fraction from accessions of C. zanthorrhiza. High-performance liquid chromatography investigated xanthorrhizol content with the standard. Pharmacological activities were evaluated by inhibition of α-glucosidase, the brine shrimp lethality test, and anticancer activity. The ethyl acetate fraction yield varied from 8.24% (Karanganyar) to 13.13% (Sukabumi). The xanthorrhizol contents were found to be in the range 43.55% to 47.99% with Ngawi and Wonogiri promising accessions having the lowest and highest value, respectively. IC50 value for α-glucosidase inhibition ranged from 339.05 µg/ml (Karanganyar) to 455.01 µg/ml (Ngawi). LC50 value for cytotoxic activities ranged from 33.25 µg/ml (Ngawi) to 42.28 µg/ml (Karanganyar) in brine shrimp lethality test, 3.10 µg/ml (Karanganyar) to 9.85 µg/ml (cursina-III) in Vero cell, and 1.17 µg/ml (Ngawi) to 6.83 µg/ml (Sukabumi) in MCF-7 cell. In this study, C. zanthorrhiza accessions have a high in xanthorrhizol contents and cytotoxic activities that showed a high potential of studied accessions for breeding programs on a commercial scale.