Phytochemical characterization of the ethanolic extract of Kaempferia galanga rhizome for anti-oxidant activities by HPTLC and GCMS

Background The rhizome of Kaempferia galanga (K. galanga) was collected from Meghalaya, India, and its ethanolic extract was obtained by freeze-drying or lyophilization process, which was then assessed for its in vitro anti-oxidant activity and phytochemical characterization using high-performance thin-layer chromatography (HPTLC) and gas chromatography-mass spectroscopy (GCMS). Results In vitro anti-oxidant activity analysis shows an inhibitory concentration (IC50) value of 1.824 mg/mL and 0.307 mg/mL for, α, α-diphenyl-ρ-picrylhydrazyl (DPPH) and 2, 2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays, respectively. Total polyphenol content (TPC) of 23.55 ± 0.5 mg gallic acid equivalent (GAE)/g dry weight of extract and total flavonoid content (TFC) of 100 ± 1.414 mg rutin equivalents (RE)/g dry weight of extract were found. High-performance thin-layer chromatography (HPTLC) analysis shows the best separation of bands at different retention factor (Rf) values, when employing the solvent system 2-butanol/1-propanol/water in the ratio of 3:1:1 (v/v/v). Gas chromatography-mass spectroscopy (GCMS) analysis confirms the presence and identification of various phytocompounds, with ethyl p-methoxycinnamate identified as the major active compound. Conclusion Freeze-dried ethanolic extract of K. galanga (rhizome) possesses anti-oxidant activity. Ethyl p-methoxycinnamate is present as the major bioactive component (about 94.87% of the total area composition), and since it has very important and diverse medicinal properties, a freeze-drying process (lyophilization) can be utilized for its isolation and extraction.

Analysis of Ethyl p-Methoxycinnamate from Kaempferia galanga L. Extract by High Performance Liquid Chromatography

Ethyl para-methoxycinamate (EPMS) is a major compound of Kaempferia galanga L that has anti-inflammatory effect. The purpose of this study was to determine of EPMS in Kaempferiae galanga L rhizome extract by High Performance Liquid Chromatography (HPLC) and evaluated the performance of the analysis. This study included determination of system suitability, accuracy, precision, linearity and range, limit of detection (LOD) and Limit of quantitation (LOQ) and selectivity. The results of system suitability test HPLC System for EPMS analysis were as follows isocratic elution system of a mobile phase mixture of methanol: water (70:30) containing 0.1% TFA, uv detector at a wavelength of 308 nm using column C18 (150 × 4, 6mm, 5μm) flow rate 1 ml / min. From the analysis, it was found that the average EPMS content was 78.74%. Then method had linear concentration range from 5-360 ppm, with R ² = 0.9999. The LOD and LOQ were 7.0722 ppm and 21.4311 ppm respectively. The accuracy of this method that represented by % recovery was 98.02% - 101.26%. The precision of this method that expressed by Relative Standard Deviation (RSD) was 1.57%. The selectivity of this method that showed by resolution value was 2.6. Based on the results of the system suitability test and analysis performance evaluation,all parameters met the requirements.

In vitro Microrhizome Induction and Essential Oil Production from Aromatic Ginger Kaempferia galanga : An Economically Important Medicinal Herb

Kaempferia galanga L. or ‘aromatic ginger’ is a stem-less herb in Zingiberaceae having different pharmacological properties like antioxidant, antimicrobial, nemeticidal, vasorelaxant and wound healing activity. The plant is generally a vegetatively propagated annual herb; its conservation using conventional methods takes more time to get sufficient amount of planting materials for commercial cultivation. Micropropagation by in vitro methods helps to overcome the present demand for this high sought medicinal and aromatic species. At present the concern on in vitro propagation is directed to rhizome or storage organ induction for productive acclimatization and to reduce the injury during transportation. Microrhizomes are the small rhizomes developed in in vitro conditions and its induction is an effective biotechnological tool for the production of quality planting materials as they are genetically stable and disease free. The present study is discussing the role of silver nitrate (AgNO3) along with sucrose in in vitro microrhizome induction in K. galanga for the first time. MS medium fortified with 2.0 mgl-1 AgNO3 along with 6% (w/v) sucrose produced maximum amount of microrhizomes i.e., 4.52±0.11 g after 3 months that increased to 5.70±0.20 g in six months of harvesting. Here we also reports the comparative analysis of chemical constituents in the essential oil of in vivo rhizomes and in vitro microrhizome through GC-MS analysis that further reveals the superior characteristics of the microrhizomes in terms of the bioactive components ethyl p-methoxy cinnamate and ethyl cinnamate, the esters that contribute the nematicidal, antituberculosis, anti-inflammatory, antifungal and larvicidal properties to the oil. This protocol for in vitro microrhizome induction can be used for the commercial production of rhizomes and essential oil in K. galanga and the outcome of this study can be further used for mass production of pathogen-free microrhizomes and conservation for its sustainable utilization of the species.

Qualitative and Quantitative Study of Phyto-Constituents and Antioxidant Potential of Rhizomes of Kaempferia galanga, Kaempferia parviflora and Kaempferia pulchra

Medicinal plants are the potent source of biologically active compounds and have always been of field of interest for the effective chemotherapeutic agents and offering a broad spectrum of activity with greater emphasis on preventive action. The objective of this study was to screen the phytochemicals, estimate the content of phenolic, flavonoids and alkaloids compounds and determines the antioxidant capacity of the rhizomes of Kaempferia galanga Linn, Kaempferia parviflora and Kaempferia pulchra (K. galanga, K. parviflora and K, pulchra, Zingiberaceae). Qualitative analysis of various phytochemical constituents and quantitative analysis of total phenol, flavonoids and alkaloids were determined by the well-known test protocol available in the literature. The ethanolic extract of rhizomes of K. galanga, K. parviflora and K, pulchra was studied for antioxidant activity on different in vitro models namely 1,1-diphenyl, 2-picryl hydrazyl (DPPH) assay and Nitric oxide (NO) radical scavenging method. Phytochemical analysis of ethanolic extract of all three plants revealed the presence of flavonoids, alkaloids, saponins, phenolics, carbohydrate, and tannin. The total phenolic, flavonoids and alkaloids content of ethanolic extract of K. galanga, K. parviflora and K, pulchra rhizomes were 0.813, 1.146; 1.047, 1.237, 0.285; 0.755, 0.822, 0.975/100mg respectively. All extracts showed dose dependent free radical scavenging property in the tested models, which was comparable to that of ascorbic acid. The present study concluded that the crude extract of K. galanga, K. parviflora and K, pulchra rhizomes is a rich source of secondary phytoconstituents which impart significant antioxidant potential. It is expected that the important phytochemical properties recognized by our study in the indigenous medicinal plants will be very useful in the curing of various diseases when taken along with our food.

THE DETERMINATION OF ETHYL P-METHOXY CINNAMATE IN KAEMPFERIA GALANGA L. RHIZOME EXTRACT HARVESTED IN RAINY AND DRY SEASONS

Objective: Kaempferia galanga L. rhizome (KGR), has been empirically used in Indonesia, particularly by Javanese, to cure inflammation. KGR contains various secondary metabolites which explain its pharmacology activities, among them is ethyl p-methoxycinnamate (EPMC). However, due to the different seasons of our country, the yield of extraction is often unalike. In this work, we determined the percentage of yield (w/w), the water content (thermogravimetric method), and the concentration of EPMC in the Ethanol extract of Kaempferia galanga L. Rhizome (EEKG) harvested from the rainy (EEKG-R) and dry seasons (EEKG-D). Methods: The sun-dried rhizomes were cold macerated for 3x24 h with 70% ethanol, filtered, and the solvent was evaporated at 40-45 °C until a viscous extract was obtained. The determination of EPMC in the extract was carried out using the RP-HPLC standard addition method. Detection was set at 308 nm; injection volume 20 µl; flow rate 1.0 ml/min. The column used is C18 (length 250 mm, internal diameter 4.6 mm, particle size 5 µm). Results: The yield of EEKG-R (harvested in the rainy season) = 14.56% w/w, water content = 4.37%, and the EPMC = 0.01%. Meanwhile the yield of EEKG-D (harvested in the dry season) = 5.79% w/w, water content = 18.76%, and the EPMC = 0.001%. Conclusion: Different climates affect the percentage yield and the quality of the extract. In our work, the EEKG-R (harvested in the rainy season) revealed a better quality compared to that of EEKG-D (harvested in the dry season) This study gives important information to standardize and optimize the harvest time of KG rhizomes for drugs development, which are strongly influenced by seasonal differences.

Introduction pages

Notulae Scientia Biologicae (http://www.notulaebiologicae.ro), Issue 4, Volume 13, 2021: The papers published in this issue represent interesting novelties in different topics of life science. Among the exciting researches or reviews, we invite readers to find news about: Haemato-biochemical alterations and acute toxicity study of hydro-methanol root extract of Anacardium occidentale in cockerels; Diversity of butterflies (Lepidoptera: Rhopalocera) in Bhubaneswar, Odisha, India; Molecular detection of Trypanosoma species and haematological alterations in four trypanosome-infected Nigerian horses; Cryopreservation of aromatic ginger Kaempferia galanga L. by encapsulation-dehydration; Comparative study of some physicochemical and biological properties of effect host species variation on the relationship Saharan parasitic plant Cistanche violaceae (Desf.) Beck.; Comparative leaf anatomy of ten Nepenthes L. species (Nepenthaceae) from Peninsular Malaysia; Seed germination response of Indian wild pear (Pyrus pashia) to gibberellic acid treatment and cold storage; Molecular and morphological characterization of Globodera rostochiensis (Wollenweber, 1923) Skarbilovich, 1959 from Egypt.

Immunomodulation Effect of Kaempferia galanga Ethanolic Extract On In Vitro Lymphocyte Cell Proliferation

<p><em>Kaempferia galanga</em> L. is a traditional medicine with antitumor properties, as indicated by its immunomodulatory activities. This study aimed to determine the effect of <em>K. galanga</em> on lymphocyte cell proliferation activity as an indicator of immunomodulatory properties. This study was conducted at the Indonesian Research Center for Veterinary Science (February to April 2018). The immunomodulatory activity of the extract was evaluated with an <em>in vitro</em> splenocyte proliferation assay. The assay was based on cellular enzymatic synthesis to transform the XTT from formazan tetrazolium as an indicator. The <em>K. galanga</em> extract was obtained by 96% ethanol extraction. The test was conducted in an aseptic condition, consisted of five treatment groups with three replications each. Three groups of splenocyte cell culture, each with extract concentration of 2.5 µg.ml^-1, 25 µg.ml^-1, and 250 µg.ml^-1, as well as a positive (Concanavalin A/Con A) and negative (cell only) control. The cell suspension (10x10⁴cells/ml) was distributed on 96-well plates and cultured following the treatment groups. The same five plates were made for five days of observation and retrieved daily by observing an Elisa reader at 450 nm. The extract of <em>K. galanga </em>at 2.5 µg.ml^-1, 25 µg.ml^-1, and 250 µg.ml^-1 significantly (P &lt;0.05) promoted splenocyte proliferation compared to control. Therefore, it was expected that <em>K. galanga </em>has a high potential to be used as immunomodulators. Hence, further investigations should be done to clarify the mechanisms of the immunomodulatory effect of <em>K. galanga</em> as an antitumor <em>in vivo</em>.</p>

In vitro Anti-inflammatory Properties in Various Extracts (Ethanol, Chloroform and Aqueous) of Kaempferia galanga Linn Rhizome

Introduction: Kaempferia galanga is a medicinal plant belonging to the family Zingiberaceae: ginger family. It is treated as a folk traditional herb. Anti Inflammatory property refers to the ability of a substance to reduce inflammation or any of its 5 cardinal signs. Aim: To assess and compare in vitro the anti-inflammatory properties of various extracts (ethanol, chloroform and aqueous) of Kaempferia galanga L Rhizome. Materials and Methods: Protein Denaturation Inhibition was carried out in vitro and statistical analysis was done using ONE WAY -ANOVA and Duncan Multiple Range Tests. The test was done in triplicates. Results: Chloroform extract of Kaempferia galanga rhizome has the best anti-inflammatory potential followed by Ethanol and Aqueous extracts of the rhizome. Conclusion: With further in vivo and clinical research, the chloroform, ethanolic and aqueous extracts of Kaempferia galanga can be recommended as a novel, innovative and potent anti-inflammatory drug in the market as it’s natural and doesn’t have side effects.

Cryopreservation of aromatic ginger Kaempferia galanga L. by encapsulation-dehydration

Kaempferia galanga L. is an endangered multi-purpose medicinal plant in Family Zingiberaceae, the rhizomes of which are used for several ayurvedic formulations. Encapsulation-dehydration (ED) method was optimized for cryopreservation of shoot tips of K. galanga. Shoot tips (STs) bearing the apical meristem dissected from the established in vitro shoot cultures were preconditioned in MS+0.4 M sucrose prior to encapsulation in calcium alginate and the beads subsequently transferred to MS liquid+0.3 M sucrose for 3 days afterward dehydration inside the laminar airflow for 4 hours upon rapid freezing in LN and rapid thawing produced maximum 62.2% survival and 46.7% regeneration rates. Shoot regeneration was observed from the apical meristems exclusive of intermediary callus phase. The plantlets regenerated from cryopreserved STs transferred to the field were phenotypically analogous with the mother plant.

Analysis of bioactive compounds present in Kaempferia galanga rhizome collected from different regions of East Java, Indonesia

Kaempferia galanga, also known as kencur in Indonesia, is a medical plant that is traditionally used in various human diseases therapy. Ethyl p-methoxycinnamate (EPMC) is an essential bioactive compound in kencur that plays a role in the management of diseases. Kencur is widely spread in East Java. Thus this study aims to identify and analyze the bioactive compounds in kencur rhizome from a different region in East Java. This study was conducted from June 2020 until November 2020. The samples were collected from 12 regencies in East Java, including Banyuwangi, Blitar, Gresik, Kediri, Lumajang, Madura, Malang, Mojokerto, Nganjuk, Pacitan, Ponorogo, and Trenggalek. A sample from Wonogiri (Central Java) was used as a comparison because its cultivation procedure has been standardized. The altitude of sample accessions location was 15-995 masl (m above sea levels) with the rain intensity of 500-2000 mm3. Based on soil analysis, Banyuwangi had good soil characteristics with high total nitrogen, available P2O5, cation exchange capacity (CEC), and organic carbon. The bioactive compounds were analyzed using gas chromatography-mass spectrometry (GC-MS). The results showed that all the samples contain three major compounds: EPMC, pentadecane, and (Z)-ethyl cinnamate. The highest content of EPMC was obtained in Wonogiri (79.8%), followed by Malang (78.28%), Blitar (77.23%), and Trenggalek (73.33%). It can be concluded that the kencur samples from Malang, Blitar, and Trenggalek have a high content of EPMC, thus could become the source of kencur used in further medicinal research.